S.C-loç,É,

('

Non -Llpopolys aocharide Protecttve Antl gen s of Vi brio

Cholerae

Dharam Pal Sharma

I

ô*--rr-rçtr. I C".c. d å'¿, f .'ì,*¡

Department of Mic¡obiolog and Immu¡olo6l

Univenityof Adelaide

Adelaide 5000

Australia

A Thesis Submitted for the Degree of Doctor of Phibeophy

lYlarch 1990

. , - . , ., :,

', ,, .¡;'-.,¡5; ¡1¡1; .j

Non-Lipopolysaccharide Antigens

:'eof Vibrio cholero,e .i t..

"+.-t¡-,;+

ÐDhararrt PaI S lït

I dedicate this thesis to rywife and to my children for tùeir hue and patienoe. Thankyou

foryour loYal suPPort

Abstract

,,hls thesig covers the investigtion of non-LPS antþene exp,rressed byboú biotypeo (Ctassical

and El Tor) and serotypes (Ogrwa and Inaba) oî,l4bb døbæ and the determi¡ation of the

eúent to which these are shared e¡ 6fi'ain-specific, since rhic will be a criticat determinant of

theirvaccine potenriât. The established inf¡nt mou6e modelwao used to iludy both virulence

and protection The non-LPS antigens are eryressed both þ Claosical and El Tor drains tested

eæept for four old bolaæs of the El Torbiotype. The protective activitie¡of antibodies to the

Bhåred antþns correhted with their capacities to hhibit the å¿ vifro atræhment of vibrb

organisms to isolated murine enterccytes.

Suboequentsbrdies were di¡ected to*,a¡ds identi$ingtìe non-LPS antiget(s)- Su¡diee

from orreßeas suggested foxin<oregulatedpili GCP) is an important pilue colonþefien f¡ctor.

Considerable time wag spent in trying to find expressiea ef thi¡ laclor h víto elldthen witù the

aid of SDS-PACE, immunobloüing and electron microscopy techniques including im-

munogold-labetling itwas found to be eryreseed only by Classical strain&

The infant mouse cholera rcdel was used to evaluate the ¡el¿tive imporønce of TCP

as protectiveantþns of.V dpbæOl. Antibodies to TCPwere sufficþntto conferp¡otection

againet two Classical strains, and were mo¡e effrcient if the challengevibrios were culu¡red for

TCP ery,ression In cont¡ast, such antibodies did not protect mice aeainst challenge with any

of four st¡ai¡s of El Tbr biotype. Since two of the latær have previousþbeen shoq¡n to po*sess

non-Lps prot€ctive antrgenq the¡e results ouggest that TCP a¡e not the onþ such antigea il

thb model

A genrm containing antibodies to non-LPS protectiye antigels of V úolsac has been

use4 afær extensive absorption, to fac¡litate the cloningof genes involved in the rynthesis of

TCp A gene bankwa¡ conetructed fromV dptaae 2,17561 DNA using a mobiliz¡ble cæmll

vectorinE coli, a¡deubsequently transfer¡edþ conjugation i\lovúolqae O17. Thb shai¡

does not produce Tep ¿n vúto endlacts non-LPS protective antþns. E¡Ét pooitive clones

were isolate4 and of these, four producod TeP as deærmined by eþct¡on nicroecopic and

immunoblottinganaþs€& TCP positive O17 cloneswere 70-fold morevirulent^han fQp-¡sgp-

tive clones or O17 in the infant mouse cholera modet Only the former could remove p,rotective

anftodies by absorption and upon immunization elicit protective antibodieo specific for

non-LpS components. As a oorollary, serum containing antibodþe to rcP protoct€d mbe

from challenge with TCP-poeitive cloneo, but not with rcP-negative clones or O17,

demonstratingthatantibodies to TCP a¡e sufficient to mediate protectio¡ in thb rystem- The

data indicaæ that Ttxt ¡s both avinrlence determinant and a protective antþn in the i¡h¡t

mouse cholera model This çþrt þ the best of my knowtedge, hm demo¡etrated for the fnst

time the protective efrect of anti:TCP antibodies albeit i¡ the infa¡t lDouse cholera model

Thi¡ thesb ç6¡¡r¡ins no material which h¡¡ been accepted for the 8s'ad of any other degree or

diploma in any Univerrity ¡nd to the be¡t of Ey tûowledge a¡d belie[ tf,is thesis contains no

material psodoruþ prblbhed or wriüen þ another perso& ercePtuùere due reference i¡

made in the ûext of the tùeeis. The author oonsents to the thesb beiog made available for

photooopyiagandloan, if applicabþ and if accefled for the awad of the &g¡æ'

IÌù¡ram P¡l Sh¡ma

Acknowledgements

I wish to €fpress my fupest appreciation and gntitude to my supervísor, Dr' SÛephen R

Ãtt i¿ge for his excellent guidance and encouraçment throug[tout the cour¡e of tlis proiec¿

I am deepþ indebted to hofessor Derrick Ronley for giving me the opportunity þ conduct

thb research in the Department of Microbiologl and Immunologl and for valuable suggestions

and timeþ critbisn&

My hearúelt thanks go to: Eh. Connor Thomas for hb eryertire in eþctron micra-

co[y; Dr. Paul Manni¡g for his helpful discussions; Gafy Fenny and ct¡is cnrsaro fo¡

professional hetp in photognphy. My deep 8en8e of appreciation Pes Ûo Atbeft Horcn'oo4

Chief of the Medicat School 6¡imal House, for his ercelþ¡t mai¡ænance of tle i¡fa¡t mouse

colony without which thb proiect would not have been pæsible.

I c'uld also like 1e ¡[¡nk past and pres€nt colleagues in Labs 6 and Enætorra¡ Labo for

mâEng it such a pleasant envi¡onment in which towort I ac&nw'ledge also the receiptof a

university of Adelaide Postgraduate scholanhip duriag the period of Ey research'

Last, but by no meatrg leas! I am greatly indebted to my wife, Nen4 for her extreme

patience during the time'his thesis was being assembled.

Publications

Materiat contained in this thesis has been publbhed i¡ the following

Sharma D.p., Aüridge S,, HackeüJ. & Ror*'ley D. (19SÐ Non-lþpolyeaccharide protective

antþenssharedþClaseicålandElTorbiot¡rpes ofVibrío úobæ, Iotunalof Infætbns

Dísezrvs l55z 716-7?3..

Sharma D.P, Thomas Q.J., Hall R[L,I-er¡ine M.]vf. & Aüridgp S.R. (1989) Signiñcance of

toxinoregulated pili as protective antigeno of,V dplqw in tûe inf¡nt mouse model

Vacche. 7: 151456.

Sharma D.P., Stroeher U.H, Thom¡e CJ., Manning PÂ & Affiidgp SR (1989) The charac'

terization of a maior non-LPS protective antigen of.Víb¡bchobæ sEa¡n 21756l and

mobcu tar cloning of the TC? l¡: A dvwtø b tr^xath on dplaa ø¿ rclale¿ diarrhæt

(Eds. Y. Dkeda & R-B. SacL). Vol. VIIL Toþo: KTK publicatio¡s. In P¡es*

Sharma D.P., Stroeher U.H., Thomas CI., Manning P.A & Arridgp S.R (1990) The torin-

coregulated pilua (ICP) oî. Wbb úolawz molecular clqning of genes invoþed i¡

biosynthesb and waluation of TCP as a protective antigen in the infant mouse model

Mìøobíal Pathogencsí& In Pr€ss"

Abbrevlatlons

Ap

CFA

CFB

CP

CT

DIC

dNTP

DNA

E coli

ETISA

EM

E]IEÊ,

Gur

IIAg

ID

tDa

kn

LD

LPS

MFRIIA

MoAb

MSITA

NA

NB

ampicillin

oolonizati,on factor agar

colonization fætor broth

clone probing

cholera toxin

duodenal inocuhtbn with cecal lþtion

deoxy-nucleoeide triPhosPhaæ

deo:ryribonu cleic acid

Esrlwíc.hhcoli

enzyme-linked immunoeo¡bent assãy

elctron micrucoPY

enterotoxþnic

monosialoeYl gan glioeide

hemagglutinins

infectiou¡ dose

kilodalton

Lanauycia

leih¡l dæe

lipopolyt""charide

þ-m¡ nn e6e-frrcose-re¡ietant hemagglutinin

monoclonal antibodY

D-mannose+ensitive hemagglutinin

nutrient agar

nutrientbroth

OD

OMP

oRs

oRr

PAGE

PBS

PP

R

RBC

Sark-OMP

s

w

SDS

SE

SHA

Sm

SRBC

TBS

Tb

TCP

TSA

TTBS

ttg

pl

Vchobæ

optical deûsity

outer membrane Protein

oral rehydration solution

oral rehydration theraPY

poþacrylamide gel electrophoresie

phosphaæ buffered saline

Peyer'e patches

resistant

redblood cells

Sarkosyl-insoluble OMP

sensitive

subcuttneot¡s

¡odium dodecYl rulPhate

¡tanda¡d error

soluble hemagSlutinin

ctrepomycin

sheep red blood cell¡

Îis-bufiered salhe

tetracycline

f orin coregulated Piltts

lis-buffered sati¡e containing sodium a^&

Tlisåufiered saline 6paraining Tlreen Z)

microgram

nicrolite(t)

V¡bito ùolaac

Tbble of Contents

1 Introduction

1.1 HistoricalBacþround . . . . .'''''''

1.2 Etiologr

l.Ll Characteristics of the bacterium

l.Z2 Claseification

13 Fathogenes¡s andPathophysiologr 4

1.3.1 lngestionandinfectiousdose "" ""' 5

132 Penet¡ationoftlemucuslayer'''''''''' "' 5

1.3.3 Adherence to the epithelium of the smâll iltesti¡e

1.3.3.1 F'lagsllar Proteinr

1.3.1,2 Lipopoþsaccharide (LFS)

1.3.3.3 Hsmagglutinins (Htu)

1.3.3.4 Pili¿finbriae

1.3.4 hoduction of enterotoxin and fluid accumulation

1

1

3

3

3

6

7

I

8

10

11

1.3.5 Patholog ""'13

1.6 Ïleatment .... ""

1.4 ClinicalManiÍesations ....;... "" ""'13

1.5 Diagnoois 14

I

15

1.7 Epidemiology.. " " '16

1.7.1 lncidence " " '16

1.7.2 Aqrntic¡eservoi¡s ....17

1,7,3 Ihansmission "" "'19

1.8 Potential protective antþns of.V eholsac ' ' ' ' ' '21

1.8.1 LPS " "21

1.8.2 Non-I-PS antigens n

l.8.Zl OMPB B

I.&ZZ Flagellar (þ antþens - . . -24

l.8.Zg HAs. ............ ..U

l.&24 Pili ...25

1.&3 CT. .... .-..25

l.g Immunig ûo cholera . . .n

1.9.1 SystemicantibodyresPonsesto?clpbæ " ""n

1.9.2 Developoe¡tofintestinalslgAantibodyrcsponscs . .' .'D

1'9'3 sIgA anti-v cholqæ resPonses disPl^ty immunologiøl rcmory ' '32

1.9.4 Mechenism of sþA antbody-mediated protectbn eg¡'i¡st

Vetøbæ " "32

1.10 Vaccination asainst cholera 31

1.10.1 Pastapproachesûor¡accination . . . . . . . .34

1.10.1.1 Conventionalkilledvacci¡es ....31

1.10.1.2To¡oidvacci¡e¡.... ...35

u

l.lL,zGureotapproachestovaccination"' "'3ó

LlO.Zl Live attenuated oralvaccines ' ' ' '%

l.lO.Z2 Non-viableoralvaccines ' '38

1.lO.L3 Live oral Cholera/Ilphoid hybrid vaccine 39

40

4l

1.10.4 Conclrubn ....

1.11 Aims of Thesis

2 Materials and Methods 42

ZilBacterialstrains "" "" ""'42

22 lvlairtenence and growth conditions ' '42

Z3 Specidizæa growth media

21 Chemicak, reagents andbuffers

43

Z1,l Enz¡rme-linked immunoeo¡ùent assay (ELISA) Bufrer¡ 45

1z4.2 DetergenB """" ""46

1z1.3 Enzymes ¿¡d immunoconiugates ' ' ' 6

ZS d¡imal¡ "" "ß

u

47

2;5.l Adult mice

2,5.2 Infantmice ' ' ' '

z6 heparation of boiþd a¡d formalin-killed organisms

Z7 IsolationoflPSfuomKùol¡¡ræ.. "' ".. "'48

L8 Preparatio¡ of OMh 49

zg hoduction of a¡tisera towhole cells and to oMI)s olv úoleræ 49

2:g.l Preparation of mouse antisera

47

lu

49

I

úñ

29.2 hePa¡ation of rabbit¡ntisera 50

1ZIO Absorptionofantisera ".. ""'51

2:l}.l AbsorPtionwithlPSorOMP ' ' ' ' ' ' " " ' ' '51

ZlO,2 Absorptionwithbacteria ' ' ' ' ' ' ' ' "' ' ' "51

zll h ví¿.osssE¡B ol.V ùotsæadherence: ettachment to isolated murine intestinal

I

I

I

'tII

I

I

I

I

epithelial celb (enterocYtes).

Zll.lheparationofenteroc¡rûes "" "'51

2ill.z Invífroenterocyteadherenceassãy ""'52

Zill} Inhibiti'onofadhe¡ence" "" "53

1Zl2btwruassaysq¡itlinfantmioe "" ""'53

ZlLl VirulenceasEsyl "" ""51

51

I

I

Ll/2 Protectb¡ assrys

?Àl3 l¡olationo\VclPloæFlt. """55

?Àl4 btyito tihation of a¡tibodies toV ctpbæ ' ' ' ' ' 5ó

2ill.l Hemagglutination ' ' ' o ' ' ' ' ' ' ' ' ' ' ' ' '''' '' '56

214.2 ELISA 56

2¿IS SD5-PAQEandimmu¡oblotting .... "'57

zlS.lsDs-PACE "" "'s7

215.2 Immunobloü'ing(Westernblotting) " .. " " '5t

2|16 ColonYblotting .." ""'59

?Àl7 Constructbtof Vdplcrægenebank ' ' ' ' ' ' ' ' " "59

Ll7.t Prreporatio¡ of'V dtobæ genomic DNA

tv

59

f--

Lt7.2 DNAquantitetion . . . . . . . . . . . . . . . . . . .60

2,17.3 EndEllhgwithKþnoq,fragment ........ ..@

L17.1 Conetructbaof pPM2101 . . .60

2,17.5 Co¡¡tnrctbn of coemid gene b¡nt 6l

217.6 Mobilizationofcoomidbank .....61

2,17.7 Tlansformetb¡ """"'62

2l8 EM.... .... .."62

2,l&l ïhan¡micsion EM " ' '62

Ll&z Immunoelectronmicroecopy(IEM) .....63

3 Non-LPS Protective Antigens shaltd by Classical and

El Ïbr blotypes ofv cholerae g3.1 Introduction .... .... ........ ..""'U

3,2 Ç\anrcærization of.V døbæ gü'aine 65

33 The exbænce of non-LPS protective antiçnr in strai¡s C"{401 a¡d C4411 . . .67

33.1 hoduction of a¡tbera to 5698/165, CA1Ol and C4411 otraÍ¡s . .67

ï3.2 Protectiveactivitiesofantisera .... ....68

9.4 Disfibution of non-LFS pmotective aatþns among V clølqæ shain¡ . . 70

3.4.1 Oldlaboratory¡trains .... ........ ..70

3.1.2 RecentfeHbohte¡.... .... ...70

3.4.3 Conclusion.o.. ........ ...' ""72

3.5 Subseg of non-LPS protective antþns are serotype-restricted

Y

72

3.5.1 Demonstration þ protoctio¡ a888ys ' '72

3.5.2 Demo¡stratio¡ bY IEM ....73

3.6 Correlation between protective activities of antfue¡a and their capacities to

inhibittt víto attæ}¡rent ' ' '76

3.6.1 Invifreenterocyte adherence assry " " "76

3.6.2 Inhibitionof adhercnce " " '76

3.7 Discussion 78

4 Role of TCP as a Colonizatlon trbctor and a Non-LPS

Pmtective Antigen in the Infant Mouse Cholera Model &i

4.1 Introduction ........ "" "" "'83

4.2 Sûrdies ofVcløbæOMPs . . . . .. " " " " " " " "&5

85

85

4.23 OnPV .... "" "90

1.g F,rpressionofpiliþVcholqæstrai¡s ' ' ' ' ' ' ' '''' ' ' '''91

4.3.1 Preliminaryexperimente ' ' ' ' ' ' ' ' ' ' " ' ' ' '91

1.g2hoducti'onofTCP """" """'y2

4.3.3 Distributi'onofTCP .." ".."""'94

4.4 TCP is a vinrlence deærminant i¡ the infa¡t mouse model 98

45Auenputopuri$TGP ..""" "" ""101

4.6 TCP b a protective antþn i¡ the infaDt mouse model

4.Zl A¡at)'sb by SDS-PAGE a¡d imnu¡obloüing

1.22 Dete4ent e¡trætion of OMP preparations

vr

tuz

4.7 hotection þ anit-TCP a¡tibodies b biotype-restr¡cted 1(b

116

4.8 DiscNlssion .... .......' "'16

5 Molecular cloning of genes involved in bioqynthesis

Of TCP rtz5.1 Introduction .,,.112

5.2 heparationofcl,oneprobingeenun- ..... ... ' " " 113

5.3 p¡slimin¿¡ycbningeryeriments . . . . . . . . . o . . . . . .''' 113

5.4 MolecularclorringofçnesinvolvedinrcPpmduction ..... f15

5.4.1 Cloningstrateg . . . . . . . . . . . . . . . . .''''''' 115

5.4.2 ConstructionofthemobitizablecamidvectorpPM2l0l . . . . 116

5.4.3 Cloningthe TCP gstres ntoVcløbæ Ol7

5.4.4 Detectionofpositiveclones .... ..... ' " ' llt

5.5 Anatys¡softhecl,onesDsl-Dsl0 . . . . . . . . . . . . .''''' 118

55.r EMstudþs ................ .... ...llt

55.2 tmmunoblottingara$Pir IB

553 Moþcular analysis of cloned DI'IA ta

5.6 Confirnåtion of TCP a¡ avirulence determina¡tand non-LPS protecdive

antþen of,V dwbæ t?ß

5.6.1 TCP ¡ß aviruþncedeærmina¡t t?ß

5.6.2 rcP is a protective antiçn l?ß

5.63 rcP-producing O17 clones are imnunogenic

vu

r29

5.? Disct¡ssion . . . . . . . . . . . . . . . " " " " .. "' " "lgz

6 Genenal Discr¡ssion 135

6.1 Distributionof non-LPSprotectiveantigens ofVdplqæ . " ' 135

6.2 Role of TCP as a cobnization factor and a protective antigen t%

6.3 Molecular cloni¡g of genes invoþed i¡ the syÌthesis of TCP . . . 138

ó4 Is TCP the onþ non-LPS protective antigen of V ùolaæ il the ínfant mouse

model? .... .... ."' "139

6.5 Non-LFS protective antigeoo: ¡eler¡mt studies in otùer laboratorie¡ 139

6.6 Ftturestudig . . . . ' ' ' ' ' .. 142

6.6.1 Non-LPS proæctive antigens of Et Tor V døbae 142

6.ó.2 The nature of TCP-mediatedattachment 143

6.6.3 Vacci¡e Potenrizl of theTCP """" ""1ß

7 Bibtiography t47

vrlt

List of Ïhbles

3.1 Charæterieticsof Vdplsæstrains ........ .... "6

3,2 hotective ætivities of LPS-absorbed anti¡era to CA4OI a¡d C4411:

effect of ñrrîher abeorpti'ons with live homologous vibrioo 69

3,g Distr¡bution of shåred non-LPS protective antþne amongciÍeen streins of

g.1 The erbtenoe of eerotype-restricted non-LPS protective antigene inVùolqæ .71

4.1 DisffiutionofTCP . . . . . . . . . . . . . ..' ..' .. o'' "'n

4.2 Ant¡bodiestoTC?areprotectiveintheinf¡ntmousecholeramodel ......101

13 Antibodies to rcP are protective only ngainef challenge strains of

ClæsicalbiotyPe tgl

5.1 Prroductio¡ of TCP enìences thevirulence of Vúolqæ Ol7 tn

5.2 Absorption with TCPqrøbngOl7 clonee nemoves protective antibodiee . , 18

5.3 A¡tibodies to TCP are protective i¡ the infant mouse cholera model 130

5.1 TCP-pr,oducing cloner ate imrnunogeaic and protective 131

rI

List of Figunes

3.1 Demonstration of (Ogawa) sefotype-opeciûc non-LPS determi¡ant¡ 75

3.2 Antibody-mediated inhibition o|.V. dplaw adherence ¡n víOo 77

3.3 Capacitbs of th¡ee a¡tbera to inhibit the r¡ vútro aüachmeatof.V. cWæ

¡Eain¡ofClassicslorElTorbiot¡pe . . . .''''''''''''''''''79

g,1 Capacig of antberum to strain C4411 to inhibit the ø vùto atlæ\ment of

¡eoent%.ùfuæñeldi¡olatesofElTbrbiotype " " " '."80

1.1 SDS-PAOE and immunoblouing of V clpbræ OMPs 86

4.2 SDS-PACE aaaþeb of deærgent treeted V. ùfuac 5698 OMP preparationr . 88

l.g rmmunoblotanal¡nis ol? elplqæOMPswith a¡tiSark0MP a¡tisera . . . . .89

1.4 h,oductio¡ofTCPonCFA . . . . . - . . . . . . . .'''' "'''' " " "'yl

45 hoductionofTCPonT0G .... """'95

1.6 V chobæ 5698 grown o¡ CFA at?S C reacted with LPSobso¡öed a¡ti-O395 .96

1.7 Immunobbtting anaþb of V úolsac stfai¡s for TCP erpression 99

43 Immunoblottinganaþis ofTCP expression þvarious strains oî,V dPlaæ . 100

4.g Imm¡¡egoldlsbellingof%ctplqæ 5698 ' " o " " " " ' 1ß

5.1 heparationofcloneprobingoerum "" '114

I

5.2 ConstructionofpPM2l0l . . . . . . . . . . . . .''' "''''' 1f7

53 Colonyblotting ".. '119

5.1 Pr,oductbnofTC?þcloneDS2. ...... '. ' o.. "ln

55 Pr,oduction of TCP þ cloneo fF5 and N6

5.6 Imnunogold eþctron ntrccopy of TCP produced þ chne DSS ln,

5.7 rmmunoblot anal¡nis of whole celh ta

5.E Resr¡ctbn endonucleæe analyris of pPM2103 t25

6l hopæ"d organization of the gpDes associatedwith tùe production of TCP 145

tzt

Il

Introduction

1.1 Historical Backgrtund

-..bhæ lips, a únnl<øt fæ øúhotlou, ey,et, a løottcdbclly, wíîh limbs crønped øú ctPbd

æ tf by fvq breath clhgtts ø the wøtíor's face; f*W thA øe profu4 stalE aü eJav'ed; ød

h wìthíngs ihe palíenl qþes, thevìctím of Sìv*

Insrlptton atVldannusEer Ibmple (cited in Vougþt 18fB)

çholera (orrruneeas describedabove) ie a disease of antiquig recognieed in ancientlndian

nedical liærature. It probabþ originated in the deltaic regions of the Gaoges and the Brah-

maputra in the staæ of Bengal (non, knou'n as lvestBengal [ndia] and Bangþdesh) where for

cenürries it has beeq and still iq endemic, During the late fifteenth century Portuguese

explorers gare vivid descriptions of cholera outbreaks on tle l¡dian subcontinen$ hou'ever,

the disease did not spread wertwards until the nineteenth cenhrry (see Follitzer 1959).

In 1817, cholera broke outwith a hiÈ mortatity i¡ its endemic home; and in the next

fnre years, it spread over much of Asia and tle Middle East By 183\ the disease had spread

to Europe and America (Roeenberg 1962; Morris & Black 198Ð. From 1817 to l9/3, sa'

pandemic waves of cholera epread acnoss the world. Reliþue piþinagesr the mor¡ement of

I

troops and the sea t¡ade ¡oute centered around the endemic home all contributed to the

dissemination of the di¡ease (revien'ed by Pollitzer 1959).

During this period, two important obeervations we¡e made that greatly advanced the

understanding of the disease: one regarding transmission and the other etiologr. In 1855, Joh¡

Snow published an important and influential treati¡e on cholera epidemblogr (Snow 1855).

He clearþ established that the disease invoþed fecaloral transmission and discussed factors

promoting rhis mode of dissemination, higbliÉting the signiñcance of water (ûo be discussed

furtherinSection 1.7.3).Su@uentþ,Kochconfirmedthatcholera\r¡asindeed"anaffectation

of tle alimentary canaln (Snorv 1355). He identified the causative agent as a bacilluq

Wbrío cholaæ,which he cultured from the intestinal contents of choleravictims (Koch 1883'

1884 - cited by Polliøer 1959). This confirmed earlier obeervations made in 1854 þ Pacini of

the association of the diseåsewith vibrios (Hugh l%4).

In 1905, Gotschlich (cited þ Pollitzer 1959) isolated a new strain of.V cholcræ ftom

the cadavers of p,ilgrims rehrrning from Mecca to the quarantine cåmP6 at El Tor on the Sinai

Peninsuþ FryypL This strain proved to be different from tìe prenious lClassicaF strains and

was assigned a nelv biotype, El Tor. From ly8 tþ 1f)61, cholera was onoe again reduced to it¡

endemic form being confined mainþ to tìe B"og"l region, with small foci of endemicig

reported in countries to the east of India" I¡ 193? "paracholerC caueed by V cJølaae (El Tor

biotl'pe) was obeerved by de Moor (1933 - cited by l(anal ln4l in the Celebes from where it

gzve rise to sporadic câses in adjacent countries over the ¡ext 20year&

In 1f)61, the ser¡enth (and current) pandemic began. Unlike prer.ious pandemics, this

was caused by vibrioe of El Tor biotype @arua & CVjetanovic 190; tr(anal lnÐ.It begaû i¡

Sulawesi [Celebes] (I(anal ln4),the endemic focus described Ð d" Moor (de Moor 1938 -

cited by lç¿nal lnÐ and quickly spread to the Paciñc islands, across Asia into the Middle

East, the U.S.S.R and Africa, reachingthe North American continentby 19/3 (Weissman'd d

2

ln4).other than the united stateg no countty in Norú, South or central America reponed

cases until 19g3, when cholerawas detected in the car¡bbea¡ Coast of Mexico (Black 198ó).

The spread of the disease during this current pandemicwas facilitated by th" greater mobility

of people todây and B mnqs 6igation of refugees'

12 EiologrL2.L Ctaracterbtics of the bacterium

V cholqaeis a gram-negtive comma-shaped bacillus which possesses a sheathed polar

flaçllum of variable length. In addition, undercertai¡ culü¡ral conditions, the Presence of pili

(or fimbriae) on the organism has recently been desc¡ibed (freedy el aL 1968;Taylor et aL

lggT;trlall etøt lggg). The bacillus is a non-spore-forming aerobe or facultative anaerobe-

Biochemically, itis recognisedbybeingoxidase pooitive; itfermenb glucooe (withoutgas) and

sucrose, but not lactose; it utilizes citrate, þine and ornithine but not arginine; it is Voges-

proekauer (v") p*itiue a¡d b seDsitive rD ÙnD (Pterirtine) dito (150r¿g).

LLz Classiñcation

l¡ l71g,the çnus name Wbríowas given by Mäller. Su@uentþ, the species name'

choløæ,was given in honor of pacini¡ the fi¡st to give a vali<l description of the organism ("it"d

in Gallut lnq.The çnus Víbrbtw contains, in addition loV choløæ, otlervibrioo recently

upgraded to species level (see below)'

V chobæ strains are classified on the basis of the production of a cell wall potyrac-

charide antigen designated 01. Trro different classification systems for o gfouPs are used

(Safazafi ¿ $himada 1977;Smith lnÐ.In bolh systems O-goup 1 (Ol) strains afe respoD-

sible for true cholera infection The Ol vibrios can be divided into two biotypes, Classical and

3

El Ibr (Bauman d aL lgu), which can be differentiated in several ways: the latær can

aggfutinate chicken red blood cells (Finkelsæin & Mukeriee 19í3), and is resistant to

polymlmn B (Gangroo a 4 ú l%7) and to Mukeriees's type IV cholera Phage (Mukerjee

1963). Each biotype is further differentiated into antigenicalþ related sero$peq lnrh4 Ogawa

and, rareþ, Hikojima, based on the Prex¡ence of 3 antþenic factors (À g, & C)' Ao B and C are

foundinbothogvvlarandHikojimawhereaslnabaPossessesonlyAandCantþens@edmond

teTe).

Vibrioo thatproduce pot¡'saccharide antþens other than 01 are commonþ referred to

as NAG (non-agglutinable) vibrioo or NCIV (non+holera vibrioo; revierred by Finkelstein

lg1¡).NAG is a misnomer since, althougþ these organisms are not aggtutinated by 01 sera'

they do agglutinate in homologous antisera such strains produce diarrhea indistinguishable

from cholera (Morris & Black 1gs5). Some of the NAG vibrios causingenteritiswere upgraded

to species ler¡el on thebasb of DNAhþridization studies;in 1985, identifiedspecies included

v pøahaanotyticys,v nim¡aß,u ltilvialis an6v holTuae (Blake 1980; Morrb & Black 198Ð'

13 Eathogenesis and Fathophysiologr

Cholera is an enterotoxic enteropathy. The causative bacteria are strictly non-invasive,

remaining confined to the lumen and epithelial surface of ttre gut The essential pathogenic

events invoþe: (r) ingestion and entrance of viable v cholqæ into the sm¡ll intestine;

(2) colonization of the epithetial surface; (3) multiplication and release of enterotoxin and (4)

hypersecretion of isotonic fluid by the intestinal epithelium in resPonse to the enterotoxin'

4

1.31 Ingestion and infectious dæe

Following ingestion in either water or food V cholqæ must survive the acid environ-

ment of the stomach, the first line of defence in the host It is claimed that infectious dose (ID)

is high in healthy adults. Studies with Western volunþers have suggested that 108-1011

organisms are required to cause diarrhea n 50Vo aduls (IDso; Hornick et aL llTl;Cåshet aL

lyl4+). The sþificmce of gastric acidity as a barrier to choleraic infection is revealed by

studies which have shown that prior neutralization of gastric acid by ¿rlministration of sodium

bicarbonate dramaticalþ reduces the IDsoto td-fO6 orgaoisms (Hornicket øL llll;C-ashef al-

lf'l4c;Irvi¡e et al l98lb).

The IDso ¡motrg the inhabirants of endemic areas ¡spains unknovn, butmalnutrition

would be expected to reduce the efficacy of the gstric acid barrier. In such areas individuals

with achlorhydria are predisposed to this dise¿se (Gitelson l97l; Sackef aL 1fi2). The ID is

also lowered if vibrios are ingesæd in contaminated foo{ which presumabþ protecb the

organisms during their passage through ¡¡s stomech (I-evine A aL l98Lb)'

L3;Z hnehation of the nrrcus laYer

The viable organisms which reach the small intestine encounter two additional non-

specifichostdefence mechanisms; nemeþ, intestinal peristalsis (Dixon 1!)60) and a mucus layer

(Florey 1933; Freter & Jones 1976; Schr¡nl¡ & Verwey 1976) which the organisms mut

penetrate in order to reach the epithelial surface. V cholsae useE an arrãy of virulence

properties in overcoming these barriers. The flagetlum is responsible for motility and allows

the bacteria to respond to chemotaxins such as L-aminoacids and eimple Eugan¡which diffuse

away from the mucus surface (Freær & O'Brien 1981a, 1981b; Freter er aL l98l).

5

Several workers using animal or m yitro models have demonst¡ated that motile

V choløae can be raprdly detected in the intervillous spaces and crypts of the small intestine

(Guenrzel & Berrl' 1975; Nelson el aL 1976: Schrank & Verwey 1fi6; Freter d aL l98l).

Mutant strains that are non-motile but enterotoxigenic colonize these regions much less

efficientþ and tlerefore eúibit reduced virulence (Guentzel & Berry 1975; Schrank & Ver-

wey lgl1;pierce et aL 198f¡. Elect¡on microscopy (EM) studies have shou'n vibrios adherent

to enterocytes as well as associated with mucin (Nelson et aL lÍ1.6: Guentzel er 4I l98lr.

]b gAin entrance into the mucus gelcoveringthe epithelial surface,V clølerae releases

a potent mucinase @urnet & Stone 1947; Burnet 1949; Freter 1955b). In vìxo sh¡dies have

indicated that the forward movement of vibrios is impeded þ mucug although the organisms

can migrate through channels in the mucus gel (Jones et aI 1176). It is suggested lhat,invíw,

such channels result from the action of the mucinase. Various studies have suggested that a

soluble hemagglu':nin poosessing protease activity (see Section 1.3.3.3) mig[t cont¡ibute to

virulence of V cholqæ (revierved þ Booth &, Finkelstein 198ó), possibþ þ fulfilling a

mucinase function hvivo (Schneider & Parker lfi&;Finkelstein et aL L983).

L3.3 Adberere to the epithelim of the smll int€stine

Adhesion Becure6 tle bacteria against the effects of intestinal perisølsis and facilitates

delivery of the enterotoxin- The precise molecula¡ mechanism þ which tle organism attaches

to enûerocytes of the crlpts and villi is not entireþ clear. A number of cell surface components

(adhesins) could be invoþed These include flaçllar sheath proteins and otler flagellar

antigens @ubanlc et aI ll77; Attridge & Rorley 19334 1983b), lipopolysaccharide (Chitnis

et aL L982a,1982b), hemagglutinin sandcholqalecth (Finkelstena aL 1983), and pili (Taylor

et øL 1987; Herrington et aI 1988).

6

L33.1 ffagellar PFoteiß

The¡e is little doubt that the ftagellar structure plays an important role in the coloniza-

tion of V cholqae. Studies in the infant mouse model shoç,ed that motile strains were mone

virulent tlan non-motile derivatives obtained þ mutagenesis (GuenEel & Berry l9$, and

microscopic observations of intestinal sections shouted that this difference correlated with a

more effective colonization þ the former (Guentzel el aL lW). I¡ a laær etudy, mixed

suspensions of relatedmotilityvariantswere fed to infanf 6i66; and the motilebacteria sho$'n

to enjoy a significant advantage in terms of intestinal colonization (Attridæ & Row'ley 19834).

These reports are supported by numerous ìn vitro sh¡dies which show that motile vibrios attach

more efñciently to avariety of substrates (Guentzel & Berry lflS;Freter & Jones Ll16;Jones

& Freter 1976;Attridge & Rowley 1983a' 1983b).

Whetherthe invoþementof the flagellum extendsbeyondits roleas an agentof motility

remains uncertain, althougþ reports suggest that tìis stnicture also pla¡n a role in the Process

ofattachment Jones & Freter Qnqshowedthatf,agellatedvibrios couldbindtobrushborder

membranes h vítobutthat non-motile orqanbms could no! oren if they were compacted onto

the sub6trate þ ceatrifugation. Attridge & Rowþ (19S3a) reported that flaçllated vibrioo

which had been immobilized by preincubation with antibodies to LP$ retained the capacity

to adhere to seÊments of int€stinal tissug whereas non-flagellated orgnism.s were non-ad-

herenL The onþ aprparent difference between the two populations wÍts the presence of an

immobilized flagellun, suggesting the exisænce of a flagellar adhesin In this respect some

investþtors hãve described a flagellar sheath protein (Follet & Gordon 1fb3; HtaÃitzky eJ aL

1930). Antibody directed against rhis protein aPPears protective against chole¡a in two enimel

modele; and it has been suggested that thb protein msy serve as aD adhesin (Eubanh a al

lf17;yancey d aL lg7g). Nonetheless, EM sû¡dies indicate thatvibrios do not attach þ their

7

flagellum - the flagellum sticks into the lumen of the gut (Nelsotet al.ly76r.It could be that

the initial contact is made by th" flagellum and the organism then adheres horizontally to allo*'

greats sufface contacL

L33.2 LþPol¡naccbaride (LPS)

Some evidenc€ has accumulated to suggest that LPS may Play a role in adhesion of

Vcholsæ to intesti¡al mucæa Using the rabbit ileat-loop model Chitnis and co-workers

(1982b) have demonst¡ated that purified LPS prepared from an Inaba süah o1' V ùobae

(5698), but not from .Escå qíchia coli, car- signiñcantly reduce the attachment of other Inaba

strains to the mucosal epithelium. Furtlermore, serreral workers have shown the role of

antibodies to Lps (ogara and Inaba) in the inhibition of attachment of V ehobae strahs to

intestinal mucosa both nr vútro atd h víw (Freær & Jones 1fl6; Chitnis et aL 198241987b;

Auridge & Rovley 19g3c). Iåstly, Finkelstein and co-workers, using monoclonal antibodies

(MoAbs) direcæd against v ùolqae LFS (preumabþ directed against specific o-antiçn

determinants) were able to inhibit the hemagglutination reaction caused þ vibrios or LPS

(Booth er aL 198,6).

L333 He'mgglut¡nins (IIAs)

Alrhougþ hemaggfutination systems have been wideþ used in the study of Vcholaæ

adherence, the mechanisms of attachnent to erythrocytes (RBCs) and to intact mucoeal

su¡faces may differ (Attridge & Rowley 19s3b). Before usingRBCs aß a convenient subetituæ

for a more relevant substrate, it is therefore important to initialþ confirm that the charac-

teristics of aüachmentare simíla¡'in the two syntems (Jones el aL ll76l'Jones 1980)'

Vchfuae aggtutinate certain species of RBG ¿¡d rhis proPerty correlates with ad-

herence to brush border membranes (Jones & Freter ln6). Adhesion to the i¡testi¡ el

8

epithetium and hemagglutination may be inhibited þ different sugan (Jones & Freter lnq.

It is now known thatVcholqa¿ strains produce four distinct HAs which ca¡ be divided into

two classes: cell-associated and soluble (Hanne & Finkelsteh 1982). Three cell-associated

HAs have been described so fan a D-mannose+ensitive IIA (MSHA) produced by El Tor

strains; an L-fucose-eensitive HA (FSHA) e:ryressed þ Classical strains; and a mânno8e'

fucose-resistant HA (MFRIIA) expressed þ both El Tor and Classical strainß. The fourth IIA

is solublg po6se5ses protease activity (SHA/protease) and is found in all strains regardless of

biotype or serotYPe.

There is some evidence that cell-associated HAs of Vúobæ a¡e invoþed in ad-

herence and may represent adhesins (Guinée el aL 1985; Finn d 4I 1987) althougþ a direct

correlation between intestinal attachment and tIA e,ryression has not ahva¡rs been seen (Freter

&Jones 1976;Tep,pema daI1987).Morerecently, hvinoadherenceotudies olVchobaeto

formalin-frxed þ¡men villus cells (Yamamoto et aI 198ß) or human mucus coat (Yamamoto &

yokota lgSS) harre shon¡n that adherence did correlatewith cellular HA levels' irrespective of

bio$rye or serot¡pe. Similar inhibition patterns were obtained between adherence and hemag-

glutination reaction" indicatingthat cellula¡ [IAs -iÉt plr,y a role, at least i¡ Parl, in adherence

(Yamamoto et øL lgæ). The relerrance of these studies to the pathogenesis of the disease is

not knorvn. Finn d aL (1987) iÊolated an MSHA' muta¡t from a V ùolqae strain JBK70 (a

muta¡tþl Tor Inaba strai¡ lackingthe cholerafor gene),and the mutantwas much lets able to

colonize the ileal mucæa folloving oral administration to rabbin. This suggess that the El Tor

MSHA might be considered as a putative atlachment facÛor.

The SHAlproteåse, prwiousþ referred to ascholsaleãh (FinkelsteinA aL 198)' has

no*, been purified and charactqnzà (Finkelstein & Hanne 1982). Itwa¡ originalþ thougþt to

be an adhesive factor as it inhibited the attachment of vibrios of either biot¡pe to epithelial

ce[s both ínvitro and lr¡ urvo (Finkelstein d aL ln$). Hox,orer, there ie, at prcsent no con-

9

vincingevidence thatitcouldbe an adhesin.Althougþ tìeabove studiessugEp8tthate)rytression

of HAs could promote bacærial adhesion h vítro, it remai¡s to be seen whether any of tlese

HAs are operating as adheshs during infection. As di¡cussed belw, pili hære recently been

described onV choleræ(rrylor er øL 1987) "¡d

it is unclea¡ whetler these are associated with

HA propertiee as has been described for other bacteria pcseeeing adheeive pili on their surface

(Duguid & Otd 1980; Gaastra & de Graaf 1982}

1.33'4 PiVñdrhe

pili are non-fþellar, filamentous protein appendages proiecting outward from the

surface of many gram-negative bacteria and are aEsociated with the colonization of the natural

envi¡onment (Brinton J. 198; Smith & Linggood l9ll;Nagl d aL lffr' Theee appendages

u,er,e independentþ described and introduced as fimbriae þ Duguid el aI (195s) and as pili

by Brinton (1959). Evidence b accumulating to suggeot that the acüral adhesins of these

bacteria consist of minor protein molecules at the tip of surface Pili- This has been

demonst¡ated in both enterotoxigenic (ETEC) and uropathogenic strai¡s or' E coli

(Gaastra & de Graaf 1932). þ analog, it har therefore been assumed that pili would be

inrrohed in the pathogenesis of cholera but pili erpression by v ùolsa4 althougþ the subiect

of muchdiscussion,led to conflictingreports in thesifief ardseventies(Finkelntein & Muker-

iee 1fb3; Barua & Chatted æ1964;I-ankford & Lægsombunaûa 1%5; fteedycr øl 1!)618; Nel-

sonef aL lnq. But in the eightieg reports have appeared Gonfirmingpili expression þ

V cholsse(F.rir et aI 1982; Al-Kaissi & Mosl¡atos 1985; Eha¡a d al- 1986;kyh¡r el aL 1987)'

To date, four types of pili of V cholqae h¡ve been described" Ehara et aL (1986)

described flexible pili on V c)ølsae during the colonization of the upper emall intestine in

rabbits. This pilushasnou,been purifiedandthestnrcüualsubunitis aprotein of 16lDa (Ehara

e, aL lgg:¡, lggg) shosrn to be shared by 01 (both El Tor and Classicat strains) and non01

10

V cholsæ (Nakasone & Iwanag 1983). Antisera from convaleecent patienß contained an-

tibodies against the pili subunit whilst anti¡era raiced against pili subunib neutralized the

hemagglutinating activi$ of pff preparations @hara d aL l9æ).

Recent reports have described the discovery of a pilus stn¡chrre on the surface of

claseical ogawa 0395 (Taylor d aL lg87) confirmed by Hau d aL (1988). Thb pilus is now

refer¡ed to as a foxin co-regulatedpilus (rcÐ. In addition, Hell d aL (198) ha*'e descriH

thatVcholqæcan eryress two other morpholog."lly distinct pili besides the TCP; nameþ

Type B with a wavy morphologl and Tirpe C being more rigid" Whether or not ary of these two

t,¡pes a¡e rel,ated to or identical to that described by Eh* d aL (19%. 1988) remain¡ to be

established. The significanoe of theæ latter t)"es has yet to be elucidated" There is compelling

er¡idence that rcp may ble an inporønt coloniz¿tion factor oÍ,.V dtobæ. This significance is

discussed further in subsequent chapters'

L3.4 Pnoductbn of enterotodn 8nd f,tdd accumilation

Afær successfuþ attaching to the enterrrc¡rtes ,t\eV cløbæ organisms enter a phase

of active grcsrth, duringwhich they elaborate an enteroto.in^ This molecule comprises two

q¡pes of subunits, A a¡d B (see Section 1.8.3), the latter mediating bindiag to the enterccyte

membrane (Cuatrecasas lg73) via a receptor now identiñed as a eialidase-resistant

monosialosyl ganglrooide (GMt) (van Hgrningen et aL llll: King & van HeÏningen 1973)' It

is the otþsaccharide portion of the ganglioside to which the toxin binds and there is a

correlationbetween GMlcontentandbindingabilityof cells (Holmgren 4 aI1975æ,Haneson

et aL lgi'\. Once attachment has occurre4 the A eubuni! aD elu¿yme, then passe6 acroes the

cell membrane into the cytoplasm and activates the enzyme adenylate cyclase- This in turn

lea& to elerrated intracellular lerrels of cyclic 3'5'-adenosine monophcphate (cyclic AI\'ÍP)'

resulting in an irreve¡sible hypersecretory response @eærset d aL ln4 Holmgren ef øl

,l

{

11

l91Sa)by ".llr

i¡ the crypt and on the sides of the vifl The secreted fluid is low i¡ protein and

rich inelectrolyteincludingNât, Kr, Cf & HCol-. Althougþ thetoxin is rapidþboun4 there

is a lag period rangng from 10 minutes to t hour before changes in ion trnngPort are seen This

delaly probabty relates to tle -:me taken for the A subunit to penetrate the cell membrane

(Fishnan 1980).

h both clinical (Greenougù 1fb5; Banwell d aI lnq and experimental (Cår-

penter d aI lgæ) choþra, the fluid loss occurs from atl segments of the small bowel with a

decreasing gadient from duodenum to ileum. The resultant loss of large volumes of isotonb

fluid is responsible for the cha¡acteristic clinical Fofile of the disease. As mentioned below,

tlere is no æidence that any stnrchrral lesions ale producedby the toxin

It has been propced that the action of cåolera toxin (CT) promotes the intestinal

growth olVchobæ þ meking arr¡ilahle nut¡ients secreted þ epithelial celh (Mekalana

19SÐ. Construction of site+ piÍrcc9muta¡ts has provided tle moot convincingertidence rhet

toxin pnoduction i¡ beneficial for gron'th in the i¡¡6tinel environment (Mekatanæ 198$

These ¡on-to-inogpnic mutant strains colonize rabbit intestines less efficiently than their

parental stain¡ (Pierce d aI 19æ[

Althoug! CT is taræly responsible for the voluminous diarrhea cha¡acteristic of

choler4 two observations suggeet V dtolqae 01 produces another toxin(s) capable of causing

diarrhea Fint, there b widence that naurratþ occlu:ingCFnegative El Tor strai¡s - whetler

envi¡onmental in origin or associated with cases of diarrhea - cau¡¡e intes-:nal secretion when

inoculated int¡o certain animal model¡ (Nishibuchi d aI 1983: Sanyal a aL 1983). Secondty'

mild or moderate diarrhea wal¡ Been in about 30Vo of healthy adults fed with viable 01

orgpnismq of either biotype, that were CFnegative; theæ weÍe strains whqe CT genes had

been inactivated þ nutagenesis or d€teted þ recombinant DNA techniques (Iæ',dne et aL

19SSa). The exact nature of non-CT toxin is not known althougþ it could involve the shig-like

.t

{

ù'

I

¡

'l

{i

':

'ftI

I

I

II

t2

toxin said to be p,roduced by many V cholqæ ()1 organirms (O'Bri et el d 1984). In addition,

El Tor strains have been shoü'n ûo produce a soluble hemoþin which has cytotoxic and

ente¡otoxic activity (Yamamoto 4 aL L986),.

L35 nrthotogr

The hoot cellular rcsponse to infection is minimal, invoþing primarily a non+pecific

increase in mucus-producing goblet cells. There is no maior histological rlamage to the

gasgohtestinal tract in either mnn (Gan gutxiacf aL 1%0; Fresh el aL l9&) or in elperimental

animals (I-aBrec d aL 1965; Norris & Majno l9i8; Ghosh lnq. Biopsy specimens collected

from different regions of the small intestine re't eal an intactepitheliun There is edema of the

eubmucosq dilated cr¡¡pts and minimal numben ef infkmmatory cells in ¡[s lamine p¡oPria

The intercellular tigltjunctions and capillary endothelial cells arc also normal The brush

border remains intact and the organisms do not invade the mucosa

L.4 Clinicat Manifestations

Ctolera is a digease rangng fnom as¡impomatic infection with mild diarrhea, to a

firlminantfatal syndrome, inwhich form itis one of the moetrapidþ fatalinnesses known. The

incubation period nay vary from a fen, hours to a fen' da¡R with or without prodromal symptoms

Acutg potentialþfatat infection b comparatinely ra¡e; the large majorityof ca8€8 are mild and

usualþ indistinguishable from other forms of Eastroenteritb.

In its severest form, tle disease p¡ogresoes from tle first liquid stool to shock i\ 4-12

hours with death foll,owingwithi¡ 18 hours. It b characærizÆd by the sudden onsetof effortless

vomiting and profiue dia¡rhea (Greenougþ lnÐ which initialþ lacb the 'rice-water" aP

pearance- As the dia¡¡hea progresses the fecal fluid takes on the typical tice-watef appearance

t3

due to the preeence in the gtool of flecks of mucus discharged by th" goblet cells a8 a result of

the action of the cholera toxin Dehydration accounts for the maiority of the signs and sympomr

seen i¡ cholera These include thirst, muscular ç¡¡mpor sunken eyes, cold extremities, thrcaÙ

pulse a¡d sunken fonta¡elles i¡ babies. In untreated severe cases a patientcan lose up to twice

hiß body weigþt in liquid stools (Hirschhon d aL 1968)'

Complicatione of cholera can occur in both child¡en and adults. Theee include alæred

consciousness which in adult is manifesþd ry a detached mental statq while in children

unconsciousness or conwlsions can occur as awarningof impendinghypogþcemia Electroþte

imbalance leads to hypokalemia and hnernatremia as well as acidosig particularþ in childreo

Aspiration with fatal sequelae is a serious complication in patients with a combination of

atæred consciousness and vomiting (Greenough 199)'

15 Diagnosis

The moet rapid and effective wry of diagnroeing cholera in the laboratory is þ examin ing

a fresh fecal epecime¡ under dark-field illumination The vibriq can be recognized by their

characteris trcdæt¡¡gmotilityuåen presentin largp numbe¡s. Addition of homologous antisera

çritt immobilize thevibrios and immediaæ diagnosis can be made @enenron er aL 1964)' When

stools cannot be examined at once they are best placed in a transport medium such as Cary-Blair

medium (c"ty & Bhir 1964) or Monsur's medium (Monsur 1963).

In the laboratory, the specimens are inoculated into both (a) a¡ enrichnent medium

(alkatine-peptone water pH S.a-9.2) and (b) a selective medium CrcBS agar; Kobryashlia aL

1963). Growth from enrichment medium is sub+ulûued onto the selective medium. All

putative V cholqae coloniee that are oxidase pooitive on the selective medium a¡e then

subiected to aælutination with a poþalent 01 serum before being characterized biochemi-

t4

catry. The confirmed isolates o1 V cholqæ can then be phage tJ¡Ped for epidemiological

purposes (Gla6s 4 aL 1983>.

t6 lleahent

The treatment of cholera is simple, both iû concept a¡d execution' involving the

replacement of the water and salts lost in the cholera stools. Replacement therapy in the form

of intravenous infr¡sion of fluids was shown to prerrent deaths from severe cholera as earþ as

1831 (Latta 1831-1334 O'Shaughnessy 1831-1332). Improrement in rehydration therapy

based on accurate Deasu¡ements of salt8 lost in the cholera stool was suboequentry propupt"d

as a ooffect and totalty effective tfeahent (watten d aL lg59). Whe¡ promptly apflied this

replacement therapy reduced the adult mortality rate to less thil o'e penoent of those ca'€s

se\refe enougb to warrant hæpitalization (Hinchhon daL lnÐ' Howerrer' thic form of

therapy i8 technicalþ difficult and expensive'

successful rehydration caû be more convenþntþ achieved by ü" oral administration

of fluids 6e¡rrini¡g gfucooe and ions of sodium, potassium, chloride and bicaôonate (oral

Rehydration TherapyoRll Nalin & cåsh rnq.The gtucose facilitates the uptake of water

and electrolytes by th" small intestine and helpe to retolve hypoglycemia whiht potasaium

prevents the establishmentof hlpokare-ia (Natin & cash tyTr).The oral rehydratbn solution

(oRS) reco'rmendedby the world Health organization hee undergone severar clidcål triat'

and has been found to be safe aûd useful in the treatment of diarrhea fron all causes in all

patient golrpô (Mahel¡nabis 1981; Mahalanabb & Merson 1986)' In nrral 6pmmunities of

Bangladesh, the oR.S has significantly reduced diarrheat morbidity and mortality (Rahaman

et aL l(/1g: clen d 4L lgw). tn the last 10 years, er¡idence has accumulated sho*'ing the

importance of nutrition during diarhea oRs Í¡ccompanied with feeding ha^q been ¡hou¡n to

ra¡Édty etabilize and then rever¡e the weigþt loes aûd nitrogen imbala¡ce (Inærnational study

15

Group lgll;Bron¡n 4 aL tngr,Råbbani 1986). In the worst cåses, ho*'evet, such as severeþ

dehydrated children who a¡e comatoeed with high natee of purgng and/or uncontroll¡able

vomiting ingavenous replacement therapy is required (Greenoug! 1980)' As soon as tle

patient is stabilize4 ORS can be instib¡ted. With prompt and adequate fluid replacementthe

patient usually recovergwithin a fent daya

Antibiotics arc often adminisæred to reduce the duration andvolume of diarrhean the

most commonþ empl,oyed being tetracycline, which kills Iz. ùolqæ leading to bacteriologi-

calþ negative stools after Zlhours (Greenough d oI 196/¡: Carpenter 4 aL 196ó). Hon'er¡er,

widespread use of antibiotics has led to the emergenoe of plasmftt-D€dhtêd drugresistance in

V. chotqæ (Mhalu d aL lg7g: Glass et aL ß8f,: Tabtieng et al- l99o), making it imperative to

look for alærnative meatrs of t¡eatmenL One such ap,proæh would be pharmacological

inhibition of toxin-induced i¡teetinal secretion, the most promisiag inhibitor being

chlor¡rrom¡zine. Howeter, its attendant sedative effect would limit iu clinital usefulness

(Greenougþ & Rabbani 1986).

L:l þidemiologrL7,l IncHeree

A¡nualworldrvide incidence rates of cholera has b€€n estimated tobe nearþ 8 million

cases resulting in approxim atley l?A,t00 deaths (Black 1936). In endemic areas, cholera has

the higþest attack rate i¡ children, with a peak incidence in the 2 to 9-year old age gouP

(Glass et at- l9g2). Attack rates decline rapidþwith age and the dis€ese b ra¡e afær the age of

40 (Mccormack et aL lg69). Thb distribution pattern is thought to ref,ect the induction and

maintenance of activs imm¡aity as a result of repeated s¡virenmentd expoeurctoY dtobæ

(Mooley d aL 196¡¡). Thir is supported by obeervations that, wüen cholera outbreaks occur in

16

non-endemic regions, individuals of all age group are equaþ susceptible @aine d aL lnû),

a finding consistentwith the abce¡ce of (agedependent) naürral immunit)¡'

Since the onset of the seventh pandemic, the e¡Édemiologl of cholera has been

changing Althougþ either biotype of V clnleræ is capabb of causing cholera in iß severest

form, epidemiological shrdies revealed that the infection to e€ryere cåse ratiowas approximateþ

ten times higber for the El Tor bbt''pe (Bart d aI ln0: Shahid d aL lg84.). Th" more recent

predominance of the El Torbiotype could be due ûo the fâct that these strainß can suryive longer

in water and i¡ food (Felsenfeld 19ó3, 1t)ó5) as well as in nig[t+oil samples (B^fter aL lnq.

In Bangþd€6h, the Chssicat biotype was repla"ed by the El Tor biotype i¡ 193; but i¡

Septembe t l9g\Classical choþra reappeared as the cause of a maior epidemic a¡d h¡s since

remained as the predominantbiotype (Samadi ef aI 1983;Kha¡ er aL lg84.). In Indi4 howaner'

the El Tor biotype continues to be the principal G¡ute of di¡ea¡e-

In endemic a¡Eas the disease has a seasonal occu¡retroe which differs not only in

different countries but also in areas which are very clooe geographically (Martin d al1969\

In Bangladesh before lg12thepeak for cholera (C1assicat) was pæt-monsoonal (DecÆnber)

but in lnSftshifted to Ocrober as the ClassicEl biotlpewas replaced by th" El Tor. tilith the

reû¡rn of the classi€l biotype (sanadi d aI 1983), cholera peab twice enn¡¿ll¡r, in Decem-

ber/January and in Arril/May (Cohrell et at l98l). Yet not fa¡ nfay in Catcutt4 the peak is

p¡e-monsoonal (l(han & Greenoug! 1935). The underlyingbasb for the s€åsonal i¡cidence of

cholera remains unknoum, but one poesible contributing factor is discuesedbeloç'.

L,7.2 Aquatic nesrcrYoin¡

taditionalþ, cholera epÉdemiologists coneidered that the onþ natural reservoir of

V úobæwas the hurn¡n intesti¡e, with only a brief perid of survival poesible outside the

human body (Felsenfeld lnÐ,Honever, epidemiologcal studi€s undertaken in Australiaand

t7

southern u.s..À suggest the exbtence of aquatic reservoirs rot v clplaæ El rbr and thereby

ofrer an explanation for the perftrdic occu,,enoe of chorera cas€s in the*e areas (Blake er aL

1gg0; Bourke et aL lgfl'j). It is thougþt that, tike otlervibrioq vchobæ maybe an autoch-

thonous organism of esn¡arine a¡d brackish waters (coh*,ell a aL lgn,1987; Hood & Ness

lgg2),or at least that the organism is able to peniet and multipþ in such an envircnment for

long periods of t¡me (N"lio 1976; Blake 4 4L lg80)'

6lnS,the fi,,t indþnous case in the Norti American conti¡ent in over sixty years

occurred i¡ Texas (weissman d oL tn4). Since then cases ha'e been detected on tle Gulf

Coast of Texas and Louisiaûa (Blake e, at 1980; ghandera 4 aL 1983), a¡¿ indeed thh b nory

recognized as an endemic area The mæt recent cases occurred in Baltinore i¡ 19&{ (uo' d aL

1gs6) and in Louisiana and F.lorida in 19g6 (cD.c lgsó). Epideniologicar erudies ha'e

aü¡ibured outbreaks in us.À (Blarrc d aL rgæ) and sardinia (salna6o 4 aL 19æ) to the

ingestion of localþ caugþt conaminated seafood" In both l,ocations tlere had been no cholera

in the preceding fve yearg, arguing ag¡inst the notion tl'et the disease is Bai¡tained by regular

transmißsion hom infected indivi<tuars to water. I*tead, they support the hypothesis of the

existence of an aquatic reservoit of'V clpbæ'

laboratory shrdies hæe shown thttv clplqæin pure culture can su¡vive for prolonged

periods in warm wateß without nut¡ienß wherein survival is influenced primarity þ salinity'

pH and temperaûrre (Singteto t4 al- tgï2l'l98ã¡:colwell ø øl 1985b)' I¡ such an environ-

ment v chobæcan reach a viabre but non<urturabre stagp (colwen a aL l985a) yet regain

iß cutturable status on human Passage (Brayton cl aL lg87)' Thiß suggesb thatvchobæ cÀ'A

maintain its pathogenic potential during a period in an aqua6't s¡vironment

The seasonal incidence of choþra may be at lea¡t partty attributable 1o variations in

plankton populations. oppenheimer and colleagues (198) have sho$'tr t\atvclplqæ ca¡

associatewith planktonic oopepods. ohervations in Bangtadesh re'ealed thatthe zooplanllon

18

population decreases during the monsoon 8ea8on (May-Juþ) in responoe to a reduction of

nutrienb and alæration of calinig. SubsequentU, the poprlatbn increases duringAugustand

September, shordy before the appearance of the first cholera cases which initiate the annual

epidemic. V elptsøe hæe been shorn to produce chitinase (Dastidar & Narayanasr¡mi

196g), to adsorb to a chitin eubßtrate and multipþ on such a surface (Nalin 4 4L lng)' Since

copepods and cn¡stacea have chitin in their exookeletons (Huq et aL t9%), association with

sucb organisms rnãy therefore orplain the abunda nceoî.v cholaaebsurfacewaters ¿[ 6s¡tein

'im€s of the year (Colwell d al 1987).

L73 a¡rnnission

Hietoricalþ, moot e¡ridemiologistr have been proponentr of the concept of cholera a¡

an exclusiveþ or prinarity waterborne disease. This has been based on the 19th cæntury

ouöreals, ild in particular, as argued by Feachem (19821 on misitrterpretation of the

pioneeringworkof Snow. Snocs investigation of the ouôreak¡ in l-ondon rwealed that there

was a clusteringof cases li¡kedwith the route of one of the cit/s two mai¡ water supplies (Snon'

185Ð. As pointed out þ Feachem (1982), througþout hb writing, Snow (lssÐ maintained a

concept of fecaloral choþra tra¡smission, of whbh waterborne tran¡mi¡sion b but one special

case. But perrcn-to-person spread, as a mode of fecatoral transmission, was not overlooked

eve¡ in Snow's time (roden'ed þ Feachem 1982)'

Epidemiological evidence suggests that person-to-person transmiseion can occlu' Paf-

ticularþin situationswhere there b overcrowdingand poorhygþng such as hæpitals (Mhatu

et aL lgg4: ctiff¿f 4L lg%),prbons and refugee camp' (Monb d aL lgsz). l¡ f¿nzanin, this

node of transmi¡sion has been impticated in an ouóreak of cholera in a podiatric i¡fectbru

diseases wa¡d (tvfhalu er al" lg&4). Further opporurnity for person-to-penon t¡ansmi$ion

ooeur' atburial cerenoniegwhere local traditi'on dictate¡ the cþansingof the intesti¡es of ttre

19

deceased (erren of cholera victime) prior to burial After the ce¡emony, the participanb sharc

a meal served on a @mmunal plaæ and eating b done with fingers (Mandara & Mhalu 1980).

Recent case co¡t¡ol studþs ha\re confirmed thåt, i¡ addition to water, food can terve as

a vehicle fo¡ the transmission of cholera (fauæ d aL l9æ). Foodstuffs i¡criminated to date

include rffi' or pardally cooked shellfhh in Mataytia (Duü €/ aL lnl),Italy (Bain e 4 al- ln4)

and portug¡l (Bl"k" er aI tnT, rãuv 6sh i¡ the Gilbert Isla¡ds (Mclntyre d 4I lng), and

insufficiently cooked crabs in the United States (Bl"ke el aL 198lJ). AB al¡eådy dbcusse4 food

nay have the effectof loweringthe ID of.Vdptqæþ p,rotectingthe organbms from gætric

acidlty during their passags througþ the stomåch. Thru, an ID of 106 El Torvibrios produced

severie dianhea in volunteerswhen i¡gested in foo4 whereas the same number gven inwater

did not (Lrvine 4 al l98lb).

In the liglt of gro*'ing erridence for the existence of an aquatic reeervoir lot V dtobæ

(see Sectbn 1.7.2),Miller¿f a¿ (19SÐ have rccentty p"stuLted prinary and secondary nodes

of dbease ü.ensmi¡sion They sugøt that h prinary transmission vibrioe pass from the aquatb

reærvoir to hunans via drinking water or edible aquatic flora or huna Subæquentþ secondary

fecal-oral ü.ennmi¡sio¡ invoþes penon-to-penon epread, either directly or via cotrtaminated

food or water. This concep is in agreement with the studies of Deb ¿f 4¿ (198ó). [n areas such

as Alrstralia (Rogers cr a¿ 19S0) and the United States @lake a øl 1980), where tùere are hi$

standårds of hygiene a¡d eaniøtioq littþ or no eecondary ¡¡n¡miesion would be expected to

occur, andas a¡rsuhan outbreakmrycomprieeprimarycås€sonly. Thetra¡smission ofcholera

is therefore complex and it is not ahvays easy to determine the exact mode of dissemination of

the dbease.

n

18 Potenüal protecdrrc antigeru ofVibdo clrolerae

The identification and isolation of pnotective antþens offe¡¡ one aPProæ'h to the

problemof developingvaccinesaginrtbacærialpathogens.vinrlencedeterminantsidentifi{d

by ehrdies on the pathogenesis of infection are ob,vious candidates for protective antþens' For

examplg an ap,preciation of the role of K88 pili in the colonization of the piglet gut ry

enterotoxþe ,,ic,E col¡rúto the dorclopmentof an efrectivevaccine comprisingpurifiedKS8

(Ruüef & Jones 1973;Rutter et aL 196). Althougþ various comPonents arc thougþt to assist

the pathogeneeb of vcløbae (rce section 1.3.3) it remains unclear as to whether any car

fu¡ction as protective antigenc for human infection The erridence inpticating these vario¡u

factors as pmotective antþns isbrieny revfors'ed'

L&t tPs

The LpS of a1 serotypes oonsisß of th¡ee regions: the lipid A core oligæaccharides

and the repeating o-antþea polysaocharide subunits which represent the outermæt surface

of thevÍbrio. The lipid A b hydrophobic and b responsible for the endoto¡ic biological activity

of LFS. The O-antþen region is responribþ for antigenicvariability and its chemical compæi-

tion has been determined (Hieatsune d aL lng: IGn¡e et oL lng,l98l Redmond lnÐ'

Unlike the LpS of most gram-negÊtive bacæria, the 2-keto-3deo:ryoctonate which lints the

core sugaß to the lipidA is absent i¡VùolqæLFS; instead fn¡ctose is present (Janet aL

1973;I(abif 1932) a¡d thb presumabþ eerves the same linlÉgg functio¡'

Observations made in a varÞty of erperimental models have sho\pn that antäbodies

directed against the LpS deærminan ts olV cholcræ harre the potential to mediaæ protection

Thb fu the casewith "open" systeme euch as the infant mouse model (N"oh & Ro*'þ 1972) or

the DIC (duodenal inoculiarion with cecal tigation) model in adult rabbits (Jensen er 4L 19æ)'

2l

AntÍbodies to LPS are al¡o sufñcient to mediaæ protection in the moet commonþ employed

"clooed" E''stem, the rabbit ileal loop model (Svennerholm 195; Chitnis d aL 1982a),

As discussed beloç, (Section 1.&3), it is generalþ aocepted that resbta¡ce to clinical

disease is mediated by antibodies directed aginst antþns of the bacterium ¡ather rhrn tie

todtr The rehtive significance of antibodies to LPS determinanls ¡pm¡ins uncertain, althougþ

ci¡cumstantial erridence suggerb that LPS may be a protective antþn for human infection

First, field and clhical testing of cholera vaccines has occasionalþ rrcsulted i¡ serotype+pecific

protection (Mosley d aL ln}; Cash et al- 1974a); since atrtigeos associatedwith LPS are tìe

onþ ones tnovn to difier between the serotypeq thi¡ protection was presumabþ mediated þ

antibodies directed agnin"tlPs. Secon4 a p'urified LPS væci¡e conferred E¡nsient protection

upotr adult recipiene in a Bangtadeeh ñeld trial (Joo 194). Finally, epidemblogic sûrdies have

identifred inverse correlationsbetween inc¡eased levels of a¡ti-LPS antibodies anddecreased

incidence of disease (Mosky d aL 1969; Glass d aL 1985)'

L&2 Non-LPSantþre

Until recently, little interest had beer shorrn in the possible vaccine siÊnificance of the

non-LpS oomponentr of Vdøleræ. Studies in the infa¡t mou6e model had indicated that

antibodies to ouch oomponenür were sufñcient to mediate protection and that in thb ryastem at

least these antiibodies appeared to be more eflicient h this respect than antibodies specific for

LpS (Neoh & Rocil ey ITIO;Aüridge & Ron'ley 19S3c). The naûrc of the prtative non-LFS

protective antþens remai¡¡ undeñne4 but ca¡didateewould itrclude outer membrane proteinr

(OMPs), flagellar a¡trgenq tIAg a¡d ptt

n,

L&¿l oMk

Ctaracterization of the outer membranee of Vclplqæ begpn comparativeþ recentþ

(IGbif 1980; IGlly & Parker 1981; Manningcl aL lgsl Jonson d aI 1989). Slh- oMPs of h

vino grantn organisms are anabZed þ electrophoresis in poþacrylamide gek i¡ the Pnresenoe

of sodium dodecyl sulfate (SDS-PAGE), approximatety 8-10 maior proteins rangng in ap'

parent molecular weight from 27-94 kilodaltou (kDa) can be resolved" Maior proæine include

3-4 proteins in the ratrgp of 45-48 tD+ uÀirù p¡obably repreeent the porins (Kabir 198q

K.lly&Parker1981;Manningøf',L]lg94l(abir1983;Iang&Paþa1987;Jonsoner4I1989),

a 40 kDa prcteio' ompT and a 3g kDa proteiq ompu (petereon & Mekalanoe 1988; Jonson

er aL tggg). Other oMps include a 35 kDa OmpA-like protei¡ (Atm et al" 1986; Jonson ¿Ú a¿

19S9) and a 25 kDa prote¡n, OmpV (Stevenson d aI 1985; Fohlner et al l9ú41986b)'

Recentwork indicates that the oMP profile of V dtobae is significantly influenced þ

the gro$rth conditions empþed. The moet gtrikiog example of cr¡lu[edependent protein

s¡,athesb is the productioa of pili such as TCP (Section 1.3.3.4). Sciortino & Frnkehæin (1983)

reportedth atVdtolqaerecorrered f¡om the intesti¡es qf infantrabbiu expessednovelOMh

when oomparedwith h vito grotttorganisms. Cr¡lture in irondepleted medium regulted itr a

similar OMp proñle, suggesting that thb was a convenient means of sürdying Prote¡tr8 syn-

thesized in respons etohvir.p conditions. Ea¡lier etüdies had sho'c¡n thatV ùobæ OMh are

immunoggnic i¡ man (IGbir 1983; Seârs d oI lg8/). lVith the exception of TCP and the

flagellar stn¡cture, the cont¡ibution of any of these OMPo to pathogenesis (and thus the

potential protective signiñcançs) is unlnowa'

B

L&22 trlagenü fiI) antþere

St¡rdies in two differentcholera models rabe the poesibility lhar'V eholøae mi$tb€ar

a protectiveantþenassociatedwith the fl"ællretnrcture. Eubanla¿t aI (tg77)reportedthat

an antigen present in a crude flaçlta f¡action was able to elicit prrotective antibodies in a

modifrcation of the infant mouse model This antigen was not present on a noa-flagellated

mutant straiq nor did it seem to be ascociated with the fLællat core protein Subeequent

erperiments revealed that the sarne component was a protective antigen in the adult rabbit

tigated ileal loop õy6tem (Ya¡"ey et aL 1979). Althougb this antigen remaín¡ undefine4 tieee

¡ûrdies are of considerable interest in vien' of the poosibility ^hat the flagellum carries a¡

¿g¿phment factorwhich Playg a role in colonization (6ee Sectbn 133'1)'

L&2.3 IIAs

There is paucity of evide¡ce to suggest that anti-tlA antibodies are ¡xotective.

Chaicumpa & Atthâsfuhtha (199) reported that an anftody to a cell-associat€d HA from

Vdplqæ El Tbr Ol7 was marginalty protective i¡ the infant Douse c;hoþra nod€L Other

sû¡dies in the same systeq howorer, led to the conclusion that the O17 strain lacks non-LPS

protective antigens (Attridge & Rou,þ 19s3c). Finkelstein & Hanne (1982) found that Fab

fragments of antibodies againstpuriñed SHA/proteasecouldinhibitthe hvíto attachmentof

V ctplqæto infant rabþi1 sm¡Îl bwel Suboequent e.rperiment¡ failed to demonsEate protec-

tive activity associatedwith such antibodies (Booth & Finkelstein 19Só). A clearer appreciation

of the likeþvaccinesþificance of úevarior¡sHAs producedþ Vchfuæ mustarraitfurther

studies. lvith the cloningof the gene encodinga v dpleræ tIA (Franzon & Manning 1986) it

mãy 5. possible ro determine whether or not HAs possess protective activity in cholera

7A

1.&¿4 P¡n

At the co¡¡me¡¡oement of theee sûrdies, ¡orhing was knoq¡n of the prrrtective cig-

nificance of a¡tibodies directed against V dplaæ pili nâinly becar¡se the question of pilru

expression remained coDt¡o/enial Recently, howerrer, Taylor et aL (1987) described cultural

conditions which allorred TCp synthesis by Vctwbac and shoil,ed tìat these pli plty"d a

critical role in coloniz¿tion, at least in the infant rnouoe model Ð "n logwith other sy8tem$

one migþt speculate that anti-pih¡s antiHies could be protective againrt V drcbæ by

¡s¡ü.alizjng the colonþ¡tion capacity of the bacteda Thus, pilus vaccines prepared from

diarrheagsnic strains ol E coli and given parenterally to pregnmt so$'E and coctB harre sig-

nificantly reduced ¡eonaøl deaths ftom d¡arrheal disease (Rutter & Jones 1973;Moryatd aL

lflg;Nan, d aL lng). hotection was ehoq'n to be mediated by anti-pilus antibof passiveþ

transmiüed ir maternar colootnrm @uüer & Jone¡ lglS Morg¡n¿l ø1" t9l8¡ Nag et aL 198).

pilus vaccines have also been effective in preventingerperimentzlMorælh h,ús infectbn i¡

qos,B (Ilamont & Bcbgo 198Ð. There is, therefore, a rtrong possibili$ thet TeP win achier¡e

the stafirs of a Protective antþen'

L&3 CT

Theother maiorantþen producedbycholeravib¡ioe isthe CT(revieu'edbyFinkelst€itr

1fl3; Finkelsæin & Dorne¡ 19SÐ. It has been purified to homogeneity (Fhkelstei¡ &

Lospa¡uto 1969,tnqRichardson & Noftle lg0), and its subunitstrucü¡re and mechanism

of action elucidated (Lonn¡oth & Holmgre ¡ 1973;Hohgrea & Lonnroth lnq' cT i8 a¡ &4

kDa polyneric protein composed of two protomeÉ: a¡ A (ZI YDa) or aderylaæ cyclase

etimulating compotrent (chobragen) and a B (5S kDa) protomer (choleragenoid) associated

soleþ with binding (I-ospalluto & Finkels tÐln lnlPierce 193; van Heyningen 'gm'

The A

25

subunit comprisee two PoþpePtide chaing At(pkDa) and A2 (5 kDa) (Saüler 4 aL 197!'Ln

et aL 1976)while five identical subunits (11.6 kDa eaeh) make up the B prrotomer (Gill 196;

Nakashima 4 aL ln6). The r¡arious physiologicål manifesatbns of the toxi¡ on different

tirsues, including the hypersecretory r€sponse elicited in the intesti¡at e¡úthelium, have been

found to be based on a singte moþcular mechanism. Folbwing the binding of the B subunit to

the Gvt ganglioside of the cell rembra¡e (Eee section 1.3.4), the A subunit is cleaved and

ente* the celr, activating adenyrate cycrase and leading to elorated cytoplasmic levels of cyclic

AI\,íP @ierce 4 aL lnl: Oill lØr'

CT b hl#ly immunoÊÞnic with nearly all the antibodies directed 4gainst the B subunit

(svennerhotm 1980; Hotmgren 1981). Ninety percent of North American volunteers

developedrises in senrm þG antitorin afreranexperimentalcholera infection andlevebwere

etill detectåble two yeafs latff (I-evin e 4 d 1981c)' Approximaæþ 6t% o1' votunteers

manifested rises itr eIgA antitoxin in i¡teetinal flur4 but the r€sPotts€ tetrded to be relativeþ

ehort-lived with antibody levets dropping one month after challenge (cited by l-e'n^e et al-

1e83).

Sûdies in various animal modets have i¡dicated that, whether passiveþ acquired or

induced þ active immunization, antibodie to t[e toxin caa protect ¡gninst eryerimenøl

chobra (roriewed by Finketrtst\ lnl, tflS; Finkekteh & Dorner 1985; Holmgren cÚøL

1g5b; yoohiyama & Brown l9sÐ. In contrast, volunteer sû¡dies have consistently failed to

demonstrate the invoþeme¡t of such a¡tibodies in ¡esistsnce to clinical infection (c-ash d aL

tgl4a;:If,l/trle¿f al 1y7g,1981a). I¡dee4 a rece¡t report makes it cþar that immune re¡Ponses

to the toxin a¡e notnecessary for protection in man (Levine ef 4¿ 198sb)- onþ two of the three

choleravacci¡es currently Þing developed have the potential to generate a¡ti-toric immunity

(eee Section 1.10.2).

?ß

1.9 ImmunitY Ûo cholera

It is nos, clea¡ that cholera infectio¡ gives rise to suboø¡tial protective imnunity to

subeequent expæuru to the disease. This has been convincingþ demonstrated by the fact tlat

the i¡cidence of the disease raprdly declines with i¡creasing ag. (see Section 1'7'1), and that

recurrent i¡fecti,on¡ a¡e fare in the communities whe'te the disease ie endemic (Ñennerholm

1980; Glass d oL lgg2). In addition, I-e'ine 4 4L (lgSta) reported that, three years after an

inducedcholeraice¡risodgvolunteerswe¡ìe abletoreristchallengewitha strainof homologoue

or heterol,ogou eerotYPe.

The nahrre of the protective immune response remains unclear. Available erridence

suggests that anftodies directed aginet determinana of the bacærium rather than the toxi¡

are primariþ responsibh for immunity to clinical dbease (cash á aL l9l *a;l-€'v lü!le d aI lnÐ'

In addition, despiæ the ris€s in serum antibodiee elicitedþ parenteralvaccines, such formule-

tions hare prfled inefiective in conferring protection Thb i¡dicates that the location, and

poesibþ the isotype, of the a¡tibodies is aho cfidcal Evident! the IgG and lglvl antibodieg

inducedby parenæral inmunizstion do not reach the gut in sufficient concentration to make

an effective co¡tribution to enteric defence. It b now çneralþ accepted +hat atr effective

vaccine win need to be oratty administered (sectbn r.10.2) to generate antibodies in the

intestine to prevent col'onizatb n o1 V dPbaa

L9,1 SSnsúeuic antibody rcsponrcs toV cholerac

Infectbn with, Vcløtqæ is associated with a ri6€ in titer of a varie$ of circulating

antibodies, incrudingvibrocidar a¡tibodieo (Fint*tstein lg6e Mosley 1969) and antibodies to

cT (Pierce d aI lnq,IJ,S (Clements d aL tg$?4,svennerholm et aL l984b) a¡d oMP (Sears

ct aL lgu). Esrty studies demonstrated thatvibrioci¡tat antibodywas acquired eåriy in life i¡

n

endemic a¡eas; p1lels increase with ag. and remain elertated, presumably æ "

oonsequence of

repeated environmental exposure to Vùfuæ (Mosley 1969: Glass aaL l98S). SharP in-

cneases in tiæn of both vibriocidal and antitoxic antibodier have also been demonstrated in

volunteer studies and in infected individuals from non+ndemic areas (Cash cú aL tÍl4c;I-wine

et øL lgg¡c; Snyder d aI lgSl; Clemenb el øl^ 1982). Hoç'errer, in contrart to the situation ¡n

endemic a¡ìeas, kvels of such a¡tibodies in these individuals are not sustai¡ed (I-f5tin.e d aL

1981c; Clenenß 4 ølI1982).

As mentioned above (Section 1.9), the circuLating antibodies generated þ inactivated

parrenteral vaccines do not confer signiñcant protection against the non-invasfueV dpbæ.

Nevertheless, earþ epidemiological studies rwealed an inverse correlation between

vibriocidal antibof titen and the inci&¡ce of choþra in the field (Mosley 1969).Althougþ

such a relationrhip was not apparent in avolunteer study in which vibriocidsl antibodiee we¡e

elicited by parenæral immunization (Cash etaL lll4a), such antibody rcsPonses wele as-

¡ociated with resistanoe to challengp in a rccent evaluation of an o¡al cholera vaccine (hcket

et aL,I¡ pne$). Þidentþ, ifvibriocidal antibodiea arbe as a consequence of oral presentation

ol V ctplaæ antigenq they reflect the occurreace of a protective lq(al immune fesPonse-

Mbrioci¿al anti6y responses may therefore ofÍer a convenient and helpful index of im'

munogencity to ass¡st the developrment of improved choleravacci¡es (hcketel&L,h press).

Since V etpløæis a non-invasive pathogen producing dbease at the gut mucæa onþ, itwould

be likeþ that the secretory imnune system i¡ the mrior protective mechanism in limiting

infection and the severity of the di¡ease.

a

Lg.z Dwebprent of intestinat sIgA antibody rcIþßrcs

Induction of the intestinal reslþûse occurr primarily in the Peyer's patches (PP;

\uoodruff & clâfke lggT;Mestecþ & Mcchee 1gs7). These are dome-like ss'ellings on the

epithelial surface of the gUt which serve as a staging area for mþating þmphoid cells to come

in contact with luminal antigens (Husband & Gowans 198). This interaction begins with

uptake of antigen by specialized epithelial cells residing o'/er the pP (Bhalla & ou'en 1983)'

These cells were initially characterized by the presence of luminal surface microfolds rather

than microvini lendingthem the name M celrs for microfold cells (oren & Jones ln[\.\vhen

microrilli were observed on these cellsr the name persisted and now refers to imembranous"

epithelial cells because of the very thin rim of cytoplasm separating the intestinal lumen from

the þmphoid cells below (wotf & Rye 1gs4).In addition these cells are devoid of pnoteoþtic

enzymes and correred with scant mucus thus designed to sample and transport antigen without

degrading it (reviened by sneller & Strober 19g6). Absoôed subotances from the lumen are

pinocytosed by the¡e M cefls and are transmitted to underþing PP in an undenatured form' to

be suboequentþ preeented by accessory cells to T and B lynphocytes (sminia et aL 1982;

Wilders er aL 1983)-

The factors which deærmine whether bacteria present in the gutwill be "sampled" þ

M cells remain unclear. uptake þ abeorption may be facilitated by specific membrane

receptors or gþcoproteins shown þ lectin-binding experiments to be present on the surface

of M cells (orn en 1gg3). Further research is necessary to determine whether specific receptors

would enhance selective antigenic endocytoeis and transport onren et aL (1986) has recently

demonstrated that viable, but not inactivate4 V choteræ are indeed inæ.t"d by M cells and

trancferred apparentry intac! to mononuclear cells in underþing l¡'mphoid follicles. They

suggested that critical surface antþens or structures' apPafently mis¡iag from inactivated

29

bacæria, are required for uptake (ornen et øL 1986). Inman et aL (19%) have shown that the

nature of the surface poþaccharide can influence the rate of E' coli samplingby M cells'

Follouring antigen sensitization, the primed l¡rnphocyæs leave the PP via efferent

l¡rmphatics without further difierentiation (Craþ & Cebra t97l)' They then pass th¡ougþ

meeenteric nodes and enter the thoracic duct f¡om which they mþate entering the s¡ætemic

ci¡culation to home back to the gut lymph nodes. The concept of a common mucosal immune

system linked þ emigrating IgA Pfecun¡of cells has been supportedby e:perimental studies'

These harre sho*m thattùe circul,atingintestinalþ-Primedþmphoblasts notonþ return to the

sub-epithelial tissue6 of ûe gut, but migrate also to the lungs and genitourinary t¡act as well ae

to exocrine glands (salivary, lacrimal, mammary) (McDermott & Bienenstock 1fl9;

Bienenstock & Befi¡s 19g0; Bienenstock d aL r9g3). This ensures that the activation of

lymphoblasts in a particular mucosal sile can ¡esult in the deplo¡rmentof appropriate effector

cells th¡oug[out that mucosa as well as other mucosal surfaces'

Evidence has ¡ou, accumulated to indicate that humans also have a common mucooal

immune Bystem. primed þmphoblasts harre been detected in the peripheral blood afær earþ

intestinalexpooureto antþen; in addition, oralimmuniz¿tion resultsin thesecretion of specific

sIgA antibodies in saliva, tears, milk a¡d nasal secretions (roriewed by Mestecky 198Ð' l¡ this

contex! þcke et aL (lggÐfound thatSwedish volunteers immunized oralty with a combined

bacærial-toxoid vaccine generated peripheral blood l¡'mphocytes which differentiated into

specific cholera þA antitoxin and anti-I-PS antibody-producing cells during a period or'ín vino

culture. Other studies ha\¡e demonst¡ated that oral immunization of volunteefs or natural

disease in cholera induces ar¡n¡v chotsa¿ antibody respomes in sativa and breast milk

(Jertbom et 4L lg8fj) also reflecting the gut mucosal responses is part of the common mucosal-

associaæd immu¡e s)tstem.

30

Recent erridence shoun that cell surface molecules selectiveþ e:çressed in mucoeal

organs are required for lymphocyte honing to theee eites (Streeter e|øL 198ß)' To enter

intestinal tissues, þmphocytes must reach the pootcapillary venules in PR other þmphoid

aggregates or the muco6a, and must adhere to tìe endothelium to begin their migration- It is

nou, clear ft¿t this is due to seþctive and specifrc interactions between surface determinants

of þmphocytes a¡d the endothelial cells (referred to as hiÉ endothelialvenules, HEÐ of the

pootcapillary venules of the þmphoid tissues. Surface determinants that permit these interac-

tions ate found on both the tymphocytes and the endothelial cells. The determinants invohed

in mucosal tissues differ from those invoþed in peripheral lymph nodes (reviewed in Mõller

1e8e).

tl is beco,ming increasingþ errident that the þA resPonse is T cell dependent (revieu'ed

in Meetecþ & Mc6hee 19gz), particularþ in the induction and differentiation of IgA+ B cells'

They harre been ohourn to induce B cells to ss'itch from e:pression of surface IgM Ûo surface

IgAaswell ag inf,uencingthe proliferation and ærminal differentiation of B cells throu$ the

production of inærþukins IL4 and IL-5, respectiveþ (revien'ed þ Mestecky & McGhee

19sÐ. The dimeric IgA produced ry these cells in the lamina propa is transported into the gut

lumen through the columnar epithelial cellsvira a speciñc receptor, secretory component' which

is located on the basal surface of these cells (Mostov et aL l98/ù and produced by epitheliral

celts of avarieg of other secretory organs @randtzaeg 1gg5). The secletofy componenton the

sIgA nolecule confers some resistance to proteoþfu, il important characteristic because of

the environment in which sIgA exerts its activity (tindh 1975)- Recently, it has been

demonst¡ated that intes"îal antitoxin formatio¡ and pnotection against toxin challenge after

oral immunization with CT is thymuedependent and tikeþ to be under Tcell control (I-ycke

et al- 1987a).

31

1.9.3 sIgA ant¡-v c.holerae rcsPoßtes displ¡y irnrunobglcal nnlmry

The intestinal sIgA response to cholera is of relativeþ short duration, lasting for a fern¡

weeb to afeç,months (Sennerholmel aL lg8ø¡b).This contrastswith protective immunityin

cholerawhich appears to last for severalyears (Levine 1980; Glas6 et oL 1982). This paradox

has been recentþ resoþed by experimental and clinical studies which document long-term

sIgAmemory to cholera antigens (Ñennerholm¿f aL [9$4a;Holmgren & Lycke 1986; lq"b

etú 1987b). For example, women in Bangþdesh who had been gry"o 2 oral immunizations

with cholera B subunit 15 months eadier respondedwith intest¡"al IgA antitoxin production

Eofe rapldly, and to a lourer dose of antigen, than a concurrentþ teste4 p¡atioueþ unim-

munized gfoup of volunteers (þcke & Holmgren 1987). Similarly, 2years after initial oral

immunization of mice, a second dooe of antþn evoked a rapid intestinal antibody resPonse

which corretated with heigþtened resistance of gut loops to CT challenge (þcke & Holmgren

19s7).Inaddition,immunologcalmemofyhasbeenehol¡rntobecarriedþBcellsandthatit

is possible to adoptiveþ transfer long-term memoryþ these cells isolaædfrom donoranimals

1 year afær priming (þcke & Holmgrel 1989). Ttese studies all show that the intestinal

secretorylgAsyEtemhasaveryefflrcientimmunologicmemory.

Lg.4 Mechanismof sþa antibody-rediatcd Protectbn agdnstv choleme

Various studies hatre denonstrated that the biological function of sIgA is to prevent

initiation of infection þ organisms using the mucosal surface as their primary location of

attachment during deyelopment of disease. rilhile sþA is neither opeonic nor p*s€sses

bacteriocidal activities (re'iened by underdown &, schiff 198ó), it can efficbntly cross-link

antþns, prerrent adhesion and inactivate toxins. sIgA can render bacæria mucophilic (Mag-

32

trusson & Stiernslrom 1gg2). It is well knou¡n that vibrioo are associated with mucus ínvivo

(Jones d aI ln6; Nelson et aL 1976)'

The specific mechanism by which the mucooal immune system functions is poorþ

understood. purified sIgA antibodies har¡e been shoum to prorent attachment of bacteria to

mucosal surfaces. Intesthar loops prepared in orarþ immunized mice were partially prctected

from the effects of live cholera organbms in elperimental v ùobæ infection (Fubara &

Freter lg13).The protection appeared to be due to the action of specific antiMies excluding

the organisms from attaching to the gutwall. steele et aI (1974) reported that purified sIgA

di¡ected agúîÊtv e]þlqæLßwas prrotective in the inf¡nt mouse cholera model; subsequent

sûrdies in this sy'tem suggested that the antibody function necessary for protection is the

capacitytoinhibitbacterialattachment(Attfidge&Rou{ey1983c).

severat shrdies have shq¡n that þA antitoxi¡ antibodies induced by e:perimental

v chobøeinfection, orbyoraladministrationofcf areassociatedwith pmotection ef ialsstinal

loops asainst CT challengp (Pierce ef 4L lnE: Taman¡ & Broum 1985; Vaerman d aL 1985\'

sIgA can neutalize toxin (svennerùolm 19SO). I'.ryckeet al (l9s7a)have recentþ demonstr¿ted

that IgA antitoxin antibodie¡ qrnthesized h vito þ lamina propia cells have protective or

neutrarizing activity. Evidence suggests that the essentiar mechanism is þ preventing the B

subunits from binding to the crrar ænghoside recepto¡s on the epithelial cells; indeed moot

antitoxin antibodies a¡e directed ¡grinst the B subunit rather than the toxic A subunit after

both i¡fection and oral immu¡ization (Holmgren 1981; Lindholm eï4L L983)'

ILl,

Þ

I

I,1,

fl

II

I

I

i

33

f

I

li^

1.10 \äæination against cholera

L10.1 Ptst appmaclcs Ûo rraccinatbn

A century has ela@ since the first choleravaccine was adminisæred to human beings'

yet there is still no acceptable, effective vaccine for cholera (revietved by Finkelsæin 1984)'

The history of vaccination 4gaiast cholera began in 1885 when a vaccine composed of broth

cultures of tiving cholera vibrios was iniected subcutaneously by a spanish physician named

Jeime Ferrán y Clua (Bornside 19s1). In 1892, lValdeman Mordecai Haffkine, a Russian'

derreloped a resimen of two subcutaneous inoculati,ons of a vaccine which had been nat-

tenuatedn þ culture at 39 C Althougþ the vaccine caused malaise, ferter, pain and swelling

these side effects were considered acceptable and the vaccine euitable for admintst¡ation

(Bornside 1932). Honreveç due to ethical coneiderations, livevaccineswere then replaced by

rìe killed vaccines of Kolle and otherworkens (Pollitzer 1959).

1.10.L1 Conventionat Hlled vaccines

Theee vaccines $,e¡e prepared þ inactivation" either þ pheno[ formali¡ or heat

treatmen! of suspensions of Vchobæ groun on agar or in liquid media. only in the 19608t

with theadventof cont¡olled fieldtrialsasa meansof er¡aluatingvaccineefticacy, diditbecome

apparent that such vaccines were not eufficiently effective to have an impact on the incidence

and prwalence of cholera (reviened þ Finkelstein 19&4). Althougb some formul¡ations

conferred higþ lwels of protection, the immunity quickþ waned (Joo 194; Feeley & Gan-

garosa 19g0). Furthermore, killed parenteralvaccines were generatþ more effective in older

people, for whom the vaccines wene thougþt to booet environmentalþ acquired natural

immunity (Finkelste tr lg6}).More frequent admi¡ist¡ation of vaccine was required to observe

I

It(

I

I

v

simlar rates of protection in young children (Joo 194; Feeley & Gangarosa 1980)' the group

at highest risk of acquiring infection'

The oral administxation of killed vaccines had been considered ser¡eral time¡ prior to

1960 (Polli Eet 1959;Freter & Gangarosa 1963), but the impetus for such an app'roach was

provided þ a report that college students fed heat-killed vibrios mounted both serum and

intestinalantibodyresponses (Freter& Gangarosa 1g63). Howerrer, subsequenttestingshowed

that the oral route of administration was not as effective as parenteral vaccination in conferring

resistance to a clinical infection (Cash et al' 1974b)'

1.10.L2 Toxoid vaaines

The recognition of the fact that cholera is a toxin-mediated disease (De 1959;

Finkelstein e Lospalluto 1g6g), suggested the feasibility of developing protective toxoid

vaccines, an appfoach which had proved successful ia somþ¿tfing diphtheria and tetanus' As

discussed above (Section 1.g.3), data fromvarious animat moders indicated that antibodies to

the toxin could mediate protection againsterperimental cholera, promptingthe development

of toxoid vaccines for clinical apprication. A formarized toxoid (which was stabilized s'ith

gþcine to prevent partial rerrersion to toxicity) wÍ¡s prepared (Ohto mo 1977\ and tesæd in the

philippines (Noriki tgïl)but had no impact on the incidence of infection. A gluraraldehyde

toxoid prepared by Rappaport and colleagues (194) was tested in Banþdesh' Prolection was

observed up to 24 weeks, but the cumulative protection ovef a one-yeaf Periodwas marginal

(Curlin et at 197ó).

Volunteer studies suggest that resistance to clinical infection is prwided by antibacterial

ratherthanantitoxigimmunity(ca"hetaL|974a;I.er,inedaL|979).ofaladminist¡ationof

three doses of cholera toxoid failed to prorride any proteclie¡ against cholera challenge,

whereas previous clinical infection conferred enduring resistance to re-challengewith strains

i

35

of homologous or heterologous serotyPe (I-evine et aL l!79; 1981a)' More recentþ, lær'ine

et aL (l9ffjb) haye shon¡n unequivocalþ that resistance can be elpressed in the absence of

antibodies to cholera toxin.

L10.2 Cument approachcs Ûo vaccination

The disap,pointing results obtained from fietd trials designed to er¡aluate the efficacy of

inactivated parenteral vaccines contrasted with the realization that clinical infection is ibelf a

very effective immunizing pfooess. Epidemiologic frndin8s (Mosley er 4L Lgflf¡; Glas et aL

19g2) ande:perimentalchallenge studies involunteers (Cash er øL lg74a;l'erttneet aL l9Sla\

have demonstrated tùat cholera infection is followed þ poten! long'lasting immunity for at

least three year' (I-evine d ar lggla). current k¡o*'ledge about intestinal immunologl

(Section 1.9) indicates thatthe oral route is superior to the parenteral rr¡ute for stimulatingthe

formation of sIgA antibody in the gut mucoea Indeed, this knowledge, coupled with the

collective failure of parenteral vaccines, provided a strrong impetus for the development of

chorera vaccines suitåble for oral administ¡.ation Tr¡o main approaches have been followed

over the past 10-15 years, with a thiÌd option being develo@ mo¡e recently'

L10.¿1 üve attenuatcd oral mccines

The basic approach employed in the dei'elopment of these vaccines has been to

inaptivate the genes for toxin production, leaning i¡tact thoee bacterial comPonena required

for gut colonization. The prototype vaccine was t\ev choløae strain Texas star-sR which was

attenuated þ chemical mutagenesis (Honda & Finkerstein rg9). This mutant produces tle

B but not the A subunit (ATl* ) of cholera toxitr. Involunteer studies, itwas found to colonize

and proliferate in ttre gut and to elicit serum vibriocidal antibody responses comparable to

tiose observed followingnaûrral infection Production of slgAaminstsetreralvibrio antþens

%

was denonstr¿ted (I-evine et 4I lgf!|¡). Hou'ever, in spite of the Prcsence of the B subunit

antitoxin responses were poor (Iævi ne d øL l9g3, 19s4). Although the strain was well tolerated

by a maiority of volunteers, ?AVo e:rperienced mild dianheq a side effect which precluded

further work wittr this st¡ain (Lerrine et 4t. lg8p¡). It did Plry a role, however, in e:ploring the

feasibility of mimicking infectionderived immunity þ means of an oral attenuated vaccine

(I-evine d ar lgu), thus p*ing theway for the geneticalþ engineered st¡ains to be discussed

belou,

The ñ¡st geneticalþ engineered candidate vaccine strains were described in 1984

(Kaper d 4L lguíÐ19S4b). St¡ains olV choleræ knou to cause disease and to give rise to

protection in volunteen (Lerrine d 4L lng: Le'ine 1980; Levi¡e d 4L l98La) were used to

prepare a series of derivatives from which the genes encoding both the subunits of the toxin

(AT-) or just the A subunit (A-B* ) were deleted by oiædirected mutageneßß (Y:apr ef d

19844 184b).

Volunreer eh¡dieswith JBK70 (A B-) and CVD 101 (AB+) shou'ed that these strains

colonizedwellandeliciæd goodvibriocidal andantitoxic(cvD 101) reeponse& Furthefmo¡e'

JBK 70 (AB) vaccine strain gæe significant protection (89Vo efficacy), illustrating that

resistance is notdependent upon tie generation of antitoxic immunity- Howerrer, þeû strains

were reactogenic (r.evine er at. rgßb), causing mild diarrhea similar to that seen with the

prctot)?e Texas StarSR dercribed above. one poosibility is that the Prccess of colonization

pa sepnovides sufÍrcient irritation to the epithelial surface that mild diarrheawill inevitabþ

occuf in a propofion of recipients (snith & I-inggood l97t; Finkelstei¡ 19&4; Wanke &

Guerrant 1gs7). Alærnativ ety, v ehotøae mifit secrete multiple toxin molecules, so that

deletion of the genes encod.ing the well-recognized cholera toxin might not completeþ

abrogate diarrheagenic potential (Iørine d aL l98Á;b)'

37

I-errine et at (lgæar lgssb) have performed further volunteer trials to address this

issue. Deletion of the El Tor hemoþin/cytotoxin genes did not decrease reactogenicity of

JBK70 or CVD101 (Isrin e et al- 1988b). In addition, studies with (X/D103, an AB + derivative

of a Classicål Inaba strain 569B which lacb the geneß for the El Tor hemoþin, confirmed that

ttis molecule was not the cause of diarrheagenic activity (I-evine er^L l988z). Hou'ever,

althougþ CVD103 caused diarrhea i¡ l2To of volunteerõ, a mercury-resistant derivative

appears more p,romising CVD1O3-HgR was well tolerated by ?A of 25 recipients, while

retaining the immunogenicity and protective efficacy of cvDr03 (Irvine ef øl 1988a). Further

clinical evaluation of this strah is clearly warranted. At this stage the precise mechanbm

responsible for the reactogenicig of these derivatives remai¡s unclear (Kaper 1989)'

L.I|O.LZ Non-viable or¡l vaccines

Evidence thatantitoxic and antibacterial immunity actsynergisticalty to Protectagainst

e:rperimental cholera (Svennerholm & Holmgre n1976)prwided the impetus for the develop

ment of a combined bacterial-toxoid vacci¡e. Althougþ protective antitoxic immunity has been

shou,n to be directed ptrmarily egrinst determinanß of the B subunit (Holmgre¡et aL 1977:'

Holmgren & Svennerholm 198Ð, the targets of proæctive antibacterial antibodies remain to

be elucidated particularþ with respect to clinical infection (see Section 1.8). Accordingþ the

combined vaccine comprises killed whole bacteria a¡d the isolated B subunit of the cholera

toxin (Clemens d aL 1986).

This vaccines has now been extensiveþ evaluated. Administration has been shos¡n to

stimuliate local sþA antitoxin and anti-LPS reEponses in voluntee¡s in both endemic (Senner-

holm ef aL l9g4a)and non-endemic a¡eas (Jertborn 4 aL 1984). No side+frects were observed

in studies with several population groups (Black e! aI 1987; Svennerholn et øL l9ah;

Migasena et aL t9g9), or in a Bangladesh field trial (Clemens eÍ aI 198Ð. Vibriocidal serum

38

responses were lor' @lack et at. t987; Clemens ef 4t 1987; Migasena e¡øL 1989) when

comparedwith that inducedwith the attenuatedvaccine strains discussed above (I-evneet aL

19SSb). Nevertheless, it has proved to be prctective in volunteers (Black 4 aL 1987) and in a

recent field trial in Banþdesh (clemens d oL 1988, 1990). To confer a7o7o protection rate,

it had to be gryen in tno doses to adulte in Bangfadesh over a three year obsewation period

(Holmgren d 4L 1989: Clemens d aL ßm)'

The combined bacterial-toxoid vaccine clearly represents a maior advance over the

killed parenteral vaccines, in that the protection conferred is enduring. As a vaccine it is not

yet ideal, howwer; its main deficiency is that protection in young children waned rapidþ with

time aftervaccination (Clemens d aL lg8f,, 1990; Holmgren et al- 1989).It is also debatable

as to whether the inclusion of the B subunit is justifiable from the vieu'point of vaccine

production coat A comparison of the protective efficacies of the combined vaccine and a

vaccine conprisingiust the bacterial component indicates that the advantages of the former

are t¡ansient (KaPer 1989).

L10.23 üve oral CtolerqÍBrPhoid bùrid vaccine

In an att€mpt to combine the supe¡ie¡ immunogenicity of a livevaccinewith the eafety

of an inactivated formul,ation, an attempt is nou' underway to clone a protective antigen from

V chobæ into the attenuated, live oral typhoid vaccine Satmonella typhi Ty2la (Germanier

19g4). The latter is now acceptedas atr effective typhoidvaccine, thesafety of which has been

demonstrated h numerousvolunteerstudies and fþld trials (Germanier 19&4). Althougþ the

protective antigens of. V cholqac remained undefined with regard to clinical infection, cir-

cumstantiål evidence suggested that antibodies to LPs could confer immunity (Section 1'8'1)'

Accordingþ the genes responsible for O-antrgen synthesis nV dtolqæwefe cloned (Manning

et aL 19116) aod transferred hto T21a (Moronaet4¿ 1988)'

39

one of the resulting clones, EXó45, has nou' been shoutn to be safe and immunogenic

in man (Forrest d øL rggg). Io "

recent cholera challenge t¡iar¡ vorunteers immunized with

Ð(ó45 and then challengedwith pathogenic v cholcræ excreted sþificantly fenrer bacteria

and suffered significantly less diarrhea than unvaccinated cont¡ols (Tacket daL,In press)'

Althougþ this study showed that antibodies ûo the oantigen ol v cholqae are sufücient to

confer protection agninst clinical disease, a greatø consistency of immune responses lltiu be

required before such avaccine warrants extended evaluation'

LllI4 Conclusion

To achier¡e the greatest public health effect in endemic areas an ideal choleravaccine

should be (a) safe without side effects, (u) easy to administer, (c) inexpensive in vie$/ of the

target population and (d) higily protective preferably after one dooe. clearþ none of the

vaccine candidatesiustdiscussedmeetallof these criteria. Because of its safetyandlong-lasting

protective efficacy, the combined inactivated vaccine is the onþ suitable formulation for

widespread use in endemic areas. Unfortunateþ, hou'ever, althougþ safe' the cost of this

vaccine, and the fact that murtþle doses are require4 rimits its utility. A geat'" drawback is

the factthatitprorrides only shortterm protection inyoungchildren, the groupwith thegreatest

risk of infectioo AlthouÈ the attenuated live oral vaccine being derrel"p"d by l-evine's gouP

elicits stronger immune nesPonses and would be much cheaper to produce' two problems

remein. Fint, the issue of residual reactogenicity is notyet satisfactorily resohed" secon4 the

safety and efücacy of such a vaccine remains to be demonstrated in young child¡en. As poinæd

out þ lGper ( 1989), rapid progress in vaccine reæarch makes it unlikeþ that another 100 years

will pass without a successful vaccine against cholera

40

1.11 Aims of Thesis

Atthe commenoementof these ¡tudies theprotective antigens or.v ùolsøe remained

undefined. Altlougþ circumstantial er¡idence suggested that antibodies to LPS a¡e prrotective

against human disease, the inactivated whole cell vaccine possess€s these antigens yet remains

inadequate. The preoenoe of non-LPS protective antþns on at least one Vclølqæ strain

(5698) appeared to merit further invertigation, to at least ascertain.the distribution of these

components emong recent field isolates. Such a stuf represents the maior aim of Chapter 3

of this ttresis. Clearþ, if such strains share non-LPS protective antþne, frEther study of theee

antþns miÉt evenûralþ identi$ trew oomponents of vaccine sþificance- Duringthe oourse

of these initial e:perinents, ehrdies þ Mekalanos' group (T¡ylor d aL 1987) resolved the

cont¡oversy surrounding pilus production by V choløæ. The critical involvement of TCP in

infant mouse pathogenesis offered a ne,s' approach to choleravaccine development It seemed

important to confirm these finding¡ and to determine whether TCP represents a protective

antþn ¡¡ this model This, together with other attempts to identify non-LPS protective

ant' ns ofV dtobæ, is the focus of Chapter 4'

ffis ¡im of the ñnal eryerimental chapter of this thesis is to clone genes involved in the

bios¡rnthesis of TCp. The derivation of clones eryressing TCP would allow unequivocal

confirmation of initial findingp concerning the sþificance of these pnli * virulence deter-

minants and protective antigens in the infa¡t mouse model

4t

Materials and Methods

2.! Bacterial sfains

M*, of the st¡ains used are shor¡,n in Table 3.1, which lists biotype, serotype, virulence,

sounee andyear of isotation (wherever poosible). All are resistant to streptomycin (100Fg/nl);

when neoessa¡y, such ree istance was selected þ growth on st¡eptomycin+ontaining ( 100 pdml')

nut¡ient ag'r Otqr; Difco, Det¡oit) plat€e Other straiûs used but not listed in Table 3'1 are

detcribed and discussed in Chapters 4 and 5'

2.2 Malntenance and grcrvth condiüons

permanentetoclg were stored in the þophilized state in 107o skim milk at 4 Ç and also

frozen at -70c in a mixh¡re of 0.4 nI SOvo (viv) dycerol plus 0'6 nl lvo peptone (Difco'

Bacto-peptone). strains wefe reconstituted s'ith nutrient broth (NB; Difco, Detrcit) as

required. Storking stock culürres wefe 6¿iatrined on NA stabo stored at rr¡om temperature

for periods of up to eightweels. For rr¡utine uee, the stabs were streaked onto NA pl'ates which

were then incub atÃat3l CovernigþL scrreralof the resultingcolonieswere used to inoculiaæ

10-ml aliquots of NB, which u,ere then shakenwithvigorous aerationat3T c unless otherwise

42

specified" such culhrres t¡picalþ attained a concentfation of 1-2 x 109 bacteria per Dl afær 3-4

hours.

Asloppy 4gar orrerlay techniçe (Stocker lg4g)was employed to ensure thatthe st¡ains

remained hrghly motile because motile strains have been shou¡n to be more virulent in infant

mice (Guentzel & Berry 1975; Attridge & Rou,ley 1983a)- A NA platewith 100-200 isolaæd

colonies was overlaid \tith t}-lzmls ef sloppy aga¡ (2 parts NB to 1 part melted 2Vo NA¡

prewarmed to 45 C). The Plates $'efe left at room temperature for 15 minutes to allou' the

overlay to se! and then incubated a¡ 37 C. Those colonies comprised of motile bacteria

gradualþ derreloped a halo as the organisms swam into the sloppy agar' MicroscoPlc obser-

vation ofbroth cultures seededwith thaloed'ornnon-haloed" colonies confirmedthe reliability

of this technique.

23 Speciatizùgrowth media

Water used to prepare media was deionised and filtered, using the Milli RO60 system

(Millipore corporation, u.s-A,). Media used to sû¡dy the produclion of pili n v chol¿ræ

stlains were the follorving

typtone waÛer (T"e"dy el aL lgæ)t lgBacto tryptone and 0'5gNaClwere dissolved

in 100 ml of water.

Iþpticasesoybroth(Farisetal.1982\:obtainedfromBal¡moreBiological

I-aboratories @BL), Baltimore.

FepSone salim brcth @vans et øt.lglS):ZgBfictopeptone and 0.5g NaCl dissolved in

100 mlg of water.

hptone sallne agar (Ev¡ns et al. 1fl8)z Peptone saline broth to which 2gor' Agaf

(Difco) was added.

43

Colonlzation Í¡ctor bnoth (CfB; Evans et al. 1979): consisted of 10g casamino acids

(Difco), 1.5gyeast extract (Difco), 0.059 Mg$Oa and 0.0059 MnClz in 1 liter of water'

colonizatlon f¡ctor agar (c:M; Evans er at. t979): prepared by adding Ba cto ag'tr (27o

wft) to CFB medium-

'ICç Medium (Ehar a et aL 198ó): consisted of 10g Bacto try¡rtone, 29 yeast extract

(Difco), 59 NaC[ 39 NaHCOI 29 monosodium-L-gfutalnate' 0.2gthioproline (sigma)' lmM

EG?\ (Sigma) and 20g Bacto agar in 1 liter of water'

LB Medir¡m (Miller 1fl2): was Pfepared by adding 10g tr¡ptone (Difco), 59 yeast

extract (Difco) and l0gNaCl to l liter of waterwith adiustme¡tof pH to 6'5'

V cholqac strains urere inoculated into the above media a¡d incubated at different

temperatures forvarying periods (see Chapær 4)'

When ap,propriate, antibiotics were added to broth and solid media at the follou'ing

final concent¡ations: ampicillia (Ap; Sigpa) 100pg/nl; kanamycin (Ih; Sigma) 50 t'üml,

tetracycline CIc; Calbiochem), 10 rgûl for.E coli a¡d a pdñ fot V eholqae (Chapter $;

str,epomycin (Sm; Glaxo), 100 P g¡mt

2.4 ChemicalE neagents and buffers

Reaçnts were prepared with deionised and filæred Milli Qwater as described above.

Chemicals were anaþical gade. Pheno! sodium dodecyl sulphate (SDS) and sucrose were

from BDH chemicals. l}is was Thizma base from Signa EDTA was fiom Sigma Bovine

sefum albumin (BSA fraction Ð was fncm Flow l-aboratories, Roclville, Md). cesium

chloride (technical g¡ade) was from May & Baþr'

The follorrringelectrophoresis gaÃereagentswereobtainedfromthesou¡cesindicated:

acrylamide and ammonium persuþhaæ (Bio-rad), ultra pure N,N'-mettrylene-bisacrylamide

u

fron Boehringer Mannheim, lVestcermany. The subotraæ for immunoblottingwas 4-chloro-

l-naphthol (Siena).

Buffers and solutions were also prepared with Milli Q deionised water. Saline was

o.85ro 1wfu) NaCl in waler, while phosphate buffered saline (PBS; prl7'2) comprised of 8g

NaC[ 0.2g KCl' 1.159 NaZHPOa and 0.2g KHZPO¿ in 1 liær of water.

L4.L Enzyre-thlrrd irrnunosorbent assay (EtISA) Bufiers

2SmM tis-HCl þH ?.5): was made þ dissohing 3.039 TLis base in 900 ml of water

and adiustingthe pH tD7,'with lM HClbefore makingthevolume up to l liter'

Ihis-saline.Adde(TSAc-oatingbuffer): preparedþdissotvingT.T2gNaCl (132mM)'

3.039Tris (25 nM) pH 7.5 and 0.5gNaN¡ in 1 liter of water.

\ltrashing bufier wlth tlton X-1011 prepared þ dissolving43.83gNaCl 3'03gÎis' 5ml

titon x-100 in 4000 ml of water and adiusting the pH to7.6with ap'proximateþ 18 ml of lM

HCI before making the volume to 5 lite¡s'

O¿S M tiethanolamlne: 3T.Sgtriethanolaminewas neutralisedwith 2;S\ÁHCI (ca 60

mls) to pH7.6; 1g NaNf was added and the volume made up to 1 liter with water'

BSA[Ibeen: l6gNaC[ 2gNal|[r 0.4gBSA 100 mls of o.25 M triethanolemine' and 10

mls of lO% Tttæn 20 in 2liærs of water'

hzyme Dtluent (for dilutin gentymelattibody conjugate): 8g NaCl was dissolved in

g00 ml $ratet, follou,ed by the addition of 50 ml of 0.25M triethanolamine pH 7-ó and 2mls of

500XMg* * lZo+*(SOOnÀ,f MgClzll.zímMZnClz),0.2gBSAand0'2gNaN¡; thevolume .

was made uP to 1 liær.

45

2,42 IÞtergents

The follou¡ing detergents were used in an attempt to solubilise oute¡ membrane

preparatioñ of V choleraez

ionic:Sodiumdeo:rycholate(BDH)&sarkooyl(Ciba6eþ);

non-lonlc: Ti.iton x-100 (BDÐ, Tliton x-114 (serv4 Feinbiochemica, Heidelberg)'

Tneen Ð (Signa), Bfij 53 (Serva), Nonidet P40 (BDH), Urea (BDH-Analar), deærgent test

kit No ?ßYs}(Calbiochem Behring Diagnostics, La Jolla, California);

zwltterionlc Zwittergent detergent test kit No. 693030 (Calbiochem)'

L43 nnzlm and inrnrnorcoq¡qgatês

The following enzymes were obtained from gigme: deo:ryribonuclease I (DNAse I)'

ribonuclease I (RNAse I), þozyme and hyaluronidase. honase and ProÛeinase Kwere from

Boehringer-Mannheim.

Restriction endonucleaseo Banr{l[\ r/;oR:l, San3A aDld J(bal were purchased from

Boehringer-Ma¡nheim. DNAmodisingenzJmeswerepurchasedfromNen,EngfandBiolabs

(t4 DNAligase) andBoehringer-Mannheim (DNApolymeraseI, Klenow fragmentof DNA

polymerase I and moleculiarbiologr grade, calf intestinal alkeline phoophatase)'

Horseradish peroxidase-coniugated goat anti-¡abbit and goat anti-mouse IgG were

obtained from Nordic Immunological Reagents (Tilburg the Netherlands)'

25 Animals

2-S.l Adult uice

Adult female closed-colony inbred outbred I-{C mice urere obtained from tle Central

fl¡imal House of the university of Adelaide- These micewere bred under specific pathogen-

ß

free conditions and subcequent$ mored to a conventional animal room These micewere used

for preparation of antise¡a and as a source of enterocytes for h vìtro studies of bacterial

adherence.

L52 Inhntmice

Abreeding colony of conventionalized I-AC mice was maintained in the Medical School

Animal House of the University of Adelaide. Each breedingcage containedfour female mice

and one male, and the breeding stock was replaced at six-monthly inærvals. The infant mice

wereusedatabout5 da¡,sof age (weig[trange restricted toL4-275g) forstudieswiththeinfant

mous€ cholera model (see belov).

2.6 Preparation of hiled and formalin-killed organism s

(i) Boiling Bacteriawere gro\r,n in NB washed ¡n'ice in saline, then resuspended to a

concentration of approximatery 1010 per mL Aviable countwas performe4 followingwhich

the suspension was heated to 100 c for 2 hou¡s The organisms were then washed three times

in sali¡e and resuspended in såline to lìe original volume; aliquob wele Pl'ated onto NA to

ensure thatnoviableorganisms remained. o.LTo 1w&) Nal.I¡was added to thesuspeneionwhich

was then stored at 4 C.

(ii) Formalin-inactivaüon. An equal volume ol l7o (vfu) formalin was added to a

suspension of V cholqæ (at approximately 1010 Per ml ¡o PBS pH7.2). After incubation at

37 C for 60 minutes the bacteria were washed three times with PB.S and resuspended in saline

to the original volume; aliquon $'ere plated on NA to ensure that no viable oraanisms

remained. The suspension was stoned at 4 C with 0.1% (wfr) NaN}

47

2,7 Isolation of LPS fiom V cholerae

LpS was isolaæd from several strains during the course of these studies fo¡ the

production and absorption of antisera, and sensitization of RBCs for hemaʀlutination assays'

LFS was extracted from cells with hot 90% 1wft) phenolÄn ater using the procedure of Wesþhal

& Jann (1%5). Briefþ, a 100 mlbacterial saline suspension (20 ng/ml dryweigþt) was heated

to 6g q An equal volume or.9TVo phenol'rrater (pren'armed to 68 C) was mi:red into the cell

suspension and stirred continuously at 68 Cfor 30 min. The miÉure!\¡as allouted to cool and

the resultingphases reparated þ centrifugation in gþss buckets (1500 rp', 20 min' Coolspin'

MSE). The aqueous phase was collected and stored at 4 C The phenol phase was re-extracted

urith half the original volume of preurarmed water (68 C) for 30 min. with stining and the

phases aFin separated þ centrifugation. The aqueous phasee we¡e combined and dialysed

æainst 5 liters of water, overnight at 4 C Insoluble material was pelleted by centrifugation

(5009 for 5 min ¿1{ e i¡ an SS34 rotor, Sowall). The LPSwas precipitated from the supernatant

with 5ó votumes of cold ethanol containing 250 mg of sodium acetate powder. The precþitate

was collected þ centrifirgation (am0g for 30 min- at 4 C in a Sorvall GSA rotor) and

resuspended in 25 ml of distilled water containing 5 nl\d MgCIZ A small quantity (ap

proximately 1 ng) of DNAse I and RNAse I was added and incubated for 60 min' at room

temperature. This was folloured by the addition of pronase (1-2 mg) and further incubation

for 30 min The mixture was again centrifuged at low speed, (30009 for 10 min' at 4 C in an

ss34 rotor), before deposition of LPS þ ultracentrifugation (100,000g1or Zhr at 4 C; ó0 T:r

rotor in a Beckrnan I-SA0). Each pelletwas then suspended in 5 ml distilledwater, of which

0.25 mlwas used for dryweigbt and 0.1 ml for estimation of proæin þ l-ou'ry's method (I¡wry

e14t. LgSl)usingBSA as standard. Lps preparations obtained iû this manner usually contained

rnore t\a¡ lVo contaminating protein and were tìerefore su$ected to a second phenol

48

extraction. This then reduced the ler¡el of protein contamination to <lVo. All preparations

u,ere stored at 4 C in the presence ol.o.lTo lwrt) Nal'lr

2.8 PreParation of OMPs

preparation of oMI)s was car¡ied out as previousþ described by Mannin get øl' (1982)'

Bacteria u¡ere cultured in NB attl cfor 18 hr, han'ested by centrifugation in a sorvall GS3

nctor at 8,0009 for 15 min, and reeuspeaded in 50 ml of PBS, pH 7'L Af'¡e¡ disruption in a

French pressure cell (Aninco; siherspring MD), the þatewas clarifiedþ centrifrrgation at

10,0009 for 20 min, and the oMp in the supernatantwas recovered by ultracentrifugation at

100,üx)9 for 90 min at 4 C (45Tr rotor; Beckman)' Each of the resulting pellets wa's

resuspended in 5 ml of distilledwater; after pooling protein contentwas determined using the

nethod of l-o*'ry er al. (1951).

For eolubilization of oMPq 1 ml of oMP (2 mg/ml) and 1 ml of 50 nl\'f lis-Hcl (pH

?.5) with or without detergentwerc incubated at 37 cnot t hr. After incubatio¡' the mixture

w¿rs spun at 100,0009 for 30 min at 4 C The pe[etwas washed twice with rris-HCl and finaþ

resuspended in l ml of lxI-¡¡g(sample) buffer, aliquotted and frozen atâC

2.g Prtduc{ion of antisera to wlrole cells and to OMPs of

V cholerae

Lg.L heParation of muse antisera

Hyperimmune antisera to whole cells were prepared by intraperitoneal immunization

of mice with increasing doees (either rd-ro7 or to7-ro) of üve bacteria; animals were given

fortnigþtly iniections for 10weels. Bloodwas withdrau¡n from the retro-orbital plexus under

ether anesthesla lo_12 da¡n afær the last immunization and allw'ed to cloü 6era wefe

49

sepafateq fittered (pore sizæ,0.72 pm; Millipore Corp, Bedforrd, Mass), and stored at 4 C or

at -?nC in aliquots containing 0,O2Vo (w&) NaNa

For the preparation of antisera to OMPs mice were sequentially immunized in-

traperitoneally on d4¡s 0, !4,21and 28 with 10, 30, 100 and 120 pg of OMP extracted with

buffer alone (control) orwith ZVosf|rkæyl (SarkOmP, seeSection 4'2;2) respectiveþ' Mice

were bled 10 da¡rs afær the last immunization and sera collected as described aborte.

L92 heparation of rabbit antisera

Adult rabbits were obtained from the Central fl¡imal House of this University for

production of antise¡a towhole cells þ a combined subcutaneous/intravenous immunization

pr.tocol Equalvolu mæolH?osodiumalginate andwashedcholeravibrioo (101d in normal

saline)weremixedand0.25 mlof themiÉurewasdepositedsubcutaneousþ; withoutremoving

the needrg butusinga neu, syringe,0.r5 mrof.o.4%ocaclzwas injected intothe Bame site. This

pÍocedure was repeaæd a! three other sites. At the same time 0.5 ml of the washed bacterial

suspension was adminisæred intravenously. At 34 dap intervals thereafter, increasing doses

of live v chotsæ1r07-ro9nt in 0.5 ml saline)were given intravenousþ. The rabbitswere bled

by cardiac puncture under anesthesia 10 dãlts after the last immunization' The senrm was

collectd poole4 filtered and stored as described above.

The method used to prepare antisera to OMPs was essentialþ that of Boulard &

Lecroisey (19g2). 5698 oMp was elect¡ophoresed in a[ 16 tracts of an 11-207o SDS-PAGE

After destaining tle gelwae inmedi¿æþ cut into individual t¡acks containingall the proteins

and kept at 20 c The rabbit was immunized witlout aduvant þ sc injections of the gel

homogenates of one track on day 0, three tracks on day 10 and 6 t¡acks on day 17. The rabbit

was bled 10 days after the last iniection as described above.

50

2.lO AbsorPtion of antisena

Antisera were varior¡sþ aboorbed with LPS or oMP isol¡ated fuomV clplqæ' or with

intact bacteriq as described below'

Z1O1 Absorption ç'ith LPS or OMP

Antiserawere absorircd three or four times with LPS (0.1 mg/ml) or OMP (1'0 ng/nl)

as required. Alternate absorptions were incubated at3' Cfor 2 hr and at 4 C orernigþü the

latær ûo allow the remorral of less avid antibodies. After the first of each absorption series the

suspension \[ras slnrn at low speed to remorre the visible preciflAæ; with subeequent absorp

tions, LPS or OMP wefe remorred þ ultracentrifugation at 100,0009 (Bechan IJI80)'

2J:o;2 AbsorPtionwith bacteria

Unless specified otherwise, antisera were abno¡bed three or four times with live or

boiled vchobæ 1s x tolorhl) with alternating incubation conditions as descriH above-

After each abeorption, bacteriawere removed by low speed centrifugation (4,000Ð, "od

at the

end of the absorption series the serum was sterilized by Paseage througþ a0'72 pm fi'lter'

2.L! rn vitro assays ofv cholere adherencq attachment to

isolated murine inæscnat epithelial cell s (enteroc5Úes).

LLLI PreParation of enterocYtes

The method used for enterocyte preparation is a modification of that descriH bry

curman er øL (rylg). The mice wefe killed þ cervical dislocation, and the entire small

intestines excised and flushed$,ith 50-100 ml of ice<old PBS (PH 7'2)' A1ær trimmingaway

51

adhering mesentery and peyer,s patches, each intestine was slit longit'dinally and rinsed in

pBS before being cut into !-2 cmrdpu; these \\rere incubated in ice+old PBS (containing

smMEDTA)withmildagiøtionfor15min.ThestripswerethenwashedincoldPRSand

incubated in preurarmed PBS containing 1 mM GrC)IL 1 mM MgCIZ 1 mg BSA anrd? mg/nl

hyaluronidase for 50 min at37 Cwith intermittent shaking

The resulting enz¡¡matic digest was filæred througþ a nylon sieve to separate the

epithelial cells from tissue segments and large cell aggregtes' The filæred cells were sedi-

mented by centrifugation at 100gfor 3 min at 4 G washed twice in pBs and resuspended in 2

mro',*vo(vfu) percoll (pharmacia uppsala sr¡¡eden) conraining3 nlr{ dithiothreitol (signa)'

The suspension was then layered over a discontinuous percoll gradient (1 ml layers oî'70To,

6oTo ardsovo a,,da}-mfiayer or.4ovo1vfuD in screur+apped siliconized glass tubes. The cells

were centrifuged at 1009 for 10 min at 4 c in a bench centrifrrge (MSE) with a suringout rotor

for separation of enterocytes from þmphocyte* The purified enterocytes were washed twice

\rrith PBS and resuspended in 1 ml of RPMI 1ó40 medium (Flou',)' The total cell countwas

determined in a hemocytometer, and the concentration was adiusted to l-Zx 105 cells/ml by

addition of RpMI 1640 medium. Viability was assessed þ exclusion o10.2vo (w&) trypan blue

andwas generallY ffiTo.

LLLZ InYit¡o enÛeroc5úe adherence ass¡ay

For each a.ssay 0.1 ml of bacterial suspension (1-3 x tOT lmt in PBS) was added to 1 ml

of freshþ prepared enterocytes (r-2x105/hl in RpMI 1640 medium) in disposable plastic

centrifuge h¡be3 (sterilin, Middlese¿Engfand). heliminary e:rperimenß had indicated that a

ratio of 10 bacteria per enterocyte was optimal for assessment of bacterial adherence. Afær

incubation at37 Cfor 15 min, ttre enterocytes $'ere sedimenæd þ centrifugation at 1009 for

3 min, and nonadherent bacteria removed by three 10ml washing with PBS' The pelletwas

52

rcßuspended in 1 nt of incubation medium and homogenized at full speed for 15 sec (Ultra

Thrraa Janke & Kunkel, Staufen, Federal Republic of Germany). Suitable dilutions of the

homogenate were made in normal saline and plated on NA plates containing Sm. Percentage

adherence was calcul¡ated by relating the total number of bacæria adhering to the enterocytes

to the number added iûitirallY.

LLLS Inhibition of adherere.

An assay was developed to assess the capacities of various antisera to inhibit the

attachment of vibrioo to enterncytes. Bacteri¡a were pretreated with the test antisenrm at

various dilutions or with normal mouse serum at a final dilution of 1:10 for 10 min at 37 c

They u,ere then added to the enterrcc¡rte suspension for determination of residual adherence

capacity as described aborre'

2.Í2 In vivo assays rvith infant mict

The infant mouse cholera model was first described þ Ujüye d 4L (196) and sub-

requently eÉended þ Chaicumpa & Rowley (1972)who demonstrated thatspecific antisera

could protectmice from operimental cholera This model has been used i¡ these studies to

a'sess the vinrlence of different strains ol. V ehabæ and to determine the efñciencies with

which various antisera a¡e able to provide protection against e:perimental cholera (Attridge

& Rou,ley 1983a 1983c).

The infant mice used in tìese assays (Section 25.2)were removed from their parenß

about 5ó hours before use, to permit emptying of stomach contents. Unless stated otherwise,

the challenge strain u'as grorl,n in NB at37 C to a concentration of apprrcximatelt 109/mt

Depending upon the challenge dose, the organisnÁ¡ were either simply diluted or alternativeþ

53

spun out of growth medium and resuspended in PePtone saline (PS, a O'lVo YN solution of

proteose-peptone [Difco] in saline). After appropriate dilution' 0.1 nl aliçots wefe ad-

ministered orally to infant mice using a smooth-tipped hypodermic needle (nù' Afær

challenge the mice $,ere not rehrrned to their mothers but were kept in tissue-lined plastic

containers in a laboratory incubator at 25 G

LIIL,L Virulence asñ¡alt

Serial five-fold dilutions were prepared from a PS suspension of the Ûest strain of

vchobæ, ild each was used to feed one gouP of 8-12 mice. Forty-eigþt hours after

challenge, the survival of mice within each group was noted, and these data used to construct

a plot of cumul¡ative percentage mortality versus logro challenge dose (using the method of

Reed & Muench 1g3s). Ð inærpolation itwas then possible to determine tle (48 hour) LDso

dose for a given st¡ain - that is, the number of orgaisms capable of killingsovo olt\e mice to

which it is administered.

LL2,2 hotect¡on atxnlls

Five-fold dilutions of the test serum were used to pre-treat separate aliquots of a PS

suspension of the challenge o¡ganisms After incubation at 37 c for 15 minutesr these mixtures

were fed to different groups of infant mice (g-12 per group). The bacterial concentration in

the originar suspension had been arranged such that each mousewould receive 20 LDso doeee

in the 0.1 ml challenge inoculum. Control mice received organisms which had been incubated

in pS alone, or in the Presence of normal mous€ serufll The sunrival of all gouPß was then

followed until all of the control mice had died; generalþ this occurred,42-52 hours after

challenge (whereas mice given PS alone consistently survived fotT?hours)'

tå

54

ml.

Following the plotting of cumulative percentage mortality against logtO senrm dilution

(ReÆd & Muench 1938), the protective endpoint of the serum was obtained by interpolation

as that (theoretical) dilution which could protect 50% of the mice from death- The reciprocal

of this dilution was referred to as the protective titer of tùe serum, expressed in PDso units per

2.LS Isotation of V cholerae Pili

Glass tra)¡s (30.5x30.5 cm) containing5gg ml of CFAwere seededwith 109 (nonmotile)

v crwlqæ 5698 and incubated for 36 hours at ?5 C. The bacteria were han'ested and

resuspended rn lz|mM Tris-HCl, PH 7.0 containing 25mM NaCL 4mM MgCIZ and 4 mIVî

caclz (laylor et at.19SÐ or in 10 nM tis buffer, pH 7.5, (Korhonen et al' 1980) or in PBS pH

7.2 (/J'-lß rssi & Mostratoo 1985), to a final cell concentration of 0'14 g/nt Cells wefe sheared

by a 5 min fi.s¿tment in a Sorvall Omnimixer (at half speed) on ice' The suspension was clarified

bycentriñrgation at2í00gandthe pelletresuspendedto its originalvolumeandfurtlersheared

þ two passages throu fi a?jl-gtnge needle. After another clarising spin' the two supernatants

were pooled, treated with 507o saturated ammonium sulphate and left ovç¡night at 4 C The

resulting precipitaæ was centrifuged (10,0009 for 30 min at 4 C) and the free pili recovered

from the supernatartþ ultracentrifugation (100,0009 for t hr at 4 C) and suspended in 2 ml

of pBS This suspension was ap,plied to a self-generating isopycnic cesium chloride gradient

After ultracentrifugation ( 150,0009 for 1g hour¡ at 4 C) three distinctba¡ds were obseNed and

thesewere co[ecæd þ puncturing the side of the tube with a rg-gatgeneedle and aspirating

with a syrinæ. Afær removal of cesium chroride by dialysis against pBS, the three fractions

\f,'ere analysed by SDS-PAGE and þ transmission electron microscopy'

.t

I.ll

È"

I

I

\i

'l

'l

If,

i

i

ì

55

I

ì|*

2.L4 In vitro titration of antibodies to V dtolerae

LI.+l Hemggþtination

For effrcient sensitization of sheep RBCs (SRBG) with LPS, it was necessary to

pre-treat the LpSwith alkali. LPSwas diluæd to a final concentration of 2mf,mland tre¿ted

with 0.02N NaOH (usi¡g lN NaOH). After overnigbt stirring at room temperaûue the pH

was restored to neutrality (þ addition of concentrated HCI) and the material stored at 4 C

with 0.17o 1wft) Nal.ll

SRBC. were washed tlree times and resuspended to a concent¡aion oî.257o (v&) in

saline. Alkali-treated I-pS was added to a final concentration of 50 pdml and tle mixture

incubatedwith continuous rollin gat37 Cfor90 minute¡. The SRBCswere againwashedthree

tines, and finalþ resuspended to lVo (vN\ in saline. Sensitized SRBG were ahn'ays freshly

prepared on the daY of use-

Serial two-fold dilutions of the test sera or normal serum were made in saline in plastic

microtiter trays (Disposable hoductg Australia). A standard serum of knou'n hemagglutinat-

ing capacity was included to check the efüciency of erythrocyte sensitization. Following the

addition of an equal volume (2ti) of. a lVo (vr!) suspension of sensitized SRBG to each well'

the trays were shaken briefþ and incubated at37 Cfor 60 minutes. The hemagglutination tiær

wÍrs exprex¡sed as the reciprocal of the higþest serum dilution effecting an RBC settling pattern

clearþ different from that seen in control wells (lacking serum).

2.L+2 Í'I rSA

ELISA was performed in round-bottomed vinyl microtiter plates (Coßtar, Cambridge'

Mass) using buffers and solutions described in Section L[.L. Tays were sensitized with oMPs

of Vchaleræ (see section 28) at a @nceûtration of 10¡g¡ml in TSA buffer (coatingbuffer);

,l¡

;I

t:I

I

I

56

l00plof antigenwas addedto eachwelland incubatedat3T Cfor 2hours follow'edby afurther

incubation overnigþt at 4 C Afær washing th¡ee times with washing bufreç the wells were

blocked with lÐpl of BSASveen for 6-8 hours at 4 Ç and then washed agnin as before. To

assay antibodies in senrm, dupticate 4-fold serial dilutions were prepared using BSAfin'een

and incubarcd overnigþt at 4 C The trayr were then washed four times and bound antibody

was detected þ application of a goat anti-rabbit gfobulin alkaline-phoophatase conjugate

(kindty supplied by Dr. Peter Ey of this Deparmen! optimal dilution of 1:15ffi) in enzyme

diluent (100 pl per well). After overnigþt incubation at 4 Ç the trays were again washed four

times and 100pIof 1 mg/ml subotrate solution (pnitrophenyl phoophate disodium [sigma] itr

10Zo diethanolamine buffer) was added to each well After incubation for 3 hours at 37 C, the

resulting color derrelopment was read at 405nm using a Trtertek Multiscan automated

epectrophotometeç with reference to cont¡ol wells which had received diluent instead of

antibody dilution The antibody tiær of any g¡"en sample was defined as the recþocal of the

dilution which produced a meatr oD of 0.150. Normal and positive serawere ahvays included

as controls.

2.15 SDS-PAGE and immumbloüing'

z,Ls'I SDS.PAGB

sDs-pAGE of oMp, whole cell þates and other protein fractions wefe performed on

Í-n%polyacrylamide gradients or straigþt l2Vo potyacrylamide çls usinga modificati'on of

the procedure of Lügæ nbetgel al.(195) as described þ Achhan d al. (1978)' Samples rvere

resuspended in lx Lug (sample) buffer and heated at 100 C for J min Priot to loading Gels

were generally electrophoreæd at 100 V for 4 houre Ql-n% gradient gels) or 25 mA constant

current (l2To pAGE gels). hoæin stainingwas achieved þ incubation wernigþt with gsntle

agitation with Coomassie Brillia¡tBlue G250 hSoVo (v¡V) methenol and lÙVo (vN) acetic acid'

57

Destainingwas achier,red usingser¡eral changes oî.7.SVo (vrt) acetic acid, lÙVo (vlv) methanol

a¡d tOVo (vfu) ethanol over 24 hours'

Molecular weigþt markers (Signa) were a-lactalbumin (14,200), ß-lactoglobulin

(bovine milk; 18,400), trypsin inhibitor, soybean (20,100), tfyP6inoggn (24,000)' carbonic

anhydrase (29,000), gþceraldehyde-3-phosphatedehydrogenase (36,000), ovalbumin (45'000)'

bovineserumalbumin(óó,000),phosphorylaseB(97,400)'ß-galactosidase(116'000)and

myosin (Ð5,000).

L\5.2 lrnrnrnoblotting (Westem Hotting)

After SDS-PAGE the proteins Ìtere electrophoretically transferred to nitrocellulme

(schleicher & Schüll, Dassel) for 2 hours at?Ð}mA in a transblot cell (Bio-rad). The transfer

buffer used was 25mM Iiis-Hcl, pH 8.3, containing 192 nI\d gþcine altdS%o (vfu) methanol

After transfer, the nitrocellulose sheet was incubated for 30 minutes h SVo (wfu) skim milk

powder in Tpeen-Täs-buffered saline (TTBS; O.OSVo [vfo] ßveel-2f.,?Ã mM Täs-HCL0'97o

[wþ] NaCl) to block non-specific protein binding sites The nitrocelluloee was tlen reacted

with antiserum by the method of Towbin et aL (1979). This was then follou'ed þ overnigþt

incubation of the nitrocellulose at ¡oom temperature in a 1:21000 dilution of a horseradish

peroxidase+oniugated goat anti-mouse or anti-rabbit IgG' Binding of p'rimary antibody was

detected by 5-10 minutes incubation at room temperature with the substrate (10 mg 4-chloro-

l-napthol [Sigme] dissoþed in 3.3 mls 99.57o methanol and mixedwith 16'5 mls of Täs-buffered

saline (IBS; 20 mM Tris-HCl, O.9Vo fw9l Nacl) containing 30 pl H2o2) as described by

Hawkes et al. (1982).

58

2.L6 ColonY blotting

Anitrocellulose disc (9 cm diameter Schleicher& Schäll, Dassel)waspliacedonto agar

plates containing the colonies to be screened. once the colonies had adhered to the disc (1

min), itwas removed and placed, colony side up, on a piece of \l¡hahan 3 MM papef ; bacteria

u,ere lysed ín siruwith 0.5 M HCI and left in the dark for 30 min, according toHautkeÆ et al'

(19g2). The cell debris was removed from the nitrocelluloee with a iet of normal saline' The

method used for antigen detection was the Bame as that used follor¡'ing\Vestern transfer'

2.[l Construction of V cholerae gene bank

LIIZ.L Pneparatbn ofV clolerae çnonic DNA

Thiswas essentially as describedby Mannngel at. $9rß} cellswere groum overnigùt

q¡ith aeration at 37 c in 100 ml of brain heart infusion broth (BHI, Difco). Bacteria were

centrifuged at room temPefature and resuspended i¡ 2 mls cold2SVo 1wft) sucfose in 50 mM

T'is-Hcl (pH S.0). To this was added 1 nl þozyme (5 mg in 0.25 M EDTA pH 8'0) and the

miÍureincubatedonicefor 10min. Aftertheadditionof pronase (10ngin lmlof TEbuffen

10 ml\d rtis_HCl, 1 mM ED-,[A, pH g.0), the mixt.re was incubated for 1$ minutes at37 C

followed by the addition of 0.ã ml of lysis solution (svo sarkooyr in 50 nN'f rristrIcl, 0.25 M

EDTA, pH S.0). Thiswas i¡cubaæd at 56 C for 60 min with occasional gsntle su'irling' The

þate was then gently eiracted three times \ñ'ith TE buffer saturated with phenol (prepared

þ adding 1g phenol per ml of TE buffer). Residual phenol $'as removed by exÍracting twice

with diethyl ether, and the DNA solution was then dialysed orrernight against 5 liærs of rE

buffer at4C. The DNAyield was routinely S00 to 1,3t0 p/ml per 100 ml cultures'

59

LL7,2 DNAquantitation

The DNA concentration was determined þ measurement of absorption at 260 nm'

assumingan Azroof 1.0 conesponds to S0pgDNA/ml (Miller L972).

Lyl.3 End fillingwith l(lenow fragment

protruding ends creaþd þ the cleavage with EcoRl arrd&antHl were filled using the

Klenou, fragment of E. coli DNA poþmerase I. Typically 1 pg of digæted DNA 2pl l(h nick

translation buffer (Maniatis et al. t9g2), 1¡l of each dNTp at 2 mM concentration, and 1 unit

Klenou, fragmentwere mixed and incubated at37 Cfor 30 minutes. The reaction was stopped

þ heating at 60 C for 10 minuteg followed þ removal of unincorporated dNTP and enzyme

þcentrifugation througþ a l mlsepharose cLóB column-

Ln,4 C-onstmction of PPMÍ1101

plasmid ppM2l01 was constructed as follou's (Figure 5.2). Approximateþ 1pl of DNA

of rhe plasmid pHCyg (obt-ined fromDr. J. Qsllins; Hohn & C.ollins 19s0)was digested$'ith

the rest¡iction endonuclease fuoRj, The protruding ends produced by cleavage with EcoRl

were filled using Kleno*, frament ol E colí DNA poþmerase I. The plasmid P$I''P201-1

(obøined from Dr. d Pühler; Simon et 4L lg83) was digested witi the rest¡iction en-

donucleas e1mt1ltoyield a 1.6kb fragmentcontainingtle mobilization region of the plasmid

Rp4 from psup201-1. This digest was similarþ treated with the Klenou' fragment ol E' coli

DNA polymerase I. leated pnCTgwas ligated evsrnight to treated PsuP201-1 usinga 1:10

ratio and transformed into E. coli s1?-1 Qtro, hsdR, recA; and integrated plasmid RP4-2-

Tc::Mu-Km::Tn7) selecting for ApR and TcR. The cloningof the 1.64kb mobilization region

wa¡ selected for by looking for mobilization into E. cotiKl2 Sm10 (thí thr lat, SupE; KmR;

60

and integrated ptasmid RP4-2Tc::Mu), selecting for Ap& TcR and KnR Any positive clones

ufere further screened by making DNA preparations using the three-sæp alkali þis method

deecribed by Garger eJ at. (rgg3) and digested with the restriction endonucleases PsfI and

Bøntillto shou¡ 1.61ù increase in this fragment of positive clones'

L17.5 Construction of cosmkl gene bank

Genomic fragments of approximateþ 40 kb were generaæd by controlled partial

digestion ofv clprqæzrTsírgenomic DNAs'ith the restriction endonuclease sø¿3d The

coomid vector ppM2lol was clear/ed with BamHI and treated with molecul¡ar biologl grade

alkaline phoophatase (Boehringer Mannheim Biochemicaþ sydney) to prevent self-lþtion'

The two DNAs were mi-ed, lþted overnig[t and packaged ín vito ntobacteriophage lamMa

using a Promega Packagene kiL

The packaæd phaæ were then used to infect recombination deñcient SecA) E' coli

K12 517-1. Cells harboring cosmid clones were detected by plating onto nutrient media

containing ampicillin. Greater tha¡ govo of the colonies $'ere Tcs and could be assumed to

contain V dtobae DNA"

?.!7.6 Mobilization of osnid bank

The cosmid bank of Z l7 s6l n E cotíKl2 s 17 - 1 was mobilized into th e v chob æ strain

O17 (El Tor Ogana, S-R). This was done by replica-plating the cosmid bank onto a lawn of

o17 celrs er\ r¿rof log phase culture) on nutrient medium and incubating at 37 clor 4 hours.

These coniugation plates were tìen repricated onto nutrient medium cont^iningAp (Sopg/ml)

and Sm (200 pglnl). After overnigþt incubation the colonies were re-patched onto CEA agar

containin g Ap and scree¡ed-

61

L17.7 Ïhansf,omtion

This is basicalþ a modification of Brou'n et at. (1979b\. E colí K12 st¡ains were made

competent for transformation witl plasmid DNA as follows: an overnigþt shaking culture (in

NB) was diluted 1:20 into BHI a¡d incubated with shaking until the culture reached an ODeso

of 0.6. The cells wefe chilled on ice for 20 ltrh, pelleted at 4 c in a bench centrifuge,

resuspended in half volume of cold 100 mM Mgcl¿ centrifuged again, and resuspended in a

tenth volume of cold 100 mM C-¿clz. Thiswas allon'edto stand for 60 min. on ice, afterwhich

üe cells wefe deemed to be competent, and 0.2 mlwere mixedwith DNA (volume made to

0.1 mlwith 10 mlvf 1iis-Hcl, 1 mI{ EDTA, pH S.0) and left on ice for a frrrther 30 min' The

cell/DNA mixture was heated at 42C for 2 min, then 3 ml BHI was added and the mixture

incubatedwith shakingfor l-2hr atTl} The culürrewas plated onto selection plates directþ

or concentrated by centrifugation before plating Cells with sterile bufferwere run as a control

2.r8 ElVf

Z1&1 lhansrrÉssion EIVÍ

For negative staining strains of.V cholerøe u,ere suspended in PBS to 109 cells/ml One

drop of this suspension was applied to a Formrrar+oated coPPef g.ld (300 mesh, Graticules

IJd., Tonbridgg, Eog) for one minute, washed once with distiled $rater, then stained with tVo

uranyl acetate for 1 minute. Excess fluid was removed þ filter paPer' Grids were then

examined in a Jeol JEM 1005 electron micrrcscope operated at an acceleratingvoltåge of 80

KV

62

LI&2 Imnnnoplecüun nicrcscopy (IHV[)

Colloidal gold particles (ca. ß-?Ð nm) were prepared using the citrate method as

describedþ de Mey & Moermans (19s6) and coniugatedwith Protein A (Pharmacia).

V cholcræ cells grourn on NA or CEA plates $'ere harvested and washed once in PBS

(pH 7.2) conraining lvo BSA(PBS-BSA) A drop (35 pl) of cell suspension was placed on a

sheet of parafilm. A poly-L-lyshe tfeated formrar electron microecope grid was then placed

plastic side down on the surface of rhis drop for 2 min (Mazia et aL 1978). Th" grid was then

successiveþ transferred to drops of the following reagpnts placed on the same parafilm sheet:

pBS-BSA rwice (1 min each); anribody (1:10 dilution in PBS-BSA containing O.OSVo Tveen

20) for 15 nin; pBS-BSA rhree ':m€s (1 min *h); hoæin A-gold (1:50 dilution in PBS-BSA

containing o.117o5veen 20) for 15 min; distilledwater, three timec (1 min each). Excess fluid

was removed from the grid using filær paper and the grid allowed to air dry without negative

staining In some elperimentr, immuno-gold labelled preparations were negativeþ stained

with a solution of27ow¡! uranyl acetate for 1 min atroom temperature. Gridswere examined

in a Jeol JEM 100s transmission electron microocope operaæd with an acceleratingvoltage of

80 kv

63

Non-LPS PnotectiveClassical and El Tbr

3.1 Introduction

gnrdies in various model systems har¡e shou,n that antibodies to the LPS determinants of

Vcholqæcan mediate protection against experimental cholera (tilatanabe & Verwey 1%5;

Neoh & Rowþ lgzl svennerholm 1975; Holmgren et at.1977; Auridge & Rowley 1983c)'

In consequence the poosible prophylactic potential of the non-LPS components has been

largeþ ignored. As discussed in Section 1.10, conventional cholera vaccines generally com-

prise preserued suspensions of vibrios inactivatedþ treamentwith phenol' formalin or heat

despite tìe presence of LPS antþeng such vaccines have conferred onþ short-term protection

in the field (Joo lnÐ. The reatization tìat other bacterial enteropathogens po6sess labile

surface components that can function as protective antigens (Rutter & Jones 1973; Morsatr ef

at.lng)promptedtle evaluation of theprophylacticsignificanceof theno¡-LPSdeterminanß

oî.V cholqæ.

In 1970, Feeleyhadshown thatlivevibrioswere more effective than heatedones in the

active mouse protection test, raising the possibility of heat-labile protective determinants

(F"eley lnÐ. Subsequentþ, Neoh & Rowley ([gZz)showed tlata mouse antiserum prepared

asainst vcholqøesógB, and then repeatedly absorbed$rith higþty purified homologous LPS,

u

retained significant protective activi$. Bellamy et al. (1975) went further to show that an-

tibodies present in an IgG fraction of a similar rabbit antisenrm u'ere protective and direcæd

against heat_labile non-Lps determinants. Finally, more recent studies compared the protec-

tive activities of antibodies to the Lps or non-Lps antigens ofv cholqæ 5698 and concluded

that the liatter more efücientþ protected infant mice from challengewith this strain (Atfridge

&Rourþ1983c).Thesameworke¡salsoshowedúattheprotectiveactivitiesofvarious

antibody preparations correrated with their capacities to inhibit vibrio attachment in vitro,

suggeeting that at least some of the non-LPS protective antþens may Play a role in adherence'

This finding provides a¡r encouraging analogr with studies of enterotoxþe¡ic E- coli nwhich

isolated colonization factors harre been successfulþ used as prophylactic immunogens (Rutter

& Jones ll73; Morsan et al' tY78)'

The studies outlined above clearþ demonstrated the exisænce of non-LPS protective

antþens nV cholqæ, at least in the Classical Inaba strain, 5698' Further investigation of the

vaccine potentirat of such components seemed warranted. f[s immunoprophylactic sig-

nificance of the non-Lps antþens or.v chotqæobviousþ depends on the frequency of their

distribution and the extent to which they are shared or strain specific In a previous study,

Attridge & Ron ley (19g3c) were unable to detect such determinarts in the o17 and r50 strains,

both of which are of El ror biotype and ogærra sefotype. Itwas clearþ essential to begin by

examining other st¡ains for tle preËence of non-Lps protective antigens, as the existence of

shared compotrents of this type migþt offer a ne$' apProach to vaccine developmenL

3.2 Characterization ofv cholerae strains

For this study, a total oLlT strains was used (Table 3.1). v dtolqøe strains isolated in

l9g5 a¡e referred to as recent field isoliates, in contrast to the old laboratory strains which were

fi¡st isolate d m-nyears ago. The significance of t.his distinction will become apparent later'

65

Ibble 3.1 Cha¡acterbtics oî'V dtobæ etrains

Strain

5698

c4401

ztT56t

cA411

A414041

NIH4l

8æ3

v86

Aú{1399ß

BM69

358

ol7T50

A¡\14üt3

H-1

il56eB/165

Biot¡'Pe Serotype

Ctaßsical Inaba 5'5 t 0'66x ld

Classical Inaba ?-6 x'0'26 x 103

Clasßhal Inaba 9'3 t 0'a6x 1ú

Classical Ogær'a 1.1 t 0'01x ldClassical Ogawa 9'4 ¡0'08x 103

Classicat Ogawa 1'2 t 0'01x 103

ElTor Inaba 5'7 t 0'68r1d

El Tor Inabe 8'7 t 3'a0x 10Ê

ElTor Ineba 24 x'2'40xl}a

El Tor Inaba 1'0 t 0'23x 104

El Tor Inaba 5'5 r 0'65x 104

ElTbr Ogova 9'4 ¡0'18x10s

El Tor Ogma &0 t 280x ldElTor Ogrwa 4'1 t 12¿4xlú

El Tor Ogma 1'8 x'?'40x tdEl lbr Ogæva 1.8 t 0'31x ldClæsical Inaba x non01 V¡brb > 109

Vrnrlencel So*ce (year of isotation)2

a(1ea6)

b(1es3)

(leBs)

b(1es3)

(1e8s)

b(1e41)

b(pn -19Ð3

a(1e61)

c(1985)

d(1e8s)

d(re85)

a(p,re-1965)

a(pre-1962)

c(1e8Ð

d(le8s)

d(1e8Ð

a$e2)

1 Eoprored as LDSOvalues, calculated as descrig il Section 2'l2.l. Data a¡e mean tSE

values for two to four determinations

2

^ = Dr. K Bhaekaran (Central Dnrglnstiürte, Lucknou', hdi.)t b = fh. J. Berry (Univer-

sity of Teúa¡, Austin); c = Dr. B. I(ay (International center for Diarrheal Diseæe Re-

seafch, Dhak¡, Bangþdesh); d = Dr. S.C Pal (National Institute of chole¡a and Enæric

Diseases, Calcutta, hdia).

h.. Gu*tzel&BerY (195).

6

The 5698/165 hybrid lnbriowaf¡ preparedby coniugation between v cholerøe strain 5698 and

the non-cholera vibrio strain 165 (Bhaskaran 1971), and carries the LpS determinants of the

165 strain on the non-Lps antigenic bacþround of the 569B strain (Bhaskaran l97l; Attridge

& Rowþ 1983c).

Biotype was confirmed by assessing sensitivity to the antibiotic poþmixin B (50

units/ml) and þ typing with biotype-specific Phages. serotype was confirmed by slide ag-

gtutination using specific typing sera virulence war¡ determined by the infant mouse cholera

model (see Section zlzl). The data in Table 3.1 show that 16 of the 17 strains were pathogenic

in this model, althougb strains o17 and g233 had to be passaged in infantmice to increase tleir

virulence to the levels shourn. The 5698/165 hybridwas non-Pathogenic-

33 The existence of non-Lps pnot€c{¡ve antigens in strains

C4401 and C4411

33.1 hoduction of antisera to 569ry165' CA40l and ca4ll strains

To study the distribution of non-LPS protective antþns! it was decided to prepare

antisera to three strains of V cholqøez 56981165, C4401 and C4411' The first antiserum was

p,repared by immunizing mice with live vibrios of the hybrid strain 569F,1165 as described in

section zg.l.In a pro,,ious study, Arridge & Rowþ (19S3c) ha'e confirmed the antigenic

relationship between strain 5698/165 and its vcholerae parert, in particular the absence of

5698 LpS antigens from the hybrid strain. An anti-56gB/1ó5 serum was found to protect infant

mice from challenge with 5698, demonstrating the existence of non-LPS protective antigens

in this strain (Attridge & Rowþ 19S3c). Such a sefum' therefore, prwided a convenient

means of surveying other strains for the pfesence of such determinants'

67

Antjserawere also prepared against the C4401 (Classical Inaba) and C4411 (Classical

Ogawa) strainq but these sera had to be absorbed with homologous LFS (Section 210'1) so

that polentiatly protective anti-I-PS antibodis u'ere removed' fIA assays performed on the

LpS_absorbed antisera showed a titer of < 1:| indicatingthe efficacy of this procedure.

3.32 hotective activities of antlsera

The infant mouse model was used for assessment of the protective capacities of the

antisera. Initial assays shoured that tle anti-569H165 serum was indeed able to Protect against

challenge with the parentar 5698 strain. Furthermore, the Lps-absorbed anti-c4401 and

anti-CA41l serawere also protective againsthomologous challenge, suggestingthe existence

of non-LPS pfotective antþens in these two strains. To eliminaæ the possibility that in the

laüer instances protection was due to sub-aggtutinating concentrations of anti-LPS antibodies

in the absorbed sera, aliquots of each serum were further abeorbed with live or boiled

homologous vibrios. Aboorption with live orÊanisms reduced the protective tiærs of the

L'S_absorbed sera þ 97vo or ggvo,whereas absorption with boiledvibrios reduced the tiær

by only l4vo ot3.5Vo (Table 3.2). Itwa.s concluded that str¿ins c4401 and c4411 do pæsess

non-LPS Protective antigens'

To a^sse8s thevaccine significance of tleee comPonents, itwas importantto lookat their

distribution âmong other V cholqae strains, and to determine the eÍent to which üey were

sharedorstfain_specific. Atthis stage itseemedpossible thatposseesion of such antigens might

be restricæd to strains of the Classical biotype'

ó8

rhbre 3J hotective activities of Lps-absorbed antisera to cA40l and c4411: effect of

further absorptions with live homologous vibriosa

Protective Activity

Serum

I-PS-abso¡bed anti-C440 1

LPS-abso¡bed anti-C441 1

Before firther

abeorption

1980

2000

Afrer aboorPtionwith

Boiþdvibrbs Livevibrios

l7l0 (t4%o)b 55 (ÍtTo)

a0 QEVo)t*t0 Q.SVo\

" Anti¡erum to cA40l which had a protective tiær of 1:7480 (eee ßble 3'3) was diluted 3'7

times to grve an equivalent titer of 1i2000. Each antisenrmwas tested for its capacity to

mediaæ protection 4ginst challengewith the homlogor¡¡ stfain'

htgtto in parentheses referto Pefcentage reduction of protective tiærs

69

3.4 Distribution of non-LPS pmtectirrc antigens among

V cholerae strains

3.4.1 Old laboratorY shains

In the first series of experiments, the antiserum to strain 5698/165 and the LPS-ab-

sorbed antisera to strains CA401 and CA4ll were tested for their capacities to protect infant

mice from challenge with eight old laboratory strains of V dtolerøe. T}le same spectrum of

activig was seen witl each antiserum; protection was mediated against isolates of the Classical

biotype but not against those of the El Tor biotype (Table 3.3). The protection observedwas

not serotype specific These data were encouraging for they raised tle possibility that all

st¡ains of classical biotype might possess common non-Lps protective antigens. It seemed

importan! howerrer, to check this ñnding using v cholqøe strains recently isolated from the

ñeld.

!4,2 Rsoent ñeld isolates

A second series of protection elperiments was performed using eigþt isolates obtained

from Indira and BangÞdesh. In cont¡ast to the previous finding both of the sera tested

protected not onþ against the two Classical strains but also against the six of El Tor biotype

(Table 3.3). AgÊin, hourever, protection extended to isol,ates of heterologpus serotype, al-

thougþ the tiærs o6øined lvere consistently higher in cases of homologous challenç' These

results suggested a differencewith respect to the expression of non-LPS protective antigens by

old a¡d recent El Tor isolates. To confirm this apparent dichotomy, antisera were prepared

¡gainst two of the latter strains, abso¡bed with homologous LPS as before, and then surveyed

for protective capacities against a fange of challenge strains. The absorbed sera retained

significant protective activities aginst recently isolated strains of the four common biotype-

70

rhble 33 Distribution of shared non-LPS protective antigens a¡nong sixteen str¿i¡s of

Vclpbæ

Antiserum to

Strain 5698/165 cA401a c4411 4413993 H-1

Old l¡olates

5698

cA401

cA411

NIH4l823.3

\¡86

ot7T50

687 t 50

433 x,3l333 t 38

393 x,2<10<10<10<10

1780

2500

1310

tz50<10<10<10<10

NDb

ND

NDND<10ND<10

ND

ND

ND

NDND

<10I{D

<10

ND

t3?0 x,147

l2A0 x,121

2000 t 135

1690 t 190

<10<10<10<10

Rcccnt lsolatcg

zl756t4414041

AAl}w}BM69

358

4A.l4t73

H-1

u

ND

NDND

ND

ND

I{DND

I{D

34t0

2570

5590

4450

3990

1150

t290

r830

590 t 95

1630 t 130

573 x,4O

543t&05Ð x, t2

1ã)0 r 8ó

1390 t 8ó

LZTO x, tl?

25lO x,192

2190 t 3ó8

3770 x,ÐßNDNDND

1830 I 336

ND

1160 x,7

Ðß0 x,ZTI

13f) t l(DND

ND

ND

3560 t 88

ND

NOIE Data sho$, prrotective activities (mean tSE for multiple estinates) of a¡tisera, ex-

pr,essed as S0To protective endpoints as described in Section zlLZ Antbera to strains

56gBll65and cA4ll were titraæd three times against each straiD, antisera to strains

AA139g3 and H-l wene titrated twice, and the antiserum to strain CA40l was titrated onos

Teparaæ baæhes of a¡tben¡m to strain CA4O1 wer€ tested against the tu¡o sets of isolates.

The senrm assayed agpinßt the rccent isolatÊs had a prctective titer of t:7480 against

homol,ogous vibrios.

b l.ID = Not determined"

7l

serotype combinations (Table 3.3). As with the three sera tested earlier, no protection u'as

evident when the challenge strain were old isolates (8?313 and O1?) of El Tor biotype @ble

3.3).

3.43 Conclusbn

Collectiveþ, these data indicaæ that all of the Classical strains examined, as well as

recent isolateß of El Tor biotype, share comlþnents which function as protective antigens in

the infant mouse cholera model Analogous components were notdetected on any of four old

laboratory El Tor strains. The possibility that the liatter might possess the common antigens

but that the corresponding antibodies are not sufficient to confer protection against such strains

was eliminated by the finding that absorption of an anti- 56981165 serum with live o17 vibrioo

did not lead to a reduction in protective activity (data not shown, but see Section 5.2).

35 Subsets of non-LPS prctective antigens ane

semt¡rpe-restricted

3.5.1 Demnstration by proÚection assa¡t

The data in Table 3.3 shou, thag on average, tle protective titer of an antiserum was

two-fold higber when the challenge strain wa.s of homologous rather tlan heterologous

serotype. This finding suggested the existence of serotypicalþ restricted non-LFS protective

antigens in addition ûo the shared components discussed above. To examine this Point more

&otly, two serawere extensiveþ abeorbedwith sü'ains of heterologous serotype in an aüempt

to prepare sera which would protect mice against challenge with strains of homologous

serotype onlY.

The sera selected for these absorptions Ìl,ere rabbit antisera prepared against C4411

or 569B1165; the former w¿ts extensively absorbed with homologous LPS as described in

72

Section LlO.l. Both seratrrere protective against challenç with either serotype, althougþ as

noted prerriousþ, each was more active against homologous challengp (Tâble 3.4). Further

absorption of the LpS-absorbedanti-CA4ll serumwithvibrios of heterologous Inabaserotype

(strain 5698) effectiveþ removed protective activity against this serotype, but the residual

serum retained significant protective activity ¡grinst each of five Ogara challenge strains

(table 3.4).

Simila¡ absorptions of the anti-5698/165 serum with vibrios of heterologous Opwa

serotype (strain C4411) failed to remove all protective activity against challenge with the

absorbing strain, and this sih¡ation still pertained e\ren afær further absorptions with the seme

and subsequently a different ognra strain (4414041). However, the residual protective

potential against strains of ogawa serotlipe was considerabþ less than that observed against

homologous Inaba challengestrains (Ihble 3.4). Furthermore, these protectionassays revealed

a biotype-associated difference between the various ogawa challenge strains use4 a point

whichwa^s clarifiedby subequent studies (see section 4.6).

352 lÞmnstrationbY IEXVÍ

The¡e elperiments suggested the exi¡ænce of serotyPe-rest¡icted non-LPS protective

antþens. The extensively-absorbed anti-CA4ll serum was tested for its content of antibodiee

capable of binding to strains of homologous (C4411) or heterologous (5698) serotype þ IEM'

In line with the protection dat4 significant numbers of gold particles were observed in clooe

association with cA4ll vibrioo, whereas little bindingwas er¡identwith 5698 bacteria (Figure

3.1).

73

Table 3.4 The exbtence of serotyp+restrlcted non-LPS potective antþns lnv. choleætea

Challenç Strains

Ogawa serotype lnaba serotYPe

Antberum C4411 NlH41 AAl ¿tO41 H-l 64 5698 CA¿+01 217æ1 AA1399Íl BM69

LP9'ab6orb€d

anti-CA4l1

S6gBabsorbedb

4210

3320

3320

2500

ND

2950

ND

2810

1N<n

1080

<nND

<nND

<nND

<nND

2890

ND ND ND 6110 5000 ND ND NDUnabsorbed

antþ5698/16f, ',ß70 21ocA411/4414041

absorbed 26oe 4n zgoe <n <æ 3Í!rt0 25SO 2550 æ40 2780

a Antserawere frrded only once for protecüve actlvttþs.b Lps-absorbed ant-GA4i1 wasturttrerabsorbedñßümes (thrioe d.gr Q,Zhr,andhillce d4 c, ovemþÌrt) wlüì lh/e 5698'

" AnÞ569Ey16s tested unabsorbecl as lt contains only anüboclþs to non'LPS protective anügens (see sectk)n 3'2)'

d Absortring antt569B/16s soven times with live cA411 did not remove all protectlve ac-tivity agalnst sÙalrs of ogawa serotype; Ë was fur-

ther absorbed three times wlth llw AA14041

" pD¡o of 260 and 2g0 was obserræd when the ctnilenç strains cA411 and AA14o41 were growrì under rouüne ct¡lturalcondilions (NB d

97 c). Howew[ wtren they were grown on cFA ù.zsc Üìe PÈo values uære 970 and 1380, respactively (see sæüo-n 4'6 for detalb)'

74

Figure 3.1 Demonstration of (Ogawa) serotype+pecific non-LPS determinants . An exten-

siveþ absorbed anti-C4411 serum (see text) was used for IEM examinatiot of V dtoløøe

sf'ains C4411 (A) and 5698 (B). Following growth on NA plates (37 C for Vlh),bacteriawere

hanested, incubated with 1:10 dilution of absorbed anti-C4411 serum, follor¡'ed by 1:50

dilution of protein A+olloidal gold particles at room temperahrre. Bars rep,resent 200nm.

75

4.l

t -(t

.,

.t'

ta

a

)

lr "-

*-^

-

tl -,

.ì

3.6 Correlation between protectirrc activities of antisera and

their capacities Ûo inhibit in vitro attæhment

3.ú1 Invitro enterocXÚe adherence a$¡ay

Adhesion of Vcholqa¿ to the mucosa of the small intestine is now recognized as an

important earþ event in colonization. In an attempt to confirm the previousþ reported link

between ttre capacities of a serum to protect infant mice frr¡m choleraic infection and to inhibit

the ín vito atlachment of bacteria of the challenge strai¡ (Attridge & Rowley 1983c)' an

adherence assay war¡ developed using enterocytes isolaæd from the adult mouse snall intestine

(as described in section zll). phase+ontrast microscopy showed that the enterocyte isolation

procedure yielded populations of epithelial cells, which retain their characteristic columnar

morpholog and have clearþ deñned apical bnrsh borders (not shown)' On the average 'ffiVo

viabilitywas indic¿tedþ Trypan Blue exclusion. preliminary experiments demonst¡ated that

a ratio of 10 bacteria per enterrccyte was optimal for assessment of bacterial adherence'

3;6:2 Inhibition of adherence

Three of the antisera described above were tested for their capacities to inhibit the in

vito attachment of. V cholqae to isolaæd murine enterocytes. The antiserum to strain

56g8/165was initi;ally tested against the 56g8 parent st¡ain, and the LPS-absorbed antisera to

st¡ains cA401 and cA41l were tested against homologous vibrios. Each serum produced a

concentrationdependent inhibition of attachment (Figur e3.2), confirming a previous finding

þ Attridge & Rowþ (l9g3c) that antibodies to non-LPS determinants can inhibit theinvitro

binding of sub-aggtutinating concentrations of vibrioe. Each sefum was tìen tested fo¡ its

capacity to inhibit the adherence of the seven other old isolates used in tlis study (final serum

dilution, 1:10). All three antisera consistently inhibited the attachment of each of the four

i

76

;

,'{

¡ìl^'

IÞ

I{flrÌ

!

.'rl

tI

iI

Figure 32 Antibody-mediated inhibition ol V choløæ adherence h vito'' Antisera u'efe

tested for their capacities to inhibit bacterial attachment to isolated murine enterocytes, as

described in Material and Methods (section Lll.3). Residual percentage adherence after

pretreatme¡twitì antiserum is shourn relative to adherence observed after preincubationwith

a 1:10 dilution of normal mouse serum (rt}vo). Graphs show bindingof strain c4411 in the

prcsenc€ of LPS-abeorbed antiserum to strain c4411 ( o ), binding of strain c4401 in the

presence of LPS-absorbed antiserum to strain C4401 (r), and binding of st¡ain 5698 in the

presence of antiserum to strain 56gvl65 (v). Data are mean tSE (bans) attachmentvalues

f¡om three determinations.

I

77

100

60

IJJozl¡JÉ,l¡JrF¡to

UJol-zl¡lC)Eu¡o-

120

80

40

20

30

RECIPROCAL SERUM DILUTION

10010

strains of Classical biotype, whereas the binding of the El Tor strains was not reduced (Figure

3.3; results $,ith vs6 and T50 strains are not shown but were similiar to ttrose obtained with

st¡ains O17 and 8233).

Since therewas a consistent correlation between tle presence of the non-LPS protec-

tive antigens and susceptibility to antibody-mediated inhibition of adherence, itwas predicted

that tle binding of the recent field isolates would also be reduced in tle Presence of such

antise¡a. Figure 3.4 sho\rs that this is indeed the case. These resulß strongþ reinforce the

prwious demonst¡ation (Attridge & Rowley 1983c) of a correlation between tle capacities of

antisera to mediate protection and to inhibitôn viÞo attachment'

3J Discussion

The maior finding described in this chapter is ttrat each of eight recent field isolaæs of

V choleræbearcommon non-LPSprotectiveantigens, despitedifferences inbiotype' selotyPe'

and geographic origin Of the eigþt older isolates o|.V dtobæ, only those of Classical biotype

erpre's these antigens (table 3.3); the basis for this restricted distribution remains uncertain'

It seems unlikeþ that the old El Tor strains hat'e lost such components during prolonged storage

in the laboratory since similiar t¡eatment has not affected the old Classical v cholqae strains'

Perhaps the eryression of non-I-PS protective antigens þ recent ñeld El Tor isolates results

from inæractionbetween tle two biotypes in the environment Epidemiologicalstudies have

re,vealed that the biotype mGt commonþ associated with clinical cholera cases can change

over a period of time in a given location (Samadi ef aI 1983). Although the factorc responsible

for such biotype suritching remain undefind tìere would seem to be ample opporhrnity for

interaction between vibriæ of the two common biotypes in the field" It is ttrerefore conceivable

that vibrios of El Tor biotype harle, comparativeþ recentþ, acquired the genetic potential to

express non-LPS protective antþens. Since atleast some of these comPonents may play a role

78

Flgure 3.3 Capacities of tlree antisera to inhibit the ¡n vito attzchment of V dtolqae strains

of ClassicalorElTorbiotype. Residualadherencecapacities or.suV dtol.rz,aesüains (d5698;

B, C4401; Ç C4411; D, NIH41; F.r8?33; and E O17) are shovn afær preincubation witl

each of three antisera: (top) LPS-absorbed antiserum to strain C4411, (middle) LPS-absorbed

antiserum to strain CA40l, and (bottom) antiserum to st¡ain 5698/165. Percentage adherence

is relative to that dispþed by control orsanisms preincubaæd with a 1:10 dilution of normal

monse serum (l107o). Histograms show mean residual adherence and bars represent the SE

(fr om th¡ee determinatio ns).

79

J lo o z J I o Þ

(o oo) o

C^)

J N o Þ z { I o Þ o

J l\) oo Þ J I (tl

o) (o qt o) (,l

(oo o

o) o

PE

RC

EN

TA

GE

AD

HE

RE

NC

E

o) oo)

(oo

o

5698

cA40

1

cA41

1

NIH

41

8233 o1

7

Flgurc 3.4 Capacigof antis€rumtostrainCA4ll toinhibittheionvit¡p attachmentof recent

V e;løbæ ñeld isohtes of El Tor biot¡pe. Residusl adherence capacities of six V dtalaae

srraiDs (A 5698; B, O17; Ç 4413993; D, BM69; E H-1; and R 64) are shown after

preinorbationwith antieenrm to strain C4411 (absorbedwith homologous LPS). Percentage

adherence is relatir¡e to that shown þ contnol organisms preinorbated stith a 1:10 dilution of

normal mou¡e senrm (ltMo). Histogrems shos'meiatr residual adhe¡ence, andba¡s represent

tùe SE (from two determinationr).

80

PE

RC

EN

TA

GE

AD

HE

RE

NC

EJ ]u o

(o oo) o

(¡) o

o

Þ TD o (] m 'tl

in adherence (see belou,), it is possible that they contribuæ to pathogenic potential In this

context, it is interesting to note that the present pandemic is the first to have been caused by

vibrios of El TorbiotYPe.

In addition to the non-LpS protective antigens that are shared by strains of either

serotype, the data suggest the existence of additional serotype-restricted comPonenß' Thus

the protective titers of tle five antisera used in t.his study were ahrays greater aeainst strains

of homologous serotype (Table 3.3). The data presented in Table 3.4 provide more direct

er¡idence for the existence of serotype-rest¡icted compotrents. Abeorption of LPS-abao¡bed

anti-ogaura serum with vibrios of Inaba serot¡rpe effectively removed protective activity against

this serotype, but the residual serum retained significaat protective activity against ogawa

challenge strains" Similar datawere obtained in a second erperiment in which anti-5698/165

serum was absorbed with strains of ogawa serotype. C-ollectively these e:rperiments confirm

the existence of serotype-rest¡icted non-LPS protective antigens'

As noted previousþ by Attridge & Rou,ley (1983c), a consistent correlation was found

between the capacity of an antiserum to protect infant mice and its capacity to inhibit the i'l

vito attachmentof the challenge st¡ain The useof sub-agglutinatingconcent¡ations of vibrioe

in the attachment studies, and the fact that tnany sü'ain s are virulent when administered in lo¡'

doses (ie. sub-agglutinatingconcentrations),would argue ¡gainstantibody-mediatedbacterial

agglutination as a critical mechanism of protection in this model The blocking of vibrio

adhesins is a more likeþ mechanism (Freter & Jones 1976; Attridge & Rowley 1983c). The

correlation obsewed between these two activities of the non-LPS antibodies þrotective

capacity and inhibition of adherence) is consisænt with a role for at least some of these

componenß in colonization.

The failure to remove tie residual protective antibodies from l-PS-absorbed anti-

CA401 and anti-CA4ll sera by abeorption with boiled vibrios confirms the heat-labile nature

81

of these antigens (Watanabe et at.1969; Attridge lngr.Itseems unlikeþthatrelativeþ labile

non-LpS protective antigenswouldescape denahrration duringthe preparation of convention-

al killed whole cell vaccines. Studies W Cryzand colleagues (1932) have shown that phenol-

or heat-inactivation drasticaþ reduces the immunogenicity of cholera vibrios, althoug[ for-

malin treatment appeaß less deleterious. The recent demonst¡ation of the clinical sþificance

of at least one non-LpS component (see Chapter 5) will necessitate a more careful approach

to the problem of vaccine inactivation. Alternativeþ, should such components acquire the

status of protective antigens, itcouldbe argued thattheywouldbe more effectivelypresented

to the immune system þ an attenuated live oralvaccine (see Section 1.10.21).

82

Role of TCP as a Colonization Fbctor and aNon-LPS Protective Antigen in the InfantMouse CholeraModel

4.I Introduc{ion

,rh" results of Chapter 3 have clearly indicated thatcommon non-LPS protective antigens are

opressed by four old strains of Classical biotype and eigþt recent field isolates of both biotypes,

but that such determinants are not detectable on four old El Tor st¡ains. Althougþ these

antigens remain undefined, their relative lability (Attridge 1979) suggests that they migþt be

protein in nature (ùyz et aI lg82; Attridge & Rowþ 19S3c). Candidate proteinswould be

thoee associated with ftagelliar or pilus structures, or located elsewhere in tle outer membrane

(Section 1.8.2).

Several studies implicate the flagellum as a determinant of virulence ín V choløæ,

thougþ tlere has been debate as towhether this structure functions soleþ þ providing motility

(Guentzel & Berry lg75\, or whether it also plays a role in attachment (Attridge & Rowley

l9g3a). In the present contex! Eubanks et at. (1977) have described a non-LPS protective

antigen associaæd with the vibrio flagellum which was detecæd in both Inaba and Ogawa

st¡ains of the Classical biotype. Yancey et ø1. (1979) also described a simila¡ heat-labile

non-LPS flagellar-associated antigen.

83

V choleræinteracts intimateþwith themucosalsurface of the intestine, theinitiraler¡ent

in the patlogenesis of cholera. For some other bacterial enteropathogens, this colonization

has been shq¡rn to depend on the presence of pili which mediate adherence to receptors on

host cells (Jones 1980; Gaastra & de Graaf 1982). For example, K88 pili have been shown to

be critical for the ln vito attachment and rn vivo cnlo¡hation of ETBC strains of porcine orign

(Smith & Linggeod l97l;Jones & Rutter 1972). Subeequent studies showed tlat the (heat-

labile) K88 protein could be used as a protective immunogen, eliciting the production of

antibodies which confer protection bD'inhibiting colonization (Rutter & Jones 1973; Rutter

et aL 1976\.

As has already been pointed out (Section 1.3.3.4), earþ attempts to detect pili on

V chobæmetwith variable success (Finkelsæin & Mukeriee 1fb3; Barua & Chatteriee 19ó4;

Tneedy et aL 19ß: Adhikari & Chatterjee 1Í}69). Untilvety recent$, there \ilas no er¡idence

to suggest that such structures played any rnle in vibrio adherence (rerrian'ed by Jones 1980).

During the course of these studies, howerrer, reports from Ehara et al. (1986) andTzylor et øI

(19S7)revivedinterestinthepossibleinvoþementofpiliinthepathogenesisofcholera. There

is nou, no doubt that TCP are required for efficient colonization of both the humen (Herrington

et al. 1988) and infant mouse gut (Taylor et a|.198Ð.

The orperimenß to be described in this chapter u/ere aimed at elucidating the nature

of the non-LPS protective antigens of V c]øbae. C.omparisons of OMP preparations (by

SDS-PAGE and immunoblotting) failed to rer¡eal any consistent difference which could be

correliatedwith the presenceof non-LPS protectiveantigens, andsubsequenterperimentswith

detergent-t¡eated OMPE also failed to implicate any particular protein band" I-ater experi-

menß were desþed to er¡aluate the importance of TCP as a protective antiçn in the infant

mouse model

84

4.2 Studies ofV cholerae OMPs

+2.L Anat}'sis by SDS-PAGE and imrnrnoHoüing

heliminary eryeriments were carried outwith theV dnlerae 569B strain. Both OMP

and flagellar preparations of this strain u'ere compared" EM examination of the latter con-

firmed the presence of ftagellar structureg althougþ contamination with membrane vesicles

\\'as apparent (data not shown). SDS-PAGE anal¡nis (as described in Section 215. 1) of the two

preparations resulted in identical banding patterns (data not shou¡n). As there was no obvious

difference between theq OMP rather than flagellar preparations of otler V cholqøe strains

were use4 mainþ because of ease of preparation (section 28).

In o¡der to determine whether any consistent difference could be detected at the protein

level betw æn V choleræ strains possessing or lackin g non -LPS protective antigens, the protein

profrles of OMP preparations of 8 V choba¿ strains were examined by SDS-PAGE A

representative operiment is shown in Fþre 4.1, (left). None of the protein bands could be

correlated with the previousþ determined distribution of non-LPS protective antigens. To

determi¡e whetler such a correl¡ation migbt be revealed þ immunoblotting (Section 2L5.2),

OMp preparations of the 8 strains were run on SDS-PAGE transferred to nitrocellulose and

incubated with the protective serum (antiCA4ll; Section 3.3.2). As shown in Fþre 4.1,

(riÈÐ, ho*,ever, tlis approach also failed to implicate any protein as a candidate protective

antþen.

4;22 IÞtergent extr¡ction of OMP preparations

Becauseof thelarge numberofbands seen inSDS-PAGEandimmunoblottinganatyses,

it was decided to treat OMP preparations with a variety of detergents (see Section Z4-2) tþ

selectiveþ solubilize some of the proteins and thereby facilitate further experimentation. A

85

Figurr 4.1. SDS-PAGE and immunoblo tÃf¡golV choleræoMPs. I-ane d o17; lane B ,883;

lane Ç H-1; lane D, BM69; lane E, 5698; lane [, c4411; liane G,4A14041; andlane H 'Z1756l'

oMP, \r,e¡e prepared from various strains, arrdÐ pgnuantities were analJzed by SDS-PAGE'

protein staining (reft) or immunoblotting (with protective Lps-absorbed antiserum to strain

CA4ll; right) folloured"

86

'lia

-¡r.-ÉIlIYÉ5Õts

t

-ék:F:= rtd æ

LZ;;;;Zâ t-,

r'r!å¡Ë

5lE¡tf tÇ--à-1-"*:--ài¿i¡¡-"o 'È

*. ü .æ'*'

ABCDEFGH ABCDEFGH

comparison of tle pfotein profiles by SDS-PAGE of the insoluble oMPs (pellets afær

ultracentrifugation, section zs) with tlose of the untreated oMk showed that sarkosyl

appeared the most promising of the ten detergents examined, in that it selectively extracted

proÞins from 5698 OMP (Frgot" 4.4 lanes c & D). The number of protein bands was reduced

to six (Mr values raßgng from 2145 kDa) (Fþre 4.2, lanes C & D)' By comparing the

capacities of detergent-treated or cont¡ol (treated with buffer only) OMP preparations to

remove prolective antibodies by absorption, itwas possibte to assess the protective potential

of antibodies directed against proteins presentin the insoluble pellet In one such etperiment,

Sarkosyl-insoluble 569B OMp (SarkoMP) rnmoved 797o protective activity from a rabbit

anti-5698/165 serum, whereas the cont¡ol OMP removed lt}To' This suggested that the

proteins solubilized þ this detergentwere not the major non-LPS protective antþens butwere

retained in the insoluble fraction.

This tentative conclusion was strengthened by a second experiment in which groups of

mice were immunized with Sark-oMP or control OMP prepared from the 5698 strain (see

section 29.1 ). The resulting sera were absorbed with 5ó98 LPS (as described in section

210.1) to remove potentiaþ protective antibodies specific for LPS determinants, and then

compared for protective activity aginst 5ó98 challenge. The similarity of the protective titers

obtained (pD50 2000 of the anti-Sark-OMP versus 2500 of the anti-control-OMP sera) indi-

cates that the maior non-LpS protective antigens ¿¡p ¡stained in the Sarkosyl-insoluble oMP

fraction.

Immunoblotting anal¡nis using the LPS-absorbed antiserum prepared against Sark-

oMP failed to reveal any difference in the protein p'rofiles of st¡ains possessing or liacking

non-LPS protective antigens (Fþre 4.3, lianes J, K & L). Itr addition, this serum was further

absorbedwittr boiled 5698 four times (to remove antibodies to heat-stable antþens) andwith

live Ol7 four times (to remove antibodies to non-protective heat-labile antigens) and anaþed

87

Figur.g 42 SDS-PAGE anaþsis of detergent treated V dtolqae 5698 OMP preparations

I-ane A,2Vo Nonidet P40; lane B, 3Vo Nonidet P40; lane Ç lVo Sarkosyl; liane D, 2To Sørkæyl;

lane E 2Vo soditm deorycholate; lane R lM urea; lane G, 2 M urea; lane H, ZVo Tween?ß;

lane I, control OMP; lane J, cont¡ol OMP; lane K ZVo Brq 58; lane l.lVo Brij 58; laneM, l%o

Ti.iton X-114; lane N, O.SVo Triton X-114; lane O, 20 mM EDTfu lane R 10 mM EDTA (Final

concentrations). Arrows on the left point to molecular weigbts of protein bands seen in lane

C after 1% Sarkoeyl treament These are, top to boüom, 45 kDa, 42kDa,38 kDq 32kDar?5

kDa and 2lkDa. Arrows on tle rigþt indicate standard moleculiar weigþt markers (lane M):

Ð5 lt)a, 116 kDa 97 .4 kDa,66 kDa, 45 kDa, 36 kDa, D kDa,Vl lÐa,20. 1 kDa and 14.2 kDa

88

\

-

-- -

a-<-<-

<-

d>

-

--b

.,É.','-|', æ

-.-æ-

I lrul mrr¿t-,r.' ffi

¿?-

åi¡¿ù - ù æ

il-

Ë':ry,&.--eG 1'

.rE-

:il

A B C II E F GH I J KL II il 0P0

Figure 43 Immunoblot anaþsis o[.V cholqac OMPs with anti-Sark-OMP antisera I-ane A

H-1; lane B, BM69; lane G 8ß3; lane D, AA14O41; lane E, 21756l; lane R CA411; lane G &

J,Ol/;lianes H e K 5698; lianes I & I,5698 Sarkosyl-treated OMP pelleÇ lane M, standard

moleculiarweigþt markers: arrows indicate positions - 116 kDa, 97.4kDar 65 kDa, 45 LDa 36

lÐq291ÐqZl kDa, Ð.1 l¡Da and l4.2bDa. Arrows on the leftindicate positions of specific

proteins. These are from top to bottom - 78 kDe, 58.5 kDa, 45 kDa, 38 kDa and 18 kDa l-anes

J-L tested with LPS-absorbed anti-Sark0MP while lanes A-I tested with LPS-absorbed

anti-Sark-OMP further abeorbedwith boiled 5698 (four times) and live O17 (four times).

89

.1

i,t

à,

¡I'

türÌ

't

I1,

),

lI

I

:i, W

r-+

-t-r+>

AB CD EF GH I J KL 1{

I

,t

iÞ-

again roy immunoblotting (Figure 4.3, ranes A-I). In both instances three proteins with Mr

values of 7g, 5g.5 and 1g lDa nor er¡ident by SDS-PAGE anaþis (Figure 4.2) rcacted with the

antisera (Fþre 4.3). After additional eúaustive absorptions with live O17, the antibodies

stillreactedwith o17 OMps (Fþre 4.3,lane G); Howerrer,when attemptswere made toexcise

the relevant protein bands fn¡m sDS-PAGE gels and to use tìe (denatured) proteins to

immunize mice, no protective antibodies utere generated; the antiser4 nevertheless reacted

with 5698 OMP in an ELISA (6400), showinglhe proteins to be immunogenic'

*2.3 OnPV

As mentioned in Section l.8.Zl,a?SkDaprotein (Omp$ is amaior outer membrane

prorein or.v cholqæsógB (stevenson et aL 1985: Pohlner et aL 19864198ób)- This proæin is

found in avariety olV cholcraestrains includingS 6gBtl6íand 1074 (Manning et aL L982)'t\e

ratte¡ a nontoxigenic Brazilian environmental isolate (L,evine et al- 1982). Since a 75 kDa

pfotein is present in 5698 OMP preparations (Figure 4.2), experinents were carried out to

determine whether this migþt represent a non-LpS protective antigen. The structural gene for

the OmpV protein had beên cloned (Stevenson et aL 1985) and an OmpV clone was kindþ

provided by Dr. P. Manning. 'lvhen aliquots of a (protective) anti-5698/165 sefum were

absorbed with the ompV-e:pressing clones or with the vector st¡ain as a @Dtrol no significant

reduction in protective activity were observed. In addition, antiserum raised against the clone

e:rpressing ompv was not protective. These eryerimenr indicaæd that ompv is not a

protective antigen in thi6 model

It'

tt:ri'l

ll

í

I

90

43 @nession of pili by v cholerae sûains

43.1 helininnrY elryerirents

During the cou¡se of these studies, Ehara et aL (1986) described culture conditions

suitable for the production of pili by v choleræ. These grounh conditions, &d others pre-

vionsþ reported to promote úe e:rpression of pili in vitro (Treedy et al' 1968; Faris et aI 1982;

Ehara et aL lgg6), were therefore used for an EM shrdy of pilus production by four strains:

5698 (tre strain most commonly used to cha[enge infant mice), 64 (a recent field isolate of El

Tor biorype), H-l (a recent field isolaæ of El Tor biotype) (see Table 3'1) and K23-7 (a

non-motile El Tor strain usedþ, and obtained from, Eha¡a et al^ 1986). Liquid cultures were

incubated at both 25 C and 37 C in shaking or stationary conditions, with varying incubation

pefids (16-72hrs). when the bacteriawere examinedry EM, a maximum ofl-Zpili per cell

were observed on a small minority (approximalley SVo) of vibrios

In an attemptto selectfor a piliated subpopulation of orpanisms' Eequential subcultures

urere performed; medium containing a sterile glass slide was inoculated wit\ V cholqae atö

alæt24hrs incubation, tle slidewas t¡ansfered to fresh medium and the procedure repeated

twice. However, no sþificant enrichment for piliated bacteria was achieved- Next, El Tor

st¡ains 64 and Y\B-lu,efe inoculaæd onto the TCG agar medium used þ Ehara et al' (1986)

and incubated at 3o c or 37 c overnighL A ferr flexible pili (5-7 nm in width) were seen oû

the surfaces of S-loVo of the cells, but agarn the degree of piliation was i¡adequate, not onþ

for an attempt to isolaæ these structures, but also for studies designed to examine their role in

pathogenesis.

91

+32 ProductionoflC?

In 1987, investigators from the united States reported the expression of TCP þ the

classical v chol¿raestrain o3g5 (lhylor et aL lggT). To evaluate ttre significance of such pili

in pathogenesis, these workers constructed a mutant of 0395 which carried a deletion in the

struchrrar gene for the pilin subunit (TcpA). This mutant showed a 100,000-fold greater LDso

than its parent st¡ain in the infant mouse model, and competition experiments indicated that

TCp promoæd colonization of the infant mouse intestine (Taylor et aL 1987). Althougþ the

pro*ctive activity of antibodies directed against TCP was not examined, these pili would be

obvious candidaæs for non-Lps protective antigens. Accordingfy, the classical strains 5698

and CA41l were cultured according to Taylor's protocol and examinedby EM' However' no

pili with the morphologr of TCP wefe observe4 althougþ other morphological gpes were

present (data not shorrn).

Nex! the coloníutiøt factormedia used extensively for tÏe expression of pili by ETEC

strains (Evans er aL lng) were tested for their capacities to suPport pilus production þ

v cholqæ. strain 5698 was inocurated onto cFA and into cFB and incubated at?S c ot 37

c for ?A to36 hrs. on examination by EM, thick aggregaæd bundles of pili similar to ttrose

described by Taylor et aL (1987) were observed on cells gfown at ?5 C' About 60vo ol the

orsanisms harvested from CFA expressed these pili, and about 4o%o of those grou'n in the

corfespondingliquid medium. Fþre 4.4 shows the morphologr of the pili produced þ two

v cholqæstrains when gro$'n on cEêr at 25 c rhe dimensions of the filaments were measured

by the laüice spacing of crystalline catalase according to the method of wrigley (1968)' Itr

support of the description given þ Taylor et aL (1987), individu¡l f¡laments were estimated to

be about 7-10 nm wide. These filaments aggregated closeþ in parallel to form bundles of

variable length and 0.1-0.3pm in fiemeter'

v2

Figune 4.4 hoduction of TCP on CEA V clølcræ 5698 (A B) and 217561 (C) grown on

CFA at ã C for 36 hours and negatively stained. F = flagellum; S = single TCP filament T

: bundles of TCP. Bar = 250 nm (A), 100 nm (8, C).

93

-

-

!s

T

V

Further studies showed that the TCG medium described by Ehara et aL (1986) could

also support rcP expression, althougþ piliation was inconsisænt (Figure 4'5)' The culture

conditions which most reliabty promoted TCP production invohed growth on NA at 37 C for

z h, followed by growth on cEA (pH 6.5) for vl-36hrs at 25 C At this stage itwas important

to confirm that the pili e4pressed ty these strains when gro!1¡r in this manner were indeed rcP.

To this end a serum containing antibodies to TCp was obtained from Dr. R. Hall (center for

Vaccine Developmen! Balttmore), with tle inæntion of examining the piliated bacteria þ

IErv[ This serum had been prepared by immunization with rcp+xpres sngv cholqae 0395

(Hall e\at.19ß) and upon receiptwas absorbed four times with ogau'a LPS (Section 210'1)

toremove anti-LpS antibodies,leavingbehind antibodies exclusiveþ to non-LPSdeterminants

including those ngainst rcp. IEM using this serum confirmed that the pili were indeed rcP

(Figure 4.6).

433 DistributionofTCP

A number of Vcholeræ st¡ains were examined for their capacities to produce TCP

under the growth conditions described above. In particular, a "o''elationwas

sougþtbetween

the potentialto expressTCp and the previousþ determined distribution of non-LPS protective

antþens. The results of an EM survey are presented in Thble 4.1. pili with ttre morphologl of

TCp were seen on all st¡ains of Classical biot¡'pe examined, regardless of serotype, with the

singfe exception of cA401; Taylor et ot (1987) also failed to deæctsigniñcantTCP production

by this strain. In contras! howe'er, TCp were not seen on any of 6 strains of El ror biotype'

Recent field isolates of the latter biotype, hou,erer, do possess non-LPS protective antigens

(Section 3.4.2).

Immunoblotting studies confirmed tle results of the EM suwey. The serum used in

theseerperimentswas an anti-S6gBll1'serumwhich hadbeen extensivelyabsorbed to remove

94

X'igurre 4.5 P¡oduction of TCP on TCG. V choløae 5698 grown in TCG agan at25 C 1o¡ %

hou¡s and negativeþ stained T = bundle of TCP. Bar = 5(Ð nm (A) 100 nm (B)

95

-

-

:úùt &

a'

a

Ì.

a

It ,

I1

ÒI

o

t

a

^a

ll.r

t'.a

\,

)t-

t

?

ot

A)

Figurc 4.6 Vdtobae 5698 grou¡n on CEA at?S Ç reacted with LPS-absorbed anti-O395

antiserum, follou,ed by hotein A colloidal gold conjugêþ, and negativeþ stained" T = bundle

of TCP pili Bar = TO nm (A) and 1ü) nm (B)

96

alT

?a

rË

d.'

ÒA

¡rÈ+

¡",t

J

ù

1t

l¡

a¡

l¡

IF

r\

J3

+t',

, ^ñ:ì

t ¡¡-,ù

ta atl

a't

titt È

t*a

"î

,¡-

,tt

Ihble 4.1 Disüibutionof TCP

Strain

Clasdcal

5ó98

c4401

cA411

ztTs6l

A414041

5698/165

0395b

Erpressiona

+

+

+

+

+

+

E lbr

BMó9

4413993

H-1

64

K?3,C

ol7

a Production of TCP on CFA at?S Cfor 3ó hrs'

b OggS is a Cla¡sical Ogma kindly prodd bD' Dr. RIt Hall' Center for Vaccine Derrelop

men! Baltimo¡e, US.A"

c K23isanElToreguastrainkindþp¡ovidedbyth.ìrf"Ehara,IngtihræofÏhopicalMedicine

Nag¡saki University' JaPan.

n

antibodies to irreler¡ant bacterial components as a prelude to the attempted cloningof non-LPS

protective antigens (see section 5.2). IEM had confirmed that this serum contained antibodies

to TCp (section 4.6). The absorbed senrm was initialry tested agninst two classical and four

El ror sü.ains (two recent fietd isolates and two old laboratory strains) grown under conditions

which prrcmoted (cFA ar?s C)or fePressed (NB at37 C)TCP production' Figure 4'7 shou/s

the results of subjectingthe bacteriar suspensions to SDS-pAGEand immunoblotting when

grown on cFA at?s cboth crassical st¡ains produced a protein which could be detected by

the antiserum. The molecular weight of this protein (20 lDa), and the the fact that itwas not

produced during gro\¡,th in NB at37 C (Fþre 4.7), is consistent with it being the st¡uctural

subunit of rcp. None of the El Tor strains synthesized this protein neither at37 c nor at 25 c

in any media

In a second experimen! nine V ctpbøest'nin s were cultured on CFA at 25 c and tested

fo¡ TCp eryression using the same antisenrm. A cnrde TCp preparation (section 213) was

used as a positive control while 5698 grown in NB at37 C served as a negative cont¡ol for

immunoblotting (see Figur e 4.7). As expecte4 the antiserum reactedwith a 20 kDa protein

expre*sed only by the classical strains (with the exception of c4401) whereas no reaction was

er¡ident with the El Tor strains The TCP preparatioû waÍ¡ strongþ positive, supporting the

conclusion that this protein represents the pilin subunit (Figure 4'8)'

4.4 TCp is a virulenoe determinant in the infant mouse model

Itwas important to confirm the conclusion that rcP is avirulence determinant in the

infant mouse model (Taylor et aI lgïi). To this end tle virulence of the non-motile variant of

5698 (selectedby the sloppy agar overlay technique as described in Sectiot2.2'¡ was assessed

followinggrowth under conditionswhich supported or repressed TCP production' Thisvariant

98

Figurt 4.7 Immunoblottingmaþis of,V clnleræstrains for TCP eryression. The follwoiag

strains were emamined after culnrne on CFA at 25 C for 36 hours, (lanee A-F) and NB at 37 C

(lanes G-L): H-l (El Tor biotype, lanes A & G); 4413993 (El Tor, B & H); CA411 (Ctassica[

c & D; 5698 (Classicâl D & Ð; O17 (Et Tor, E & K); and 8233 @l Tor, F & L). They u'ere

resuspended in electrophoresis sample buffer and su$ected to SDS-PAGE The pr,oteins were

t¡an¡ferred to nitrocellulose and tle blot¡ developed with 1:10ü) rafrùit anti-5698/165 CP

serum (knoum to contain antiiTCP antibodbs) fotloured by goat anti-¡abbit IgG coupled with

horse radish peroxidase and substrate. The arrorr indicates the pooition of the molecular

weigÞtmarker, ß-lactogfobulin (bovine -ilk), 18.4 kDa

99

a

A B C D E F G H I JK L

lTgure 4A Immunobloüing anal¡ruis of TC? e:pression þ varioru strains oî. V chobæ. A

crudepreparation ofTCP(tanesÀ L) orcellefiracts fromnine V dplcrae strainl (lanesB-K)

we¡e immunoblotted ruing the serum dÊscribed in the bgend to Fþre 4.7. St¡ains (biotype,

rerot¡ipe, lane position) teoted were: H-l (E¡ Tor, Ogau'q B); 4413993 (El Tor, I¡aba, C);

4414041 (C1assical Ogara D);217561 (Classical lnaba, E); O17 (El Tor, Ogaw4 Ð; E233

(El Tor, Inaba, G); C4411 (Ctassical, Og¡wa" H); C4401 (Classica[ Inaba, I); 5698 (Classical

Inabq J & K). Strains were cultured on CFA et?S C for 36 h an4 in the case of 5698, also in

NB at 37 C for purposes of comparison (Lane Ð. The a¡ros' indicates the position of the

moleculiarweig[t marker, &lactoglobulin (bovine mitk, 18.4 kDa).

100

+-

f¡rP

A B CDEF GH I JK L

11t

wa6 chosen in order to enmine the effect ot TCP in the abeence of the

flagellarstructure (Attridgs & Rowley 1983a).

Elpression of TCP by non-motile 5698 led to a 100-fold increase in virulence; or-

ganisms grc$,n on CEA atäSChad an LDso ol l.TI r 0.4 x 106, compared with L?5 x 0.11 x

108 for those grc$'n in NB at37 C.

45 Attempts to PurifY TCP

Given the pivotal role of TCP in colonization and virulence (tylor d aI 1987; Her-

rington et øL l9B8), and þ analogr with the pilus colonization factors of ETEG it seemed

probable thatTCpwould represent a protective antigen of.V choleræ. Accordingf¡ attempts

*,ere made to puriry these structures, in order to assess the protective activity of antibodies

directed ageinst them.

FollowinggrowthonCEAat25 Gbacteriawere resuspendedin differentsolutions (see

Section Zl3) prior to shearingto remove pili. UsingPBS (AlKaissi & Most¡atos 1985) ot ILS

mM 'Fiß HCI (Thylor et at- 1987), the pili couldbe subsequently precipitatedwitl am-onium

sulphate and pelleæd at low speed centrifugation, whereas with 10 mM tis buffer (Korhonen

d aI lgg1), the pili resisted ammonium sulphate precipitation and could onþ be recoveredby

centrifugation at 100,0009 for thr. Regardless of the method use4 such pili preparatiotrs were

always contaminated with other protein bands when examined bD' SDS-PAGE; when viewed

by EM, LpS vesicles and flagella were er¡ident (data not shourn). In an attempt to remove tìese

contaminants, the crude preparations $rene subjected to density gradient centrifugation follow-

ing the method of Ehara et aI (lgS7), but EM examination revealed that this method was of

liülevalue.

Attempts urene 1[s¡ made to puriS the crude pili by adsorption onto guinea pig RBG

(Al-Ikissi & Moetratos 1935) but this too was unsuccessful A final atfempt was made Ûo

RA

101

separate pili from otler cellular material using deorycholate and concentrated urea according

to the method of Korhon eneî al. (1980), but tlis also proved futile. Since efforts to puriff pili

ofV cholqæhad proved ineffective, ttris approachwas abandonedin favor of less directmeans

of assessing the protective activity of anti-TCP antibodies.

4.6 rcP is a prctective antigen in the infant mouse model

It was important to determine whetìer the protective anti-56981165 serum (Section

3.5.1) contained antibodies to TCP an{ if so, whether tlese antibodies contributed to the

protective capacity of the serum. Although this serum was generated by immunization with

organisms gfou/n under conditions unfavorable for TCP elpression (NB at37 C), it seemed

poesible that a feu, TCp might harre been produced under these conditions, or induced to do

so following injection into the rabbit The presence of anti-TCP antibodies was confirmed by

IEM as shou,n in Figure 4.9 (A & B), which shou¡s binding of antibody+oniugated colloidal

gold particles to TCP. Subsequent work confirmed that, when gfo$'n on CFA at25 G the

56gÐtl65 strain produces TCP as judæd by ENr, IEM and immunoblotting anaþses (not

shown).

Accordingþ, the capacity of the anti:TCP antibodies to msdiete protection against

challenge with TCp-poeitive or TCP-negative organisms of the 5698 strain (ie., those grow¡t

on CFA atZS Cor in NB at37 Çrespectivety) was assessed in the inf¡nt mouse model The

protective activity of the LpS-aboorbed anti:ICP serum obtained from Dr. Hall (Section 4.3'2)

was also assessed asainst both challenge inocula The data are presented in Table 4'Z

Anti-5698/165 sho¡,ed a greater tìan frvefold PDSO when the challenge strain was allowed to

express TCp pili in vito (cEA 25 C). A similar but greater effective rise in PDso (æn-folQ

rr/as seen in anti-O395 l-Ps-absorbed Eerum when the same challenge strain was allowed to

102

Figure 4.9 Immunogold labelling of.V dtolqæ 5698. V dtolqøe 5698 was growtr on CEA at

25 C for 3ó hrs and the resultingorganisms reactedwittr anti-5698/165 (ArB) or LPS-absorbed

anti{4411 (C,D) followed þ Protein A-colloidal gold conjugate, and negativeþ stained" T

= bundle of TCP pill Bar = 2N nm (A), 100 nm (B), 100 nm (C) and 100 nm (D).

I

103

I{

¿

I

Ça

¡a

¡Ye -t

!.r

ao

a

ra

f,r 'a a

,,.r'

o

at,

-{l>

¡4

"1'

"tÉ

à

).dt

Jtt

¡t

Cþ' ,t

I

t rt t'

ttl rì

r¡'l

(, r

¿

-'rr

¿d

e.ft

ît

-E-

I

,{

È-

It'

ì{

f;,i

'l

It,

i

Antiserum to

Ibble a¿ Antibodies to TCP are protective in the infant mouse choþra model

Protective activity aglinst 5ó9Ba

rcP.b rcP+b

5698/165

()395 abso¡bedwith

Ogawa LFS

6,000 t 330 34,m t !4¡g

B,W t 1,400 250,000 t 14000

I Protective activity in PD50 uniqthl

b Th" 5698 challenç st¡ain was grou'n under condition¡ uùich favo¡ed (cFA ar ?5 C) or

repreosed (NB at 37 C)the production of TCP CICP+ or TCP, respectiveþ'

104

:

I

I

f^-

express T.;p invitro. This clearry demonstrated that antiircp antibodies have protective

potential. l-at'9r studies (section 5.6.2) provided unequivocal confirmation tlat TCP is a

protective antigen in this model. The appreciation of the protective potential of antibodies to

TCp offered an e:rplanation for previous data which had hitherto remained inerylicable' In

Section 3.5.l itwasshowntha! despiteextensive aboorptionswithorganisms of ogawaserotype

(strain cA411 seven times, AA14041 three times) to remove antibodies to antigens common

to the two serotypes, the anti-5698/r65 serum retained some protective activity againstvibrioa

oftheogawasefotyPe.ThiswasindicatedbythePDsovaluesobtainedwhenmicewere

challenged with vibrios grou¡n in NB at 37 C (PDSæ of ?;60 and 290 against C4411 and

AA14041, respectiveþ . Table 3.4). Hor,er,er' as mentionedin footnote (e) of this table, when

these organisms \ilere grown on cEA at?s cand used as challenge strâins, the PDsovalues

increased about four-fold (cA411, g70; AA14041, 1380). The higþer protective endpoints

obtained when the challenge organism.s had been grown under conditions conducive to TCP

expression suggested that antibodies to TCP had not been completely remove4 and this was

confirmed bfy IEM (not shown). In retrospec! this is not surprising since the absorbing

bacteria had not been culn¡red under conditions favourable for TCP eryression'

In the fevefse eryeriment, absorption of an LPS-absorbed anti-c4411 serum with

bacteria of Inaba serotype (5698) successfully removed all protective activity against challenge

organisms of the Inaba serotype (Section 3.5.1). In this instance, the initial anti-cA4llserum

did not contain antibodies to TCp detect¿ble by IEM (Frgure 4.9, c &.D). Hence, this erylains

why (as shown in Table 3.4) there wa.s no residual activity left against the Inaba serotype (PDso

<2q.

I

ri

105

4.7 Pmtection by anti-TCP antibodies is biotype nestricted

EM and immunoblotting anaþses had suggesæd that the capacity to express TCPwas

restricted to strains of the Classical biotype (Section 4.3.3)' However, some strains of the El

Tor biotype czrrystnrct'rar genes for TCp (Tayror et aL r9ç8a) and so it is possible that strains

of this biotype can produce such pitinvivo. Therefore, itwas imperative to determine the

capacity of anti:TCp antibodies to protect infant mice from challenge with strains of classical

or El Tor biotYPe.

The anti-O395 supplied by Dr. Hall was chosen for the experiments to be described in

this Section because of its greater protective efficacy against TCP+xpressing challenge bac-

teria, compared to the anti-5698/165 serum (Table 4.2). This antiserumwa^s firstabsorbedfour

times with live o3g5 (gown in NB at37 C to inhibit expression of rcp) in order to remove

potentialþ protective antibodies directed against LPS or non:TCP, non-LPS protective an-

tigen".

Protection tests confirmed tìat the protective activity of the absorbed serum was

prinariþ due to antibodies to TCP (Table 4.3). Fint, tle serum was now onþ moderateþ

protective against 0395 or 5698 vibrios cultured under conditions inhibitory to TCP produc-

tion. In contras! if these strains Ìvere grown on cFA at?s Ç the protective tite¡s increased

2ß- or3O0-fold, respectiveþ (tble 4.3). Secon4 the absorbed serumu'as onþweakly protec-

liys agâinst C4401 (which, in this study, does not eryress TCP) challengg, despite the fact that

this strain e:rpre6ses non-LPS protective antigens (section 3'4)'

ConsisæntwiththeimmunoblottingandEManatyses,whenthechallengestrainswere

of the El Tor biotype grolvll on cFA or in NB, the residual serum was unable to mediate

protection (Table 4.3). These results clearþ demonstrate tlat there is a correlation between

i

106

rbble 4i Antibodies to TCp are protective onþ againstcha[enge strains of classi¡al

biotypea

TCP status of challengevibrioo

Challenge suain TCP rcP+

()395

5698

o17 (PA )b

8233(PA )

H-lGA+)

AA1393 (PA*)

c4401

3.3.

230 t 50

430 t 30

<n<n<n<n

70t5

5,000 t 5ü)

130,000 t 11,000

<n<n<n<n95t8

" Figures shos, protective activity of absoôed anti-TCP 0395 cerum in PDsodmt Challenge

straiü were gro.*a to be quantitativeþ TCp' or TCp+ a¡ descriH in legend to Dble 4'2

b pA-ÆA+ refer ûo prerence or aboence of non-LFS protective antigens as detcribed in Table

IUT

the distribution of pili amongst ll choleræstrains and prrrtective activity of and-rcP antibodies

and that it is biotYPe-restricæd'

4.8 Discussion

The experiments described in this chapter !\'ere performed in the hope of elucidating

the nafirre of the non-LpS protective antþens of v chobrøe. In an attempt to identis

differences between the fourstrainslackingnon-LpS protective antigens and thetwelve strains

which share such determinants (Section 3.4), OMP preparations were compared by SDS-

PAGE ¿¡d immunoblotting anaþses. This approach was not helpful hotilever, since the oMP

profires of the four old El ror strains were not consistentþ different (Figure 4.1). Given the

denahrring co¡ditions required for such anaþses, it is poñ'ible that none of tle bands detected

þ immunoblotting involves the binding of protective antibodies' A number of PAGE system's

and various detergent and solvent ext¡actions of the oMP preparations \Ã'efe tested in order

to obtain better resolution of the proteins but these did not pfove fruitfrtl'

Subsequentexperimentswere performed to testwhether any of the non-LPS protective

antþens might be associated \ilith pilus structures produced by v choleræ' Atthougþ experi-

ments described here focussed upon the expression of TCR other pili were seen by EM (data

not shown). oo" of the other t)rpes see& albeit in low numbers, was a flexibre ¡¡¡¡ filament

5-7 nm in width. Ibrahim (1934) found slender flexible pili (3a nm in diameter) present in 1

of 3 El ror strains tested as well as in one classical st¡ain. Al-Kaissi & Most¡atos (1985)

reported the presence of longtwisted pili on some strains of the cla.ssical biotypewhile Ehara

et aL (1986) described the pili they observed as flexible, long ñbres present on El Tor stains'

It is possible the flexible pili seen on El ror strains in this study are the same a.s those described

by orhers (Ibrahim 1984; Ehara eÎ aL l9tl6). Very recently, Hall et aL (1988) reported a

108

rystematic EM study of the pilus types produced by v dtoløa¿. TCP u'efe detected only on

classical strains, while two otler tlpes (B and C) were seen on all strains regardless of biotype'

production of TCp by V choleræ was first reported by Taylor et al- (1987) following

grovth of bacteria in LB medium at 30 c. subsequent experiments by ldall et øt (1988)

confirmed the existence of rcp and recommended growth on cEA at 25 c for optimal piliation.

Initial experiments were designed to identi$ cultural conditions which would consistentþ

support TCP production by v choleræ strains used in this shrdy' Inability to detect such

appendages following growth of bacteria in LB medium suggests some difference in medium

constituents. In agreement with Hall et at. (r9gg) howe'er, rcP were observed when the

bacteria wefe grown on cFA at 25 C The widtl of tle pilus filaments with rcP morphologr

was estimaædtobe 7-10 nm, simil¡ar to previous ssfimates (7nm, Taylot et aL L987;56nm, Hall

eJ ol. L988).

EM and immunobloning studies (Table 4.1, Figures 4.7 &' 4.8) confirmed Hall's finding

tha! underthegrowth conditionsadopted, productionof TCPis restrictedto st¡ains of Classical

biotype. In tota! TCp have now been detected on - classical strains (Taylor et al- 1987; Hall

et aL lgæ; this thesis) but on none of - El Torbiotype. The studies performed to date do not

eliminate the possibility that sü'ains of El ror biotype will qynthesize TCP under as yet

undefined sowth conditions invitto, or perhaps onþ in resPonse to environmenlal stimuli

present in the guL Infant mouse eryeriments designed to assess the capacity of anti:TCP

antibodies to mediate protection againstEl Tor challenge strains failed to provide support for

the latter (see below).

Various methods ha¡ebeen described forthe isol,ation andpurification of differentpilus

st¡uctures produced by v choterøe (Korhon en et a|.1980; Al-Ikissi & Mostratos 1985; Ehara

et at. 1987; Taylor et aL 1987). Horvever, i¡ my hands, trone of these provided adequate

purification of TCR as all resulæd in subst¡ntial protein and LPS contamination. It is knw'n

109

that pili are extremeþ hydrophobic, consisting of a higþ proportion of nonpolar amino acids

(Korhonen ef aL rggo;Gaastra & de Graaf 1gg2) and rcp aÍe no exception (Taylor et aL 1987).

Accordingly pili tend to associate strongþ with other bacterial surface components such as

outer membrane vesicles and LpS. Attempted fractionation of pili on the basis of size and

hydrophobicityusingasodium deoxychorate-ureabuffer (Korhonen et aL 1980) proved futile'

At least some of the protein contaminants seen in the TCp preparations could have been due

to the presence of pilus-associaæd proteins as have been described fot Neisseris gonorrhoeæ

(Muir et at. rggg). As time was limite4 further attempts at purification were abandoned in

favor of alternative approaches to the e'aruation of tre protective signiñcance of rcP.

pilus colonization factors have been recognized asvirulence determinants not onþ for

E. coli(satterwhite et aL lgTg: Gaast¡a & de Graaf 19s2) but also for serreral other gram-nega-

tivebacterial palhogens (Kellogg et aL 1963; Duguid & old 1980;Abraham & Beåchy 1985)'

During the course of these studies TCP were shown to be essential for gut colonization in both

infant mice (Taytor ef øL r9gl,¡and man (Herringto n et aL rgsg). In the former studies, mutant

strains unable to produce TCp were shown to be unable to efficiently colonize the intestines

of infant mice, and were consequently much less virulent tlan the wild-type v dtolqae (Taylor

et at. rggl). Heningto n et oL (1gSS) subsequentry used such murants to assess the importance

of TCp for the colonization potential and vaccine efficacy of non-toxigenic v cholqae' ln

cont¡ast to the vaccine strain which could produce TC! the mutants lf,'ere found to be

non-colontzilg¡g¡.imm¡nogenicandnon-protective.

Initial e:rperiments defined growth conditions which consistently resulæd in TcP

production by V choløae 569B,the strain most commonly used in protection studies' Afær

confirming the identity of the pili by IEM (Figure 4.6), infant mouse tests confirmed that TCP

production signifrcantly enhanced tie virulence of tle (non-motile) 5698 strain' Alttrougþ the

100-fold difference in virulence was less dramatic than that proriously reported by hylor et aL

110

(1937) for the 0395 strain, this probabþ results from reliance uPon a phenotypic rather than

genotypic difference in TCP production. Thus a minority of the bacteria cultured under

conditions unfavorable for TCp expression migþt have been TCP-positive; alærnatively, the

population might har¡e been uniformly TCP-negative at the time of challenge, but synthesized

these pili rapidþ ín vivo. In contrast, the mutants used þ Taylor et al' (1987) were genetically

incapable of TCP Production'

Since virulence determinants such as colonization factors are prime candidates for

protective antigens, itwas of great interest to determine whether TCP might represent one of

the non-Lps protective antigens of. v choreræ. In this respect it was encouraging to find that

the protective anti-S ilgÐlLilsserum (Section 3.5.r) contained antibodies to TCB as judged by

IEM (Figure 4.9) ¿¡¡d immunoblotting analyses (Figure 4.?). The first evidence in support of

the hlpothesis that TCp can function as a protective antigen came from infant mouse assays

designed to evaluate the protective potential of these anti:TCP antibodies- Sera containing

such antibodies were markedly more effective in mediating protection if the challenge or-

ganisms were expressing TCP at the time of administ¡ation (Table 4'2)'

Subsequentprotection tests indicated that antibodies to TCP could mediate protection

against challenge strains of Classical biotype only (Table 4'3), a finding consistent with the

inability to detect the production of rcp by El ror strains. This suggests that TCP is not the

only non-LpS protective antigen in this model, since recent field isolates of El Tor biotype

possess such components (Section 3.4). Attempts to identiS non-LPS protective antigens þ

gene cloning are described in the next chapter'

111

Molecular cloning of genes involved inbiosSmthesis of TCP

5.1 Intnoduclf,on

1¡ryrten attempts to isoLate non-LPS protective antþns from OMP preparations p¡oved

unsuccessful, it was neeessary to dwbe alternative approaches to further chatætet'ae tlese

componen6. To üb en4 the shrdies described in the previous chapÛer were performed 1o

examine the protective sþificance of pilus ¡tnrcürs produced by V cløleræ. Concurrcntly

attempts were beg¡n to isolate non-LPS protective antþns using the techniques of gene

cloning and the¡e studies form the bæis of the pnreseot chaser.

Initialeryerimentsweredesþedtoclonegenesencodingnon-LPsprotectiveantigens

of6 dtotsacsógg and to express them in an E æti tæipfut strain. As discl¡s8ed belw, this

approach proved unsuccessful At about that time, ?ylor 4 4L (1987) reported their s$dies

of TCR fr,omwhich itwæ clearthatsurfaoe expression of TCP andotherOMPswas dependent

upon a functional úoxR gene, Since the E coti rwi¡Åent strain used in rhi¡ 3f¡dy lacked this

getre an alærnative cl,oning strategl was devisedwhich allowed ctoning of genes inrroþed in

TCp production The availability of TCP-producingclones atlo\red unequivocal oonñrmatbn

of the conclusions that rcp acts as both a virule¡ce deterninant and protective antigen in the

infant mouse choþra model

ttz

52 Pnepffiation of done probing serum.

Aclone.probingoerumwasrequircdinordertodetectbyimmunoblottinganyclones

oryressing non-LFS protective antigens, and a protective rabbit anti-5698/165 serum (Section

3.S.1) was selected for this purpoße. Before u¡e the serumwas extensiveþ absoöed to remove

antibodiee to componente other than non-LPS protective antigeng thereþ reducing the

likelihood of detecting "false-positive" clones druing the screening pmocess- Since non-LPS

prrotective antigens a¡e heat-labile and absent from old El Tor st¡ains such as o17 (Section

3.4.1), the senrm was absorùed four times with boiled 569B,t165 (to remove antibodie¡ to

heat+tabþ antþns), three ''.nes with live 01? (to remove antibodies to non-protective

heat-labile antiçns), and ñnalþ two times with live E. coli K12 strain DHl (to remove

antibodies to the.E coli rccipterl strain).

Aliquots wer€ kept after each absorption and tested i¡ an ELISA using 5ó98/165 and

Ol7 OMps as antþns (Figure 5.1). The abeorbed Eerum had ELISA tiæ¡s of 7,300 and 49

agninst 569Fl¿65 and O17 OMh, respectiveþ. This represenls l47o and <0.lWo of the

orisinal activity against both antþens, respectiveþ (Fþre 5.1). In oo¡tras! the protective

activity of the residual serum rvas virtüatly unchanged (protective fi1sr ¡grinet 5698 challenge

fell from lZffi to 11f)), indicating selective retention of the antibodies of inærest The

abeo¡bed semm is heoceforth referred to as anti-5698/165 CP (clone-probing) serun.

53 PreIimfutsry donirry exlDeriments

Initial attemptr to clone non-LPS protective antigens invoþed construction of a 5698

gene bank iîE corietrain DHI ruingthe coemidvector pHCI/9. Details of this procedure are

not given because tùese etudþs were not successful A total of 4o0 colonies were screened þ

oolonyblotting(Soction Zt6),usinga 1:1000 dilution of anti-5698/165 CP seruE'" Sifeen of

113

Flgurr 5.1 preparation of clone probing serun An antfuenrm prepared in rabbits against live

5698/165 hybridvibrioq and lnom to contain protective anúodies specific for TCP (Section

4.6), was extensiveþ abeorbed to remove a¡ftodies of irrelevant specificities (Section 5.2).

Abeorptions 1-l were with boiled 5698/165 vibrioe, 5-? with live o17 and 8,9 with live E coli

K12 Strain DHl. The unabooñed and the aliçot of the absorbed sera at various stages were

tesred aginst56gB/165 oMp (tcfq ) and 01? oMP (dghq ) in an ELxsA assay. o represents

unabsorbed serum, ELISA tiær of u¡abeorbed serum against O17 OMP ßn,1N (not ehoum

in Fþre).

tt4

6

5

4

5

4(f)

Io

ulÉ,t--

F

U)

=1¡¡

t

3

Io

ulÉEt-

Ø

u¡

2

1

2

1

5

NO.

7 Io 1 2 34

ABSORPT¡ON

the clonee were positive and su$ecæd to further anaþie. Aliquots of unabsolbed anti-

silg}lrilsserum were absorbed (æ per Section zl0.z) 4 ':mes with each of the 16 positive

clone¡ orwith poeitive (5698) or negative (8. @tí)cont¡or strains and subeequentþ tested for

removal of antib@ activity þ both EusA and infant mouse protection test'. Althougþ there

waE no correr¡rtion between neduction in ELISA titer and loss of protective activity (data not

shom), 9 of the 16 clones reduced the protective titer by 75Vo oÍ mofe' Hou'ever' when mice

$,efeimmunizedwiththeseninecl.onesnoneoftheresultingantiserawereprotective(data

not shourn).

In an attempt to erplain these dbappointing results, the nine clones were again sub-

iected to immunoblottingwith cp senrm, some tweþe weeks after their original detection-

Despite etorage of the clones in gþcerol at -lOC (Section 22), all were now negative and thus

no l,onger opressing antþens of inærest Thb ap'parent in8tsbility was att¡ibuted to the f¡ct

that the E. colirecipient strain DHl lacLed areo4'bacþround required for clone stability'

However, cubsequent ¡tudiee by Metalanoe and oolleagues made it clear that surface exPfes-

sion of TCp and otùer oM¡¡ is dependentupon theV chobæ úoxR gene (Tylor et aI1987;

Mi'er & Mekalanoo 19ss). since no guch gene is present in, E coli, an alternative cloning

stratesrwas devised"

5.4 Molecular cloning of gerrcs invohad in TCP prcduction

S4.1 Clonhg stuat€' r

The innovation thatmade poosible the cloningof genee involved in rcP EÉthesiswas

the selection of avdtobæ strain, or7, as the recipient of a cosmid gene bank initially

constructed tn E. ætí. The O17 strain poss€ss€s thefaxR gene required for eryression of TCP

(Manning penonar communication), but does not produce these pili under cultural conditions

which prorcte TC? expression by strains of classical biot)"e (Table 4'1; Hall øaL 198ß)'

115

Moreover O17 tacks non-LPS protective antigens in general (Attridge & Rowley 1983c;

Section 3.4.1), facilitating detection of clones opressing such components'

V clplcræ z;llÍillwas selected as the donor st¡ain sinoe iteryresses TCPwellûn vúrro

Cfab1" a.1) and, being a recent field bolate, it also offered the poosibitity of cloning any other

non-LPS protective antþens it night PosEess (section 3.4.2\- Cosnid cloning an.d ínvütto

packagry of donor strain DNA into bacteriophage lambds (simon d aL t983) was used as a

meians of obtaining DNA fragments of sumcient size to clone the tc? oPeron'

5.42 Coretructionof the mobilizable coffiü vector pPM2llÌl

The cloning stratßg¡ adopted required a mobilizable vector, because of the need to

reintroduce cloned V cløbægenes via an E cdiintermediiaæ recipient back ntoV ùdøæ'

Accordindy, ¡[s 6obi lizable (Mob) reþn of the b¡oad host-range Plåsnid RP4 (from plasmid

psltp2gl-l) was introduced into the cosmid vector pHC?9. This constnrction is shou¡n in

Figure 5.4 de1¿ils of the construction afe g¡v.n in Section L17.1'

5.43 Cfoning tbe TC? genes into V clolerae O17

A gene bant wæ coDstructed by Paftiatty diæstiog t\e 217561 DNA (preparcd as

described in section zl7.l) with the restriction endonuclease san3L to obtain fragnents

approximately 40 lb in size, which were then cloned in to t\e BønHI siæ of the vector pPM2101

(see section 217.4). After overnigþt lþtion at 4 C the DNA miÚure was packaged into

bEcterbphage lamda (section Lll.Ð. The packaged pnaæ wefie then used to infect E coli

K12 strains 517-1, from which the cloned DNA was mobilized i¡to Vdtfuæ O17 by plate

mating (Section 2;17.6r. The resulting cosmid bank was stored on nutrient agu Plates

conøiningAP at 4 C

116

X'lgure 5.2 Constn¡ction of pPM2101. The cosmid pHC79 was cleaved with EcoRI and the

protrudingends filled usingKlenow fragments of DNApoþmerase I. Plasmid SUP201-1 was

cleaved with BantHI, the fragment containing the RP4 Mob region was isolated and tle

protruding ends filled as above. The cleaved pHCÍ/9 and the Mob fragment was ligated

overnight and the ligation transformed into ¡Eain 517-1 selecting for Teß. The correct.'

constructs were then selected by their ability to be mobilized into strain SM10 selecting for

fetR pnU ?r}l) KanR (counter-selection) agai¡ s¡ S 17- 1.

tt1

EcoRlPstl

EcoRl

Clol BômHtHñd

Soll

CoHnd

^Q:--è

BomHl

BE

Þ^

Pstl

ClolH¡ndlll

BomHlco9

IY

So

IV

Cleove with EcoRl.

End fill with Klenow frogment'

P3tl

Ligote.

Tronsform into stroin 51 7-1 ond select for Tet

Cleove with BomHl.

End fìll with Klenow frogment

R

RR

Select for mobilizotion into stroin SMlO screening for Tet ond Kon

(Ecoñl) MOB (EcoRl)

BomHl

Soll

let ñ

pHC79

ßl(ÞttQ

g\o

psuPz01- 1

cot (an R)

a'ofn,^

b t

ÉèE

oo

pPM2101

o¿ +

¿o'

144 IÞtectbn of Positive dones

About g{Ð oolonies were patched in duplicate onto CFA plates and incubated at25 C

for3óh topromoteTCp eryression (seeSection 4.3.2r. Oneeetof coloniee was transfered Ûo

nit¡ocellulose filters and þed în sín using the method of Henning el øL $r7\' Colony

blotfing (Section 216) with CP serum identifred 4 strrongþ- and 4 moderateþ-positive clones

from abankof abouts00 (Figure 5.3). These 8 clones atd2negative clones were deoignated

DS1-DS10 (containingplasnids pPM2lÛ2 to pPM2111, respectiveþ) and subiected tovarious

analyseg

55 Analysis of the clones DS1'DS10

55.1 nVf studiË

CIones DSl-DSlg, togetherwith O17 (recipient strai+ negative control) aúZl756l

(donor s[,ain, positive control) were inoculated onto CEA and incubated et 25 C for 3ó h¡*

EM examination of the hanrected bacteria rwealed that onty the four strongþ-positive clones

(DSZ DS4, DSs, DS6) eryreseed pili with the morphologr of TCP (Figuf€$ 5.4 e 5'Ð'

Indiyidual TCp filaments ?-10 nm in di¡meter were seen to aggrw tß closeþ in parallel to

form bundles 100-æ0 nm in width. The remaining clones and the O17 recipient strain failed

to pr,oduce euch pili under these growth conditions. The donor strain zllÍ6leho$'ed a higþer

level of TCP e:pression than the four O17 cl'ones'

IEM analysis confirmed that the CP senrm çp¡rainss antibodis which bound to the

pili produced by the four Ol? clones, as visuatized by the bindingof protein A-colloidal gold

particles (Figure 5.6 A & B). The pili produced þ clones DS| DS4, DSS and DS6 were ehoun

ro be TCp by IEM using LPS-abco¡bed anti0395 serum (Section 4.3.1 Figrre 5.6 C & D)'

Neither Eerum sþificantly labetled O17 or the other 6 clones.

118

Flgup 53 Colony blotting Appnoximately 800 colonies of a cosnid bank derived f¡om

Vc]øbæ /j756l (ClaEsical Inaba) were screened for tùe Presenoe of non-LPS antþn*

Figuf€ shor16 poeitiveþ reacting clones (indicated þ anowr) whilst * and - signs i¡dbaæ

poeitive (Zl7fil) andnegative (O17) oont¡ols.

119

I

I

!

I

i

.t

j¡

Flgure 5.4 hoduction of TCP þ clone DSZ Poeitive control (2,1756l) and clone DS2

(O17þPM21031) were grown on CEA (with Ap for clone) at25 C for 36 h, harvested in PRS

andappliedtogfidsforEMinspection(Section2l&1). AandB = Zl756t; CandD = DSL

The bars in A and C = ãX) nm and in B and þ = t(þ nm.

tm

È¡

Io

oa

,T

A {a

\t

'n

rt

Tr

"---

'

#

jtf-

IJl

tr

t't,,

:1

Ilgunc 55 hoduction of TCP þ clones DB5 and DSó. Clones DSs (OU[PPM2106D a¡d DS6

(O17üIPM21üID wer€ grour¡ on CEA (with Ap) at25 G harvested in PBS and tran¡fened to

grids for EM inspection (Section 213.1). A and B = DSS; C and D = DSó. The ba¡¡ in A

and C = 5ü) nm, and in B and þ = l(þ nm.

tzt

<T

AF

r+;ãl

F

V

T

V

--

¿

,ì

#o:

\ /, <'T

'¡tt'

--

Figurc 5.6 Immunogold electron microscopy of TCP produced by Clone DS5

(O17[IPM:¿1060. Clone DS5 was growa on CEA (with Ap) at 25 Ç and han'ested in PBS and

t¡ansfered to gdds for IEÌvl" Bacteriawere reactedwith the anti¡erum (anti-5698/165 CP for

A and B; LPS-abeorbed anti-O395 for C and D) and then protein A<olloidal gold as described

in Section Zl&Z The ba¡s oorrespond to 100 nm in eæh case.

tn

-oI

aI

o

.4.

a'/aa

t'ÈUota

oa

ç?aC;¡

¡ao

ti

it .:

t-'.a

O

a

a

I

a,

t

D a''a

o aao

a

a

ratl:-

a. Ia

aþl

TI

¡;, o

-

a

5.5¿ lrr¡mnobbttiryamlFb

For inmunoblottinganalysb, the ten o17 cloner, two positive cont¡ols (strains z1756l

and 5698) and one negative contror (o1?) were cultured on cFA at 25 c fo¡ 3ó h" The resulting

growth was ha¡rrested and the suspensions sorub'ized and subiected to Sffi-pAGE prior to

s/esterû brottingwith cp s€nrtrL In agreementwith the EM observations, onþ the Dsa Dgt'

DS5a¡dDs6cloneqtogetherwirh zl1s6latd56gB,wereposirivebyimmunoblotting(Figre

5.7). In each casg the presence of a 20 kDa pnotein' corfesPonding to the TcpA pilin subunit

(Þylot a aL 1987), w¿s detected'

153 Mohculnr ana[Ffs of cbned DNA

Inordertocha¡acterizetheDNAcariedbyth"spooitiveclones,cnrdeplasmid

preparationeweret¡ansformedinto.EætiK|2stfaitrsM10(seeSectiolZ|7.7,¡,whence

prasmid DNA sui'ble for restriction anarysis could be obtained (re. circumventing the

pr,oblemofDNAdegpdingenzymesproducedby V drcbæ)' This anaþb rwealedthateaph

of rhe 4 TCp-producingclones carried DNAwhich included theltbal fragmentr (Fþre 5'8)

characteristic of the region encoding TCp (D,yrot et d 19ssa). This suggested that moot of the

gpnes requiredforbioe¡nthesis of this pilus hadbeen clonedi¡to asingte coomid' Mobilization

of the plasmids back inlo 01? confirmed that the cloned DNA conferred all of the properties

originalþ associrated with the positive clones'

t8

Ftgurr 5.2 Inmunoblot anaþb of whole cell& llthole cells of the various strains wene grosin

on CEA (plus Ap for the clones) at}SCfor 3ó h. Theywere reruspended in electrophoresis

eample buffer and rubiected to SDS-PAGE The proteins were transferfêd to dtrocellulce

and the blots devel,oped by inorbatbn with 1:100 rabbit anti-5698/165 CP serum, foll'owed

by goat anti-rabbit Igg coupled with ho¡se¡adish peroxklase and ñnalþ eubstrat€ (Section

2.15.2). The sanplee are: A O17; 8,5698; Ç DS10; D DS9; E DS8; R DS7; G, DS6; H,

DS5; L DS4; J, DB3; K DSz; I, D81; M,z'17ft1. The arno$' indicates the poeition of the

molecul¡rr weigþt marker ß-lacrogfobulin (bovine nillç 13,400).

I?A

(-o

A BCD EFG HI JK L]'l

ü

Flgune S.t Restdction endonucleaoe analycb of pPM2lÛ3 (DS2 clone). 5ü) ngof DNAwas

digprtêdwithxbøI at37 Cfor t hr. Electlophoresis of the digest"d DNAwas carried outat

¡oom temperah¡e on horizontal 0.8% 4garoee gel The size of ¡estrictbn fragnents wel€

calculated by comparing their rel¡tive mobility wilh that of .&oRI digot"d fuíIltts st¿btilis

bacteriophage SPP1 DNA"

t25

oN=ô-o-

ÀÀat1

8.377 .20

6.05

4.90

3.55

2 .63

1 .731 .61

1.291.19

2\ kb

5kb

3kb

2kb

bÀÈ--.t

5.6 Confimúion of TC? as a úmlerre determirrant and

non-LPS prctective antigen ofV choletæ

5.61 IC" b a virulenæ deÛerninant

The availability of TCP-eryressing 01? clones provided an opportunity to confirm

prerrious conclugion¡ concerning the rrcle of TCP as a virulence determinant and protective

antþen in the i¡fant mouse model (Section 4.4 & 4.6). Three such clone$ we¡e found to be

o! average, seventy rimes more vinrlent than the O17 recipient strain and a TCP-negative O17

clone @ble 5.1), confirming that production of TCP is sufficient to enhance virulence- Itr

additiotr, protection tests indicated thatantibodies to rcP are suffrcient to confer protection

in this model

5,62 TCP b a protective antígen

The fi¡st eryeriment compared the capacities of TCP-pooitive and TCP-negative Ol7

clones to remove p,r,otective antibodie¡ þ absorption. Rabbit anti-5698Äó5 ¡erum was

abeorbed four times with TCP-negative Z1756l (grourn in NB ehaking culture at 37 C) in an

attempt 1q eþtain a serum whce protective activity was primariþ, if not exclnsiveþ, due to

anti:TCp antibodies. protection tests suggesæd that rhic aim had been achiwd since the

absorbedserumhadapDsoof l55again¡tTCP-negative ZlTÍúlbut4pg0agahstTCP-pooitive

7;1756l (gown on CEA at 25 C). Moreoveç the protective activity agrinst the CA4OI st¡ain"

which poßsesses non-I-pS protective antþns (Section 3.4.1) butdoes notproduce TCP Clhble

4.1), fell from 5,0(X) to ll0 as a result of these abnorptions. Five 2-mt aliçots of the abnorbed

serum were then firrther absorbed with one of the o17 clones or with 21756l as a pooitive

cont¡ol; the bacteria used for these abeorptions were groum on CFA at25 C to optimize TCP

erpression. Dble 5.2 shorrs that 2,17561 and each of three TCP-eryressing O17 clonet

t?ß

Ihble 5.1 hoduction of TCP enhances thevirulence oî'V dnleræOl7

Plâsmid TCPstaûsa VinrlencebSaain

ot7

DSz

DSs

DSó

DS9

pPM2103

pPM2106

pPM2tVt

pPM2110

+

+

+

2.82 x,0.10¡ ld5.66 t 0.40x ld4.38 t 0.13r ld3.Ð x,0.60 x ld3.61 t0.ßxld

" rcP stetns indicates the ability to produce TCP on CFA at 25 Cin?A\'

b Virulenoe is expreosed as the LD50 for infant mice; figur€s show mean t SE of 3 deter-

minations

tn

lbble 52 Absorpion s,ith TcP-expreseing o17 clones femoves pfotective antibodies

Abaorbingstrain

zl756t

DS2

DSs

DSó

DS9

TCP status hrotective activity vs Zl7 ftl

<n

<n

<n

<n

3980 t 130

+

+

+

+

' Th. antisemm uedwas raised in rabbite against the hybrid strah 5698/165 and had been

absoöed 4 times vtr\zllÍdrcr @roum in NB at 3? C) to learre residual protective an-

tibodieswhichwere directed primad¡y ngninstTcP' Alfo¡uots of thb eerumwhich han a

prrotective titer of 4290 t 3Ðwhen assõyedaginst Zl756lrcP+ organiemc,were then

fir¡ther absoröedwith the st¡ai¡s shou¡tr Residual protective acrivity against Zl756lrcP+

was then deærmined.

t8

r€movd all protective activity, wherea' the negative clonewas unable to do so. since the o17

host st¡ain has no non-Lps protective antigenq this result directly demonstrates the protective

efücacy of antibodies to TCP.

In a second experimen! the a¡ti:ICP sefum obtained above was tested for protective

activity "gainst O17 (negative contr ol), 277561 (positive control) and four of the clones'

challenge bacteria were gro!\,n on cFA to promote TCp production and tle results of the

protection assÍrys are shown in Thble 5.3. The absorbed serum did not protect mice from

challenge with o17 or the TCp-negativ eol7 clone, in support of prerrious results (section 4.7)

obtained using a different source of antibodies to TCP. Significantly, ho$'evef, protection was

oboervedagainstchallengewiththe TCP-producingOlT clones @ble5.3), shouringunequivo-

calþ that antibodies to TCP are sufficient for protection in this model'

5.6.3 ÏtCP-produßing Ot7 ctones ¡¡p irrrrunogenic

A final e4erimentwas designed to determinewhether immunization with TCP-produc-

',gorl clonee would elicit the production of antibodies to TCp. Rabbits were immunized with

three of rhe TCp-producingolT clones (DS¿ DS5 and Ds6), one negarive clone (DS9) or the

zr4ftillstrain as a positive control. The immunizingbacteriawere grou¡r on cEA at 25 C for

3ó h,,; afær confirmation of rcp production by Er.4 the grnwth was harvested, suspended in

PBS, and stored in aliquots at -?0C

Before assEnng the resulting aatisera for their capacities to protect infant mice from

challenge with rcP-producing z1756l, it was first necessary to absolb them to remcnre

potentialþ protective antibodies to ogau,a LPS. Thiswas achievedþ absorptionwith live o17

vibrios, and the success of the procedure is verified by the fact that the absorbed sera retained

no protective activity against o17 challenge (Iable 5.4). To determine whether the sera

t29

lhble 53 Antibodies to TCP are protective in the infent mouse chole¡a model

Cïallenge stlain TCP statusa hotective fte¡

zt156t

Z1756l (l.IB)

c4401

ot7

DS2

DSs

DSó

DSg

+ l?9O x,3fr

155t m

110 t 10

<n

4(þt4O

1030 t 94

510 t 45

<x)

+

+

+

stna¡cates the ability to produce TCP on CFA at25 C for Zh.

bff" protective tiær b the PDSO aginst tle various challenge strains' The anticerum uEed

was rabed in rabbig agninsl the hybrid strain 5@Wl65and hadbeen abßoöed 4 time¡ with

zflsa-I$p- Grown in NB atn crto leave residual protective antibodies which \f,'ere

directed PrinaritY ag¡in st TCP

130

Ibble5.4TCP-producingclonesareimmunogenicandprotective

Protective activity of Ol7-absorbed anti¡eraa vs

Immunizing

strain

zl756l

zn56tc

40,000 t 450

1,180 x. lN

1,740 x.VlO

1,570 t 110

<20

DS2

DS5

DSó

DSgb

ol7c

<m

<n

<n

<n

<n

u Rabbits \r,ere immunizedwith the strahs listed, and the resultingantisera ab'¡o¡bed four

timeswith live o17 (see text). lvhen the anti-Zl756lwas further abso¡ùedwith live

zl756lrcp- @ronn in NB at37 C) to remove antibodies to non-Lps protective antigens

other than TCR the PDsowas ¡educed to 25,9(Ð t 330'

b Th" pDso of the unaboorbed anti-DS9 againet o17 was ?.360 x, lgp,.

" challenge bacteria of the z1756lor o17 gtrains wefe cultured on cHl' at 25 G and each

protection test was performed twice'

t3t

contained protective antibodies directed against TC! protection tests were next performed

using TCP-pooitiv eZl756las the challenge bacteria'

The results of these assays @ble 5.a) indicate thatthe three TCP-producingOlT clones

generated protective antibodies to TCR whereas the TCP-negative clone did not Thus TCP

is imm'nogenic when preoented on the o17 clones and antibodies directed against tlese pili

are protective. The antiserum to zrlsîlcontained protective antibodies directed not onþ

against TCp but also against other non-LPS antigens. Further absorption of the ol7-absorbed

a,,¡_zl7s61 serumwith TCp-negative zlTsîlreduced the protective titerby 35To (to25,m)'

Even after this furtler abeorption, the protective activity of the serum was much higþer than

thoseoftheseraraisedagainsttheTcP-producingolTclones,presumabþreflectingthelouler

lerrel of TCP e:rpression by the l'atter'

5.7 Discussion

Initial attempts to clone non-LPS prrrtective antigens rrcmv dtolsæ lm;toa coliwere

unsuccessful In ret¡ospec! the approach adopæd was inappropriate for the cloning of rcP

since it is nou, known that production of TCP requires a functional tuR gene (Taylor er øL 1987)

which would notbe present n E. coli. eccordingly, a nell' cloning strategrwas de*isedwhich

involved the ch¡omosomal integration of a mobilizable plasmid" A cosmid gene bank prepared

fromv cttorqæ zrlsilrwas mobilized into v ehoreræ El ror o1z viln at E coli intermediate

recipientstrain. This was achieved usinga specialþ constructed mobilizable plasmid pPM2101

(Figure 5.2), which contains al.ilkb&antHl fragment encodin gthemob region of RP4' Miller

& Mekalanoo (19gs) recently employed a simila¡ method to introduce DNA into bacterial cells

in a nonreplicating form, to demonstrate úat úoxR regulates tle expression of cholera toxin'

TCP and some oMPs in response to certain environmental sþal* v cholqæ st¡ains z1756l

132

and olzwere selected as donor and recipient strains for the reasotrs e¡tlined above (Section

5.4.1).

The plasmids carriedby the four TCP-producingolT clones contain the 2kb, 3lb, 5tb

a¡dÐlú lftøI f.n,penß which characterize (Taylo r et al.19S8a) t\etcp oPeron (Figure 5'8)'

In addition, when present i\ E coli,these pl,asmids reactwith atcp,A-specific oligonucleotide

probe, confirming the presence of this structural gene (R. Faas! U.H. St¡oeher & P'A' Manning

unpublished data). Together these finding eliminate the possibility thatwhathas been cloned

is the foxR reguratory gene uponwhich TCp expreesion depends (Taylot et al- 1987). Moreover,

theþxR gene has recentlybeen clonedandshows adifferentrest¡iction pattern (P,ru Manning

personal comm unication).

Althougþ EM and immunoblottinganal¡rees har¡e failed to detect TCP expression þ El

Tor stfains (HaU et al- 1988; Section 4.3.3),Taylor et d (198fxù found that each of five clinical

isolates of this biotype carried DNA to which a TCp probe could hybridize. using pPm2103

as a probe for the fcp operon, it has recentþ been established lhatv choløae ol7 and three

ottrer El Tor s,fiains also poosess homologous DNA (U.H. Stroeher & P.A" Manning un-

published). Further shrdy of the plasmids cloned here will be aimed at elucidating the interpþ

between plasmid and chromosomal gene$ which results in TCP production by the o17 strain'

It remains feasible that the plasmids " *ya functional regulatory element other than þxtrl such

æ tcpT (Iaylor et øL lgßa) and that this complements a defective - or repliaces a missing -

controllinggene in v cholqaeol1. Alærnativeþ, the "defectnwhich prevents TCP production

by o17 (under grorvth conditio¡s examined to date) might lie in a structural ggtrê, thougþ a

simple gene mutation would not explain the failure of all El ror st¡ains to produce TCP Finally

it is pocsible that the TCp region can unde¡go rearr¿rngements of the type recently observed

with the genes speciffing pilus production n Morax¿lla bovfu (Marrs et aL 1988)' "nd

that El

Tor strains harre become locked into a non€xPressing mode.

133

The TCp-producingOlT clones permitted further evaluation of the significance of TCP

as avirulence determinant and protective antigen in the infantmouse model The demonst¡a-

tion that TCp expression sþificantly increases tle virulence of o17 (Table 5.1) confirms the

original demonstration of the critical involvement of these pili itr infant mouse pathogenesis

(raylor et d19s7). consistentwith tle role of TCP as a colonization factor (xaylor er aL 1987;

Herringto n et aL 19gs) is the finding that antibodies to these pili are protective, presumabþ

by inhibiting adherence to the intestinal epithelium (Attridge & Rowte'y 1983c)' Thus onþ

TCp-expressing o17 vibrios could remove protective antibodies þ absorption (Table 5'2) and

generate such antibodies upon immunization (Table 5.4). Antibodies to TCP are sufücient to

mediate protection against challenge with TCp-poeitiv ezl756lor o17 (Table 5.3), confirming

previous results with the 5698 and 0395 strains of v c]øteræ (Table 4.3). The protection

obeerved asainst the TCp-positive o17 clones is particularþ signiñcan! as earlier experiments

indicated that antibodies to TCp were not protective against challenge strains of El Tor biotype

(Table 4.3), which fail to express TCP under the growth conditions examined to date (Table

4.1; Hall et at. tg88). T'rme did not permit a study of tlose moderaæþ-positive o17 clones

detected þ coronyblottingwhich did not produce TCp Erryeriment' afe planned to determine

whether these clones eryress noniTCP non-LPS protective antigens'

L34

General Discussion

6.1 Distribution of non-LPS pmtective antigens ofv cholere

pretrious studies with the infant mouse chorera model have demonstrated the existence of

non-LPS protective antigens i¡ v cholqø¿. Antibodies to some of these ¿"lsrminants were

shown to poesess protective capacity (Neoh & Rowley lno, L972; Beltamy et øl' 1975) and

their protective efficacy was greater than that of anti-LpS antibodies (Attridge & Rowþ

19s3c). The presence of non-Lps protective antigens on at least one v dtolqae strain (5698;

Attridge & Rowley 1gg3c), made it critical to determine the distribution of these components

âmongfeoentfield isoliates. If such st¡ains sharednon-Lps proæctive antþens, itwas feltthat

further study of these antigens miÈt errenhrally identify ne$' componenB of vaccine sig-

nificance.

ItwasshowninChapter3thatVcholqacofeitherbiotypeandofbothcommon

sefotyPesexPfesscommonnon-LPSantigens,althougþfouroldlaboratoryElTorstrainslack

these determinants. The reason for this restricæd distribution is unclear' It is unlikely that

these determinanb have been lost from Er Tor strains during prolonged storage in the

laboratory since classical stfains that were stored in the sÍrme ¡nantrer still express tlem'

Perhaps expression of non-LPS protective antigens by the neu, El Tor strains resulß from

environmental interactions between tle two biotypes (Section 3.7). Nevertheless, further

135

I

',1

,ll

!

Ilà

ül1

studyoftheseantigenswasclearþwarranted.SDS-PAGEandimmunoblottinganal¡nesfailed

to identi$ a consistent difference in tle oMp profiles of strains expressingor lackingnon-LPS

protective antigens and attempts to charactenzæthese determinants were unsuccessful (sec-

tion 4.2).

6.2 Role of TCT as a colonization factor and a prctective

antigen

An earlier study of the antibacterial properties of poþclon al antt-V chobræ sera sougþt

to define a consiste ntin vito correliate of antibody-mediaæd protection in the infant mouse

cholera model It was found that the capacity to directly block the i' vitro attachnent of

v chorqæprwided such a correliate, whereas the capacities .q immobilize or agglutinaæ the

bacteria did not (Attridge & Rowley 1gs3c). Experimenß in this thesis have confirmed and

extended this ñnding Each of three sources of antibodies to non-LPS protective antigens

inhibited the in vírro adherence of strains poesessing such determinants (as deñned by proæc-

tion assa¡n). In contras! these sera did not protect infant mice from challenge with, nor block

the attachment of, old laboratory st¡ains of El Tor biotype (Section 3.6.2). The implications

from such data is that at least some of the non-LPS protective components may Play a role in

colonizatiot inviw.

Numerous studies attest to the significance of pili as colonization facto¡s for a variety

of bacterialpathogens, i¡cludingnoninvasive enteropathogenswhich establish toxin-mediaæd

diarrheal syndromee similar to chorera (section 1.3.3.4). Reports publishedduringthe couße

of this thesis resoþed the controversial issue of of wheth et v cholqæalso produces pili (Ehara

4 aL rgfr6; Taylor el aL rggl; Hall et aL lg8l iJ). The e:rperiments of Taylor d aL (1987) ìvefe

of particular relevance, identifying one pilus type - rcp - a virulence determinant in the infant

mouse chorera model Attention therefore hrrned to assessingthe significance of rcP.

.tI

,{

ùì-

It'

tt

''l

I

\t,

)

I

i

#

#

I

L%

Initial work involved optimizing gron'th conditions for TCP e:rpression, and performing

EMandimmunoblottinganaþef to determinewhetrertheorpressionof rcP correlatedwith

the expression of non-Lps protective antigens. These studies ¡ervealed that ín vítro TCP

production (folloving græ,th on cFA) was obsewed onþwith the classical strains (with the

exception of cA401) and notwith the El Tor strains (Table 4'1; Fþres 4'7 &' 4'8)' That is'

whereas non-LPS protective antigens were detected on the neu'El Tor strains or'v dtolqac'

this was not the case with TCp expression. This suggests that TcR arthougþ a major non-LPS

antigeno is not the only protective non-LPS antigen' This point will be discussed further

(Section 6.4).

Subeequent experiments confirmed the sþificance of TCP as avirulence determinant

in this model It was found that non-motile 5698 vibrios were over 100-fold more vinrlent if

culurred under conditions conducive to TCp expression (section 4.4). Eryeriments described

by Trylot d al. (1987\clearþ shou,ed that production of TCP conferred a significant advantage

in terms of persistence in the infant nouse guL Moreover, Herrington et øl' (1988)

demonstrated that TCp expression similarþ promotes coronization in humans. when volun-

teers were challenged witì virulent classical 0395 those who had been previously fed with a

TCP+ryressingstrainwereproæctedagaingli¡'esswhereasthosewhoreceivedthenonjTCP

e:rpressing st¡ain were not protected

þ analog with the pilus colonization factors of ETEC strains (Iærrine er al' 1983), it

was p,redicted that the pivotal role of rcP in vibrio colonization would similarþ confer uPon

these pili the status of protective antigens Although this remains to be demonst¡ated for

human infection, the experiments described in'his thesis show that this is indeed the case in

the infant mouse cholera model. The protective efficacies of antisera shown 1e çoatain

antibodies to TCp by IEM were dramatically increased if the challenge vibrios were TCP-posi-

tive (tble 4.2). one of these sefa was then absorbed to obtain a serum whose prolective

J

,{

ñ*

It'

I¡[.llrì

lrl

tt,

I

I

1

t37

If

f^-

activitywas prinariþ, if notexcrusivel¡ due to antibodies to TCp (section 4.7). This residual

sefum was still highly protective against challenge strains of classical biotype, but did not

protßct ag¡i¡st Er Tor vibrios which fail to produce TCp under the growth conditions tested to

date (Table 4.3).

These initial erçeriments provided the first evidence that rcP is a proæctive antigen

of v chorøae. unequivocal confirmation of this point carne from subsequent studies which

followed the cloning of genes invoþed in rcP production'

63 Molecr¡tar cloning of genes involved in the qynthesis of TcP

AnattemPttodefinethenon-LPSp,roæctiveantigensorVchoba¿involvedthecosmid

cloning of DNA prepared from a recent ñeld isolate, using a cloning stratery discussed earlier

(Section 5.4). This approach resulted in the cloning of genes involved in TCP production and

eryression of these g"Des hv cholcræ or7 , astrain which norma[y l,acks non-LPS protective

antigen* TCP-producing o17 clones lltere' on average' seventy times more virulent than the

unmodifiedrecipient strain (Iable 5.1) confirmingearlier findings' MostsþificanÙ' a sefum

containing antibodies to TCp was shown to protect mice from challenge with rcP-positive 017

clones thougþ not from challenge s'ith (rcp-negative) o17 (fable 5.3). This pmovided une-

quivocal confirmation of previous experiments which had suggested that rcP is a protective

antþen in this model Not surprisingly, the TCP-producing O17 clones' but not O17' could

remove protective antibodies by absorption (fable 5.2) and upoû immunization elicit pfotec-

tive antibodies specific for non-LPS compotretrs (Table 5'4)'

It'

I

I'i

138

6.4 Is TCP the orrly non-LPS prutectirrc antigen ofv drolerae

in the infant mouse model?

TheprotectiveefficacyofantibodiestoTCPraisedthequestionofwhelherthisantþn

is the onþ non-Lps protective component in this model. This was particularþ important

because of the finding that at least one of the antisera used to determine the distribution of

such components among v choreræstrains was found to contain antibodies to TCB despiæ the

fact that the bacteria used in the preparation of this serum had been grolvtr under conditions

unfarrorabre for TCp production (Section 3.5.1). Several rines of evidence suggests that

VchotøæpGs€ssadditionalnon.LPSprotectiveantigens,however.

First, the capacity to synth esizcTCP does not correlatewith the pattern of elpression

ofnon-LPSprotectiveantigensAlthougþthelattercomponentsarefoundonrecentfield

isotatesofElTbrbiotype,suchstrainsfailtoproduceTCPasjudggdbyEM,immunoblotting

and protection e:perimenß (Sections 4.3 & 4.ó). Theee results are consistentwith the presence

of non_TCp, non_Lps protective antþens, at lea^st on recently isorated Er Tor strains. Secon4

the existence of sefotype-fest¡icted non-Lps prctective components similarb¡ afgues against

TCP being the only such antþen, since TCP synthesis is not serotype related. Thir4 the C4401

strain possesses non-LPS protective antigens (Table 3'3) yet is atypical "molg strains of

classical biotype in that it fails to produce rcp rt viúro (section 4.3.3). Finatþ, t\e z1756l

st¡ain e'identþ possesses non:TC! non-LPS protective antigens (table 5'4)'

65 Non-LPS protec{¡Ye antigens: relerant shrdies in other

laboratories

Theerperimentsdescr¡bedintlisthesishaveidentifiedTCPasthefirstnon-LPS

protective antigen or. v cholqae, at least in the infant mouse model' workers in several

I

I

139

laboratories are shrdying the nature of vibrio colonization, as colonization factors would be

prime candidatee for protective antigens'

The important contributions of Tayloç Mekalanos and colleagues have been referred

to througþout ttris thesis, and represent a maior step forward in the quest for an improved

choleravaccine. This group has recentry puriñed rcp from a flagellum-negative derivative of

V chobæO395 and shorn n that a resulting antiTCP Eerum wÍ¡s higþly protective in tle infant

mouse model, confirmingresults obtained in this thesis (Iaylor ef øl 1988b)'

To date, TCP remains the onþ defined adhesin orV eholøøe (althougþ there is some

evidence to suggest that Lps can promote attachment - section 1.3.3.2). peterson & Mekalanos

(lgss) identified naccessory colonization factorsn in the infant rnouse model þ studying

TorR_regulated getres n v cholqae. Tiansposon insertions in any of a cluster of four genes

resulted in a much reduced capacity to colonize the i¡fant mouse gut thougþ the effectwas

less dramaticthan thataccompanyingdeletion of genes encodingTCp (peterson & Mekalanos

198s).Thenatureoftheaccessofycolonizationfactorsremainstobedefined.

Recentstudies have investigated the role of HAs hv chotqæ colofiwation' Ftn¡e| aL

(1gg7) constnrcted a mutant El Tor strain which l,acked the mannoee-sensitive cell-associaæd

fIA (Hanne & Finkelstein 1gg2) and found that this mutant showed a markedly impaired

capacity to cotonize the rabbit ileum. Nevertheless, oral immunization with the mutant

p¡otected rabbits from subsequent challenge with virulent v chorøae (Finn er ar 1987\'

AlthougÞ this would seem to indicate that antibodies to ^h i s rIA are not necessary for protection

h this qrstem, furtrer studies wourd be needed to eriminate the poosibility that the mutant

produces an inactive HAwhich retains the potentiar to elicit neutralizing antibodiec

Tepp"ma eJ øL (lggi) reported tlat a motile, (mannose-resistant) HApositive, El Tor

strain and an aflagellate HA-negative, classical st¡ain showed simila¡ capacities to colonize

ligated ileal loops constructed in adult rabbits. on this basis itwas concluded that factors other

140

than hemag€rutinating activity or motility are invoþed in colonization in this model. This

illustrated the importance of the erperimental s¡ætem in shrdies of vibrio pathogeneeis, as the

conclusion reached by Teppema e t at (Lgsi)conflicts with the results of several earlier shrdies

attesting to the significance of the flagellar st¡ucture in colonization (see section 1'3'3'1)'

Attachment to the intestinal surface evidentþ occurs more readily in a statig closed system

such as a ligated gutloop; indeed, some cholera moders require a drug-mediated inhibition of

peristalsis (Freter 19554; Finn ¿f aL 1987)'

Duringthepastdecade,sevetalgoupshaveadoptedtheRITARD(removableintes-

rinal tie - adult rabbit diarrhea) model developed þ spira et aL (1981) to shrdy infection by'

¡¡dimmunityto,Vcholsæ.Theoriginalprocedureinvolvedthetemporaryobotnrctionof

the smallbowel to promotebacterial colonization and the permanentligation of the cecum to

prevent fluid resorption, although modifications to this s¡rstem have been descrbed (Guinée

et aL LgËÐ. colonization facto¡s and protective antigens have not yet been defined for the

RT'ARD moder. Li,ev crølqæimmunize much more effectively aeainst suboequent chal-

lenge ¡¡¿¡ killed bacteria (Guinée et aL 1987; Riipkem ae,aL 198?; Pierce et al' 1988)' with

the capacity for mucooal association pnrviding the onþ correlate of protective effrcacy (Pierce

d aL rggs>. protection in this moder is associated with a marked inhibition of intestinal

colonization (Guinée 4 aL rggÍ; þcke et at- rgÍ36: pierce et øL tgSs), bur the speciñcity of

the protective antibodies remains unknoç'n'

Svennerholm et¿l have found that intestinal immunization with rough Vcholeræ

mutants can protect rabbits from subsequent challenge with fuþ virulent smooth cholera

vibrioe in the abeence of any anti-LFS antibody respome (cited by Jonson eJ aL 1989)' Thit

suggests a protective role for nol-LpS antibodies. This is in contrast to their earlier observa-

tions indicating that protective capacity in the rabbit model resided exclusiveþ in anti-LPS

antibody fraction (Svennerholm 1gg0), a finding that seemed to harre been corroborated by

141

Jansen et oL (lg8S). Nevertheless, this recent work þ Svennerholm (cited þ Jonson ef øl

l9s9) extends the notion that non-Lps protective antigens might be important ' o,¡"¡ ¿¡imal

models besides the infant mouse model'

In 19g3 Sciortino & Finkelstein reported that during growth in the intestines of infant

rabbits, v choleraedisplay a different ¡nttern of oMps from that characteristic of organisms

culhrred ìnvitro. This important finding focuses attention upon antigens expressed during

gro$,thín vivo,aptentiallyreu,ardingapproachforinvestigatorsinterestedinvaccinedevelop-

ment Recentry Jonson et øt. (rgEg) confirmed that novel proteins are producedby v choleræ

during gowth in the (adult) rabbit gut, and went on to shou' that at least some of tlese are

immunogenic" of interestwas the finding that several of the proteins induced antibodies that

were detected in convalescent phase sera from cholera patients (Jonson et aL 1989)' Howeveç

none of these novel proteins aPpeaf to be TCP as kvúto growth under conditions conducive

to Tcp erpression did not induce any of t\e invirao-specific proteins nor did anti:TCP serum

rcactwitl these proteins. clearþ it will be of great importance to define the antigens

qrnthesized fi V chobae during growth in the human gut

6.6 lhrhrrc studies

6ó1 Non-LFS proÛective antþns of El Tbrv cloþrae

The data presented in this thesis are consistentwith a biotype-associated expression of

TCP, but further studies in this anea ane required" Althougþ EM and immunoblotting ap

proaches failed to detect production of such pili by strains of El ror biotype, this migbt mereþ

reflect the fact that the s''nuli for TCP s¡rntheeis differ between the biotlpes. As discussed

prerriously (Section 5.7'¡ anumber of El Tor strains have now been shown to carry DNA

homologous to that encoding TCp. Taylor d aL (rgssb) have recently reported 807o sequence

homologr between fcpA genes present in the classical strain 5698 and the El ror F,1946'

142

potential antigenic epitopes were found to be hlghly conservd suggesting that protective

antigens niÉt be shared between the pilus stn¡ctures eryressed by the two biotypes (Taylor

eføl 1988b).

The protective efficacy of antibodies to TCP was evaluated against a panel of challenge

st¡ains to address the possibility that El ror strains synthesize rcP rn wvo. These experimenb

also shoured a clear, biotype-related dichotomy horve'er, in trat such antibodies onþ conferred

protection egrinst bacteria of classicar biotype (T"ble 4.3). It should be noted tlat proÞctive

titers against classical strains are low unless the challenç organisrN afe expressing TCP (as

is necessarily tre case with El ror strains). An independent means of assessing the in vivo

signiñcance of rcp for El ror v choleræwould be to construct strains carrying inactivated

tcp¡genæ(forexample) andtocomparethe colonization of suchstrainswith theircorresPotrd-

ingwildtypeparents.SucheryerimentsarecufÎetrtlyinprogress.

In addition, an attemptwillbe made to clonethe non-LPS protective antigens of recent

El ror isolates, using the approach which allowed cloning of genes involved in TCP erpression'

Any cloned components will be investigated for their capacities to promote in vito attachment

a¡d ínyira colonization. IEM will be used to examine their di¡tribution ovef the bacterial

surface, with particuliar interest in the poosibility that El fe¡ strains produce a biotype-as-

sociated plus stnrcture as suggesæd ry :Hail etal (19gg). As already discussed (Section 6'4)'

eryide¡ce suggests r\at v choraae erytess nonJTC! non-I-pS protective antþens and this

approach could lead to their identification In this context ttre four po'sitive 017 clones which

wereTCP-negative(Section5.5.1)meritfurtherstudy.

6;62 Th nahrc of TCP'rediatßd attachrent

The speciñcity of bacærial adherence is imparted þ the recognition of target cell

receptors by bacterial attachment factors. Evidence is accumulating to suggest that the actual

r43

adhesins of gram-negative bacteria consbt of minor protein molecubs at the tip of the surface

pili rhere is nor¡ ample eryidence that gþcolipirts can act as recepûo¡s for bacterial adhetins

(revieured þ I-effler & svanborg-Eden 1gs6). Adhesion of piliated E- øli to host cells has

been shown to be mediated by pilur-"ssociated adhesins of the Pap pilus not þ pilin subunit

pøffi(uhlin a al.1985). Th" pßotein associatedwith adhesion' has been found exclusiveþ at

the tip of the pilus and ie a 35 kDa protcin (PapG; Normarkø al' 19ú; Lindbefg¿ú al'1987)

that attaches to the gfoboeeries of gþcolipids with the recognition siæ being Gatcl4€alß in

human urinary tract epithelia (t-efiler & svanborg-Eden 1936)' Other enmples of lectin-like

tip adhesins produced þ E coti a¡e a r2tDa pnotein of s pili that recognizes sialic ackl (Moch

et aI. tg87) and a 29 kDa plotein produced by t',Pe 1 fimbriae which mediates attachment to

D-mannoce (Abrahan & Beachey 1988)'

It is not tnou¡n whether the attachment of ll clplsæ to hoet cells i8 mediated þ similar

adhesins,whichmãybveryming¡componentsofthepilue.ByanaloglwithltcolÍ,such

proteins nist harre a criti€t role in adheeion oî' v choløæ to host cells' Based on the

organization of genes in the Pap operon (Lindbergdll 198Ð, Taylor etaL (1988a) have

pnopoeea a model for the gene clusrer invoþed in the biosS¡ltheris of rcP (Figure 6'1)' The

tcpA geneproduces the naior ¡ilin subunit, witb lqG rcpænting a possible tip adhesin'

Furthe¡ molecul¡ar and biological an ryses invoMng the constnrction of defined mutant strains

ie required to elucidate the nechanism of rcp-mediated attachmenL such studies will alloril

the design of vaccines aimed at inhibitin g v cholaæ colonizatio¡ þ eliciting antibodies

capable ef ¡s¡t¡alizing the binding potential of TCP'

The nahrre of host receptors invoþed i¡ V cløteræ adherence ¡pmains undefi¡ed'

T¡ylor etal. (1987) hate shown rhatvchotqæ 0395 eryressing pili could hemagglutinate

nurine RBC8 thus demonstrating that the pili apparentþ bind to receptors on the surface of

hostcells. The nature of the receptorwas notidentified but the aggtutinationwas detected

144

Figune 6.1 hoposed organization of the genes associated with the production of the TCP

pilus; (Taylor er ø1. 1988a). T\e hal fraÊment containing /cpG does not appear to be

immediateþ contþous with the remainder of the genes shown here.

145

Xbo

rN)

lx-

LoI w () rl-

]-T

-l

Xbo

l

bol

X

C)

Xbo

l

in the presenoe of L-fucoee, a sugar that inhibits most of the hemaggfutination mediated þ

v clplqæo3gs (Jones & heær rg76; Jones 1gs0). one should be aware, hm'ever, that the

characteristicsofbindingtoRBCsnigþtdifferf¡omthenah¡reof thebacterialinæractionwith

moße relevantsubetrates (Attridge & Rowley 1983b)'

6.63 \haine Potentiat of the TC?

It seems probable that future studies wilt sho$' TCP to be a protective antigen for human

chole¡aic infection. Txybr da¿ (rgssa) harre úeady suggested the incorponation of rcP into

the oral ki¡ed whole cell vaccine devel,oped þ Hotagfen's gloup (Section l'10'2J2\'u'hich

would presentþ contain [ttle if any of thiß antigen. Thb night lead not onþ to an imP¡wement

in proæctive etñcecy but also to a reductioa in coeü if the preeence of TCP permin at ba¡t a

ûnnsþnt associ¡rtion of the kilþd bæteria with the intestinal surflce i1 migþt be pcsible to

¡educe the number of bacæria per dose'

otherwa¡n to eryloittheprtative protectivepotentialof TCP include thedevelopment

of a TCP-B subu¡¡it coniugrte (faylor et aL'tgïsa) or the delivery of TcP otr atr atlenuated

carrier etraitr such as s. typhirf2la. The latter could also eryress the pnotective o-antþens'

as p,revio'sly di¡cu$ed (Section 1.10.23)' hesumably rcP would be synthesizdhvivoby

attenuated live oral vaccine candidates, althougþ the onþ data ar¡ailable to date suggest that

suchstrainselicitanti:TCpresponsesinonþasmallminorityof recipients (I-evinea 41 1988b)'

Itwould be of interestto compare the immunogenicity, protective efñcacy and reactogencity

of va¡iou¡ doeeß of TCP-pooitive and TCP-negative non-toxþnicV ffi¡w'

The impact of incorporatingTCp into poæn*ial choleravacci¡e candidates is ãs'aited

with great interesL As alrcady discr¡sse4 it is poosible that any improvement in protective

efrcac,ywill be limited to infecti,ons causod by cra$ical v ùotqae. It would nerreÍìeless

eppear thatan efiectivecholeravacci¡e b æn¡ider¡bly clæer than itwasiusta fen'years ago

tß

BibliographY

Abraham s.N. & Beachey E H. (1gs5) Host defenses against adhesion of bacteria to mucosal

surfaces. Adu. Host Def' Mech 4: 63-88'

Abraham s.N. & Beachey E H. (lgss) Binding of bacteria to mucosal surfaces. Monogr

Allerg A'- ß43'

Achtman M., Schwuchou¡ S, Helmuth R, Morelli G' & Manning P"{ (19S) Cell-cell

interactions in conjuga rngbrchøichía cori: con- mutants and stabilization of mating

aggtegús. Mol¿¡. G4' &n4' lúf: 171-183'

Adhikari p.c & chatterjee s.N. (1969) Fimbriation and pellicle formation olwb¡úo El ror.

Indinttl- MeA 57: 1897-1901'

Al_Kaiesi E, & Moetratos A, (1gs5) .preparation and properties or.vibrío cholqæ aof'fimbnal

antibdy. J. AppL Bøctqiol Stz 2l-229'

Alm RA, Braun G., Morona R. & M¡nningP.A" (19S6) Detection of an ompA-like protein

nwbr¡ocholeræ.FnusMigobioLl-cfl.37;99-104.

Attridge SR. (199) Infection by, ¿¡d immunity to, Wbt¡o chobæ' PhD thesis' Adelaide'

Ar¡stralia: The University of Adelaide'

Attridge s.R. & Rowþ D. (19S3a) The role of the ftagellum in the adherence ollñhrio

chobae- J- InfeÃ- Dús' l.47z 8&A7Z

147

Attridge s.R. & Rowley D. (19s3b) The speciñcity of wbrio choleræ adherence and the

significance of the stime aggtutinin as asecond mediator of ínvtto attachment J' Infea

Dis. t4ilz 873-881.

Attridge s.R. & Rou,ley D. (19S3c) Prophylactic sþificance of the nonlipopoþsaccharide

antigensofWbríoeholcrac.Ilnfect.Dls.l4E:93|.939.

Baine lV.B., TampienA., Mazzotti M, AngioniG., Greco D', Di Gioia M"luþF^' Gangarosa

EJ. & Pocchiari E onq Epidemiolog of cholera in Itâly in 1973' Lqtcet' 2z

1370-1374.

Banwell J..G, Pierce N.E, Mitra R.C, Brigham I(L, Caranasoo GJ" KeimowiU RL' Fedson

D.S, Thomas J., Gorbach sL, Sack R.B. & Mondal ,{ (19?0) Intestinal fluid and

electrolyæ transport in human cholera. I. Cl¡r\^ Inve'Í. 4Ì 183-195'

Bart trCJ., HuqZ.,Khan M. & Mosley \il.H. (1970) seroepidemiologic studies during a

simultaneous epidemic of infection with El ror ogava and classical Inaba víbrío

elpbøe. J. Infed. Dús Lllt S17-SZ['

BaruaD.&Cbatteriees.N. (1964) Elect¡onmicroecopy of$lTotlnbriocholøaa Ind'IMed'

Rcs. 52:828S30.

Barua D. & C\ietanoic B. (1970) cholera during the period l%l-ln}' lrlz hh"ípl6 a"d

præticeof choløøcqúrol Public Health Papef No' 40' \il'H'OT Gener¡a pp' 15-21'

Bauman P., Furniss A.L & I-ee J.V. (1984) The genus whño. In: Bergey's manual of qrstemic

bacteriologl.BtN.R.KriegVolumel.,lsteditionpp.5lS-538.

Bellamy J.F-G, Ihop J., Steele E J., ChaicumPa \il. & Rorley D' (1975) Antibody cross-link-

ingasafactori¡immunitytocholeraininfantmice.t.Infed.Dis.|321181-188.

Benenson ,{.S., Islam M.R & Greenougþ Itr W.B. (1964) RaPrd identification of Víbrto

cholqaebydartûeldmicroscopy.BulLWldHtthorg.*'8n.83|.

148

Bhalla D.K & Or¡¡en R.L (1gs3) Migration of B and T þmphocytes to M cells in Peyer's

patches of the mouse after neonatal thymectomy andhydrocortisone iniectio¡' Am' J'

Attol. l¡SLz TZ7-88.

Bhaskaran IC (1g1) Isolation of hybridvibrio strains for immunological research in cholera

l'ù; hoceedinp of ø sympaium on búqìalvaccíne* Ed" B. Gusic Tagteb,Yugoolavia:

Yugoslav Academy of Arts and Science' pp' 2A7 -?ßl'

BienenstockJ. & Befus A.D. (1930) Mucosal immunologr. Immunologt' 4lz ?A9-Z7O'

Bienenstock J., Befus A.D., McDermott M., Mirski s., Rosential Ic & Tagliabue A" (1983)

The mucooal immunological network compartmentalization of þmphocyteq naürral

killer cells and mastcells. .Ann N.YAcad sci 4t9z 164-170'

BlackR.F^ (19Só) Theepidemiologrof choleraandenterotongeúcBæhøìdtíacolidianheal

disease.ln:DettelopnølsofvaccinesmddrugsøgainsdiøtheallthNobelCon-

ference, stockholm, 1935. Studentlitteratur, Ilr¡4 Sreden

Black R.F^, I¡rrine M.M., Clements M.L, Young CR', $ennerholm ^À-lvf" & Holmgren J'

(19g2) protective effrcacy in humans of killed whole-vibrio oral cholera vaccine with

and without the B subunit of cholera toxin. hfecL h¡vnun 55: 1116-1120'

Blake P.A (19S0) Diseases of humans (other than cholera) caused by víbríos' '$n Rcu'

Misobbl 342 Al-367'

Blake p.¡u, Allegra D.T, Synder J.D., Barrett TJ., McFarland L, Cararray cT, Feeley J'C'

craigJ.P.. Lee J.V, Puhr N.D. & Feldmen RA. (1930) cholera - a pæsible endemic

focus in the united States Nen'hg.I. Med 3ll2: 305-309'

Blake P.,A,., Rooenberg.IU-L, Coeta J.B, Ferreira P'S', Guimaraes CL & Gangarosa EJ'

(1y77') Cholera in Portuga\ ln4. I. Modes of transmission Am I' Epidøtiol 1:oSz

337-v3.

149

Booth B.A, & Finkelsæin RA, (19só) Presence of hemagglutinin/protease and other poten-

tial virulence factors in 01 and nono lvibrio chobae L InfæLDis' 154: 183-186'

Booth B.A, Sciortino Cv & Finkelstein R..¿r. (1986) Adhesins ol Víbrb eholqae. In:

Míqobíallcaíw øn¿øglutínírls- E¿ D' Mirelman' Neu' York: Johq lViley & Sons' pp'

169-l82

Bornside G.H. (1931) Jaime Ferran and preventive inoculation against cholera fulL lûst'

Md. 55: 516-532

Bornside G.H. (1gs2) waldemar Haflkine's choleravaccines and the Ferran-Haffkine priority

dispute. J- HisL Men- Ahied Scí 372 39942lz.

Bourard c & I-ecroisey ,{. (19g2) specfic antisera produced by direct immunization with

slices of poþacrylamide gel conaining small amounts of protein' I' hrannoL Meihods

sÈ, m-nß.

Bourke.ATC, Cossins Y.N., Gray B.R.W., I-unney T'I', Rostron N'4" Holmes RV' Gfiggs

ER,I.arsenD.J.&IGlkvR.(198ó)Investigationofcholeraacquiredfromthe

riverine environment in Queensland' Mel- I'Atts1, t4Él. 229-234'

Brandtzaegp (lgsÐ Rore of J chain and secretofy component in receptor-mediated glandular

and hepatic transport of immunoglobulins in man. scotd I ínunnol' 222 111-14ó'

Brayton P.R, C-ohtr'ell R.R, Tall 8.D., Herrrington D' & Leryine M'M' (1937) Recovery of

wfu¡o cttoløæ 01 from stools of human volunteers fed non-culturable cells' In:

Abstruds of 23rd II.S.-lrym Choba C-onføence, Wíllia nsbta. p,zL.

Brinton Jr. C.C (1959) Non-flagellar appendagø of bacteria Nature Lß: 782-786'

Brinton Jr. c:c. (lgs) The piliation phase syrdrome and the uses of purified pili in disease

control ltz hoceedhy of îhe 13th loínt c-:otfaatce on clwlera E(t c Miller'

PublicationNo.(MH)78-l5g0.DepartmentofHeålth'BlucationandWelfare'

lVashingtoû, D.C. PP' 34-70'

150

Bror¡n KH., parryL, Khatun M. & Ahmadc. (1979a) Lactooe malaboorption inBangladeshi

village children: relation dü ag", history of recent diarrhea, nutritional status and

breast feeding Amen I. Ctí't' Nutr' 32" 1962-1969'

Brourn M.CM., Weston À, Saunders J.R & Humphreys G'O' (199b) Ïì'ansformation of

E&het¡ctti\ cotí Koobyplasmid DNA at different phases of growtl' FEMS Mbobbl

Lú. 5z 219-2,22-

Burnet EM. (1g4g) ovomucin as a substrate for the mucinolytic enzymes ol V choleræ

filtraæs. Attsl- I E'x"- Bíol' Med tl: ?A5-25L

Burnet EM. & Stone J.D. (rg47) Desquamation of rhe inresrinar epithelium in víto w v

ciøIqaeñltrates: characterization of mucinase and tissue disinægrating enzyDe' AusL

l. EA. BioL Med Sci 25: 219-nß'

Carpentar CCJ., Barua D., \trhllace CK, Mit¡a P.P., Sack RB', Khanra S'R" Wells $A'' Dnns

pB & Chaudhuri R.N. (196ó) Clinical studies in Asiatic cholera. IV Antibiotic

therapyincholera.ButLloltt:a¡HopkfuHasp.|':az2l6.D.

Carpenter cGI., Sack RB., Feeley J.c & Steenberg R.lv. (1t}6s) Siæ and characteristics of

electrolyte loos and effect of intraluminal gfucose in eryerimental canine cholera' J

Clh. Inves- 472 L2l0-1Z20'

cafy s.G. & Blair E B" (1%4) Nec/ ü'lnsPort medium for shipment of clinical specimens' 1'

Fecal specimens. I' Baaerid EE: 9(í-98'

Cash R..{., Music SL, Libonati J.P, CraþJ.P., Pierce N'n' & Hornick R 'B' (1974a) Response

of man to infection with vibrio chotqae. II. Protection from illness afforded by previous

disease andvaccine. I' InfectDn 131Ì 325-333'

cash R.d, Music S.L, Libonati J.P., Schwartz AR & Hornick R'B' (194b) Li"" oral cholera

vaccine: evaluation of the clinical effectiveness of two strains ¡¡ ft¡mans ' InfecL

Intnun- Lù' 762:7&.

151

cash RA,, Music S.L, Libonati J.P., Synder MJ., lVenzel R.P. & Hornick RB' Gn4c)

Response of man to i¡fection with Vibrb choleræ. I. Clinical, se¡ologic and bac-

teriologic responses to a known inoculum. l. Infect. Dis' l;zy' 45-52

qD.q (1936) Toxigenic VíbrüocholqæOlinfections -LouisianaandFlotrrda- M'M'WR 35t

ffi.607.

chaicumpa rv. & Atthasishtha N. (1gg) Th" study of i¡testitral immunity against v e]rcbae:

purification olv elplqa¿ El ror haemaggfutinin and the protective role of its antibody

in erperimental cholera southeas Asian L Tlop. Med- PubL HhîIL l:Ù' 72-79'

Chaicumpa w. & Rourley D. (lgzzj F.ryerimental cholera in infant mice: protective effects

of antibody. J- InfecrDic lZ5: 480485'

Chen LC, Black R.B, Sardar 4.M., Merson M.H., Bhatia S', Yunus M' & Chahaborty J'

(1930) Villaæ based distribution of oral rehydration therapy packets in Bangþdesh'

An I. TroP- Med HYg. 2*' 285-290'

chitnis D.s., sharma KD. & Ibmat R.s. (19s2â) Role of bacterial adhesion in the

pathogenesisofchole¡a.].Med.Mbúiol15:43.51.

chitnis D.s., sharma I(D. & Ikmât Rs (19g2b) Role of somaric anrigen olwbrb choleræ

in adhesion to intestinal mucos u I. Med Microbiol 15: 53ó1'

Clemens J.D., Sack D..{., Harris J.R, Chakraborty J., Khan M'R, Stanton B'F" I(ay B'4" Khan

lvf.u., Yunus M., Alkinson lv., Svennerholm A-M. & Holmgren J' (198ó) Field trial

of oral cholera vaccine¡ in Bangþdesh. I-øtcd- 2: l?A-127.

Clemens J.D., Sack D.A., Harris J.R, van l-oon E, Chalrabofty J', Ahmed E'' Rao M'R" Khan

M.R,YunusM.,HudaN.,StantonB.E,,tr(ayB.A.,\\ålterS.,EeckelsR.,Svennerholm

A"-M. & Holmgren J. (1gg0) Field trial of oral choleravaccines in BangÞdesh: results

from tlree-year follow-up' I'ancel' Lz 270-/73'

r52

clemens J.D., Stanton" BH,, Chakraborty J., SackD.A" Khan M'R, Huda s., Ahmed E' Hafris

J.R., Yunus lyf", Khat lvLu., sennerholn'{.-M" Jertborn M' & Holngren J' (1987)

B subunit-whole cell and whole cellonly oral vaccines against cholera: shtdies on

reactogenicity and immunogenicity' I' InfeclDís 155: 7945'

Clemens J.D., Harri8 J.R., Sack D.*¿1", Chakraborty J', Ahmed n'' Stanton B'E' Khan M'U.' KÂy

BA,HudaN.,Khanlvf.R.,YunusM.,RaoM.R,SvennerholmA'-M'HolmgrenJ'

(19SS) FieldtriatoforalcholeravaccinesinBangladesh: Resultsofoneyearfollou'-up

I.InfectDís- 15& 60ó9'

clements M.L I-evine M.M., Young c.R, BLack R.E., Lim Y'-L, Robins-Broü'tre RM' &

craigJ.P.(1932)Magnitude,kineticsanddurationofvibriocidalantibodyfesPonses

in North Americans after ingestion of v,br¡o cholqæ' J' Infect"Dh 145: 465473'

clitr J.L, zinkin p. & Marrelli ,{ (19só) A hoepital outbreak of cholera in Map.to, Mozam-

bique. Trø¡la R Soy'Top' Med Hyg' *' 473-476'

colwell RR, Brayton P.R., Grimes D.J., RoezakP.R" HuqS*À & Palmerl,M' (1985a) Viable

but non+ulurrable lnbtío chobøeand relaæd pathogens in the environment implica-

tions for release of geneticalþ engineered organis ms' Bioædtttologt' 3z 817'8?f,'

colwell R.R., Ikper J. & Joseph s.lv. (197 7) wbtio chobøe v pørahaønolytiLas and other

vibrioo: occuffence and distribution in chesapeake Bay' sc¡qce 19E: 394'396'

C-olwell RR, Singfeton EL, Huq ,{., Xu H.S. & Roberts N. (19s5b) Etiologr of Vibrúo

c}øløøe,Vpøahættøtyticlßandrelatedvibrioointhenatufalenvi¡rcnment.In:

BaærialDúÚlæItDiseases.Ed&Y.Takeda&TMiwatantTokyo:KÏKScientific

hrblications . PP. 273-Ð5'

C.olwell RR, Tamplin lvLt-, Brayton P.R, Gauzens A'L, Tall BD" Herrington D" I-evine

lvf.M., Hall s., Huq r¿r. & sack D.A (1987) Environmental aspects oî'vtbrb choletæ

153

in transmission of cholera

tiamsbttrg. PP.33-34.

Abstracts of 23rd ILS--Iøpan Ctwlcra C';oferarce' Wíl'

craig Sw. & Cebra JJ. $nÐ Peyer,s patches: an enriched souroe of prec'ursors for IgA

producinginmunocytes in the ¡abbiL f' EtP'Met' t34z 188-æ0'

Cryo SJ. Jr., Fürer E. & Germanier R. (19s2) E,ffectof chemical andheatinactivation on the

antþenicity ¿¡| immunogenicity or'lftrb clwlqæ Infær Imtruln ß: 21.2ß.

cnatrecasas p (rg73,) Interaction or. víbrb crørerre enterotoxin with cell membranee'

Biochanisry- t2: 3547 -3558'

cnrlin G., I-evine R, Aziz lclvf,a, Rahman AS'M'M' & verwey lv'E (1976) Field trial of

cholera toxoid. Proc. of the |IthIr c-mtr. ctwlsa ([LS'-Ipn Coop' Med' sci ProgrmtL

pP.3L4-3D.

Crrrman B, Kampe o., Rask L & Peterson P.,â1. (199) Pre¡ence of alloreactive Ia antiçns

on murine intestine epithelial cells. scøú.I. Innuttol 10: 11-15'

Dastidar sG. & Narayanaswani (r%s) The occurrence of chitinase invibrios' h¿liml' Med

R¿s. 56: 654658'

DeS.N. (1959) Enterotoxicityofbacæria-freeculturefiltraæ ollöríocholeræ' Ndwe' !t3z

1533-1534.

Deb B.C, Sircar BjC, Sehgupta P.G., De S.P., Mondal s.trc, Gupta D'N., Saha N'C' Ghosh s.,

Mit¡aU.&PalS.c(19E6)StudbsoninterventionstopreventElTorcholera

transmissioninurbanslums.BulLWld-HltILor8.6:lZ7.l3l.

DeMeyJ.&MoemansM.(1986)heparationofcolloidalgoldprobesandtheiruseas

m¡rkers in elect¡on microecopy. l¡zAàvøtcedTechníweßhMological ElætonMiso*

coPY- E/.-J.K Koehler' 3: D-nL

Dixon J.M. (1960) The fate of bacteria in the small intesrine. I- pdhoLBøctqíol 7q 131-140'

154

Duguid J.p. & old D.c (1gs0) Adhesive properties or. Enterobæterùaceac. rrz Bætøí41

úlwance, ræEtbrs mdrecognirbn E(l. E M. Beachey' London: chapman & Hall'

SeriesB 6: 185-217.

Duguid J.P., Smith w., Dempster G. & Edmunds P.N. (1955) Non-flagellar filamentous

aprpendages ffrmbriae) and hemagglutinating activity in Búqíwt coli' I' PAhIþL

Baaqíol 7ù 335-348'

Dutt,{.trL, Alwi S. & Velauthan T (1971) A shelfish-borne cholera oubreak in Malaysia

Bløts-R Soe Tlop' Med Hyg' 6: 815{18'

Ehara rvr., Ishibashi M., Ichinooe y.,Iwanag lvr", shimodori s. & Naito T (19s7) Purification

andpartialcharacterizationofñmbriaeoî'Vîbrbcholqøeol.Væcíne.52283-?ß.

EharaM., Ichinoce Y., Naito T & Shimodoris. (19SS) Fimbriae or'v clnlera¿ 01' Abstacts

of24thlointC-onføanceUS.IapanC-æperatíveMedicatscíencehogram.Clwlcrø&

ReldedDiûTlßalDúseaæsPøteJ.Tokyo,Japan.p.48.

Ehara M.,Ishibashi M., Watanabe S., Iwanaga M., Shimodori s & Naito T (1936) Fimbriae

ol'lilbriocholqøeol..oboervationoffimbriaeontheorganismsadherenttolhe

intestinal epithelium and development of a ner¡' medium to enhance fimbriae produc-

nor. TIoP- Med, 2E: 21-33'

EubanbBR.,GuenlzelM.N.&Berry r-J. Qnr Evaluationofsurtacecomponents or,r4brb

ehoer/æasprotectiveimmunogelxlnfect.Immun15:533.538.

þyans D.G., Evans Jr. DJ., CleggS. & Pauley J.A" (199) Purification and characterization

oftheCFA/Iantiçnofenterotoxigeltc&scheñchilcotí.Infø.Irranun25z738-748.

Evans D.G., Evans Jr. DJ., Ïioa w.s. & Dupont H'L (198) Detection and characterization

of colonization factor of enterotoxigenic E$heñchia coli isolated from adults with

diarrhea. Infect- Intrutn ty' 7n'7%'

I

I

d,Ë

155

Faris ,4., Lindahl M. & wadstrom T (1gg2) Higþ surface hydrophobicity of hemagglutinating

Víbriocholqaeandotlervibrios.CunMicvobioLTz35T-362.

FeachemRG. (1gs2) Environmentalaspectsof choreraepidemiolog. III. Tiansmissionand

control TroP-Dis'BulL 79t t-47'

Feerey J.c. (1970) cholera vaccines. rn. principres and practices of chorera control- Public

HealthPaperNo'40' Geneva: W'H'O' pp' 87-93'

Feeley J.* & Gangarrcsa E_J. (r9s0) Field triat of cholera vaccine. rnz chorøa andRelæed

Diørheøs. Eds. o. ouchterlony & J. Holmgren. 43rd Nobel Symposium, Stockholm

1978. Karger, Basel' PP'204-210'

Felsenfeld o. (1963) some observations on the chorera (El ror) epidemic in 1961-196L BulL

Wld HlrlL Org' 2E:' ?ß9-296'

Felsenfeld o. (1%5) Notes on foo4 be'erages and fomites confaminatedwith víbrio choreræ-

BulL lltd HtttL OÌg' 33: 7?5-734'

Felsenfeldo.(|974)Thesurvivalofcholeravibrios.In:Cholera.Eds.D.Ba¡ua&w.

Burrows. l,ondon: W'B' Saunders' pp' 359-366'

Finkerstein R.A. (1962) vibriocidar antibody inhibition (vAI) analysis: A technique for the

identification of the predominantvibriocidal antibodies in serum and for the recogni-

tion and identification of víbio choleraeantigens' J' hnmunot' sÌ 264-271'

Finkelstein R.A. (1973) Choler t CRC Crit.Iteu. Microbíol 2: 553-623.

FinkelsteinR.A (1975) Immunoloryof cholera cunTop.MicrobioLlrvnunoL 69 137-796'

Finkelstein R.A (1934) Cholera In:

Academic Press' PP' 107-136'

BactqialVaccines- Ed. Rene Germanier' New York

Finkelstein RA, erita M., Clements J.D. & Nelson ET (197s) Isolation and purification of

an adhesive factor (cholera lectinn) rromvíbrio chorqøe- rn; hoceed,ings of the 13th

I.rt

rìì'

I

It

T¡

I

I

II

#

fl

156

i,t

È-

Jt.Cüf.Chotqa.(IS-Ipn.Cæp.Med^ScLÞogrøn.DHEÏVPubLno.78-1590.Na-

tionallnstitutes of Health, Bethesda' Md' pp'137'l5l'

Finkelstein R.,A,., Boesman-Finkelstein M. & Holt P' (19S3) víbrio choleræ hemag-

gtutininÄectin/proteasehydro\zesñbronectinandovomucin:F.M.Burnetrer¡isiæd.

hoc- NatL Acad' Scí' U'SA' &): 1092-1095'

Finkelstein R.A. & Dorner E (19s5) Cholera enterotoxin (choleragen)' Phmnac Thq' 27:

37-47.

Finkelstein R.A, & Hanne LE, (1gs2) purification and charactenzation of soluble hemag- ,,

gtutinin (cholera lectin) produced by víbrio chorcrae. Infect. hnmun. 36; 1199-1208' ì

Finkel¡tein R.A & LoSpalluto JJ. ( 1góg) pathogenesis of experimental cholera: preparation

and isolation of choleragen and choleragenoid' J' F-xp'Med L3O:' 185-2frZ-

Finkelstein R.A & I-ospalluto JJ. (1970) hoduction of higþly purified choleragen and ì

t

choleragenoid. J' InfecL Dß' 1121: 563-572'

Finkerstein R.A. & Mukerjee s. (1963) Hemaggtutination: arapidmethodfordifferentiating

Víbrio cholqaeand El Torvibrios . hoc. Soc. Exp Biol- Med 112: 355-359'

Finn TM., Reiser J., Germanier R. & cryz S.J. Jr. (19s7) cell-associated hemaggfutinindefi-

cientmutantolVtbríocholqæ.InfecLImmu¡t552942.946.

Fishman RH. (1gg0) Mechanisms of action of cholera toxin: studies in lag period. I. Mqn'

BioL 54 6l-7L

FloreyH.w.(1933)observationsonfunctionsofmucusandtheeaflystagesofbacterial

invasion of the intestinal mucosa J- Pathot. Bacteriol' 37: 2Í33-?ß9'

Fo'ett E"A.c & Gordon J. (1963) An electron microscope study of vibrio flagella J. Gen'

i¡

il

f.'

TtI

I

I

i

Microbiol. 32: ?35-239'

t57

I

{

h-

Forrest 8.D., I-aBrooy J.T, At¡.idgs S.R., Boehm G', Beyer L, Morona R' Shearman D'J'C'I

& Rowley D. (1939) Immunogenicity of a candidate live oral typhoidicholera hybrid jï

vaccine in humans ' I' Infect'DLs 159: L45-146' i

Franzon vL & Manning pd (1gs6) Molecular cloning and expression in Esctprichia coli i

K-l2ofthegeneforahemagglutininrromVíbriocholqøe.hfect.Immw.52z279-?ß4.

Fresh J.W., Versage P.M. & Reyes V (1964) Intes'inal morpholory in human and experimental

cholera. Arctt-Pah' 77: 5Ð-537'

Freter R. (1955a) The fatal cholera infection in the guinea pig achieved by inhibition of

normalentericflora' I' Infecf' Dis' llz 57{15'

Freter R. (1g55b) The serologic character of cholera vibrio mucinase. I- Infect- Dis. llz

88-245.

Freþr R., Alhveiss 8., o,Brien PC.M., Halstead S.A. & Macsai M'S' (19s1) Role of

chemotaxis in the association of motile bacteria with intestinal mucosa: in vito studies'

Infect- bnmun- 342 Vl[-Vl9'

Freter R. & Gangafosa E.J. (1963) oral immunization and production of coproantibody in

human volunteers ' J' bnrnwtol' 9fr: 72A-7D'

Freter R. & Jones G.W. (1976) Adhesive properties o1 Vibrio chorøaet nature of the

interaction with intact mucosal surfaces. Infect' hnmun' t4z 246-?56'

Freter R & o,Brien pcM. (lgsla) Role of chemotaxis in the association of motile bacteria

with intestinal mucosa: chemotactic responses ol.l1brb chol'qøe and description of

notile non-chemotactic mutants. Infecr. Immutt^ 342 215 -DL

Freter R. & o,Brien pcM. (lgslb) Role of chemotaxis in the asociation of motile bacteria

withintesitnalmucosæfitnessandvirulenceofnon-chemotacticVíbriocholeræ

mutants in infant mice' btþcL Immuru 34l. 2'2-733'

158

Fubar¿ E"S. & Freter R. (1973) protection against enteric bacterial infection by secretory IgA

antibodies. J- Imtnunol' l't.lz 395-403'

Gaastra w. & de Graaf F.IC (1gs2) Host specific fimbrial adhesins of non-invasive

enterotoxi geúc4,scherirhiacoü strains. MíctobioL Rat' 4e 129-t6l'

Gallut J. (1974\ The cholera vibrios. lltz cholera' Ed" D' Barua & w' Burrows' I-ondon:

W.B. Saunde¡s & Co' PP' 17-40'

Gangarosa EJ., Beisel W.R., Benyajati C., Sprinz H' & Piyaratn P' (1960) The nature of the

gastrointestinal lesion in Asiatic cholera and its relation to pathogenesis: a biopsy study'

Arn.I. TÌoP. Med HYg' 9: 125-135'

Gangarosa E.J., Sonati ,{., Saghari H. & Feeley J'C' (1967) Multiple serotypes of

Víbrioclwlqaefromacaseofcholeral.anceLt646-648.

GargersJ., Griffith o.M. & GrillLK (1gs3) Raptdpurification of prasmidDNAbfyasingle

centrifugtion in a two step cesium chloride-ethidium bromide gradienL Bioùq¡t

Biophys- Res Conmun' LL7" 835-842

GermanierR (1934) Tirphoidfever. l,.: Bacterialvaccines Ed'ReneGermanier' orliando:

Academic Press. PP' 137-165'

GhoshH.IC (19?0) Thepathogenesisofexperimentalcholera J'Med'Misobiol 3z 427440'

GiUD.M. (1976) The arrangemenrof subunis in choleratoxin. Bioùønistry- LSz lvl2-1248'

GillD.M. (1977) Mechanismofactionofcholeratoxin. Adu'cyclicNud'Res' E:85-118'

Giælson s. (1971) Gast¡ectomy, achlorhydria and cholera. Isaetl' Med sci 7z 663-6f|7'

Glass R.I., Becker s., Huq M.I., Stoll 8.J., Khan M.U., Merson M'H.' L'ee J'V' & Black R'E'

(1932) Endemic cholera in mral Bangtadesh, 1966-1980' Am' J' Epidemiol 116:

959-970.

Glass RL, Huq M.I., Alim A"R.M.A & Yunus M. (1930) Emergence of multþþ antibiotic-

resistant wbrio cholerain Bangladesh. I- Infect. Dis' 1r42z 939-942

159

Glass R.I., Iæe J.V., Huq lvf.I., Hossain trCM.B. & Khan M.R. (19s3) Phage types of

VíbriocholqaeolbiotypeEl]brisolatedfrompatientsandfaniþcontactsin

Bangtadesh:epidemiologicimplications.J.Infed.Dirl4t:998.1004.

Glass R.I., Svennerholm AM., Khan M.R., Huda S., Huq M'I' & Holmgren J' (1985)

SeroepidemiologicalshrdiesofElTorcholerainBangtadesh:associationofserum

antibodylevelswithprotection'J'Infect'Dis'151:2%-242

Greenougþ w.B.trI. (1965) pancreatic and hepatic h¡ipersecretion in cholera. Lørceî- 2z

Ðr-994.

Greenougb W.B.II[ (rg7g) wbrb ch.oreræ. rnz hittciptes mdhactíce of InfectíousDiseases

Eds. G.L Mandetl, R.G. Douglas Jr. & J.E BennetL New York: John rililey & Sons'

pP.l672-1ffi7.

Greenougþ w.B.Itr. (1930) Principles and prospects in the treahent of cholera and related

dianheas. lnz cholqa&RelntedDisrheas. Eds. o. ouchærlony&J' Holmgren' 43rd

Nobel Symposium, Stoclùolm lnï' Base} Krager' pp'2ll2l8'

Greenough w.B. IIL Gordon R.S., Rosenberg LS.' Darries B'I' & Benenson A'S' (19ó4)

Tetracyclineinthet¡eatmentofcholeral-ancelLz355-357.

Greenougb vÍ.B.Itr. & Rabbani G.H. (1936) Antisecretory and antimicrobiat drug for treating

diarrhea rrz Darclopntant of vaccines mdDntgs øgøinstDiorrtø. Eds. J. Holmgren'

,dUndberg&R'Möllb}''llthNobelConf"StockholmlgS5'S¡¡eden:Studentlit-

teratur. PP.27O-277'

Guentzel M.N., Amerine D., Guerrero D. & Gay TV (1931) Associatiot oî'Wbrio cholcræ

mutants with the intestinal muçosa of infant mice' Scmnhg Elecfron Micraçc' 4z

Llí-t2t4.

GuenEel M.N. & Berry LJ. (1975) Motility as a virulence factor lor Vibrio chorqae- Infect

hrunun. 11: 890-897.

160

GuenEel M.N., Field LH., Eubanks E"R. & Berry LJ. (1977) Use of fluorescent antitody in

studiesqfimmunitytocholeraininfa¡tmice.hþc|htpnun.15:539.548.

Guinée p.ÀM., Jansen w.H., Gielen H., Rijpkema S.G.T & Peters P.lvJ. (1987) hotective

immuni$againstwbliocholqaeinfectionintherabbitZbl.BkH.Hyg.A.266,..

552-562-

Guinée p.A.M., Jansen lV.H. & peters p.w. (1gg5) Wbtio chorerøe infection and acquired

immunityinanadultrabbitmodelZbt'Ba&''Hyg'A259:118-131'

Hall R.H., Vial P.4., Kaper J.8., Mekalanos JJ. & levine M'M' (1983) Morphological studies

onfimbriaeexPressedbyVíbríoc}pleraco|.MictobiatPathqøt.42757-2ß5.

Hanne LE & Finkelstein R.A, (19s2) characterization and distribution of the hemag-

gtutinins producedby víbrio clnlqae hfect- Imtnun 36:' ?fr9'214'

Hansson H._.d, Holmgren J. & svennerholm L (rgii) ultrastructural localization of cell

membraneGMrganeliosideþcholeratoxin.ProcNalLAcadScLUSA.T4z3TS2-

37K.

Hawkes R.B, Niday E. & Gordon J. (1982) A dot immunobinding assay for monoclonal and

other antibodies. Anal' Biodtem LL9 142-147'

HenningU.,Schwar1¿,H.&ChenR(lg7g)Radioimmunologicalscreeningmethodfor

specificmembraneProteins.Anat-Biochøt.9|lzlS3.|57.

HerringtonD.A,HallRH.,LooonsþG.,Mek¡lanosJJ',TaylorR'IC&LevineM'M'(1988)

Ïbxin, toxin-coregulated pili, and the foxR regulon a¡e essential for wbilo cholcræ

pathogenesis iû humans t' Exp' Med l:6Ez 1487-1492^

Hirschhorn N., Kinzie J.L, Sachan D.8., Northrup R'S', Taylor J'O" Ahmad S'Z' &' Phillips

R.A'(1%s)Decreaseinnetstooloutputinchildrenduringintestjnalperfusionwith

gfucose-containing solutions' New Eng' I' Med 2792 176-18l'

16r

Hirschhorn N., Pierce N.E, Kobari I( & carpenter ccJ' (1974) The treatment of cholera

hz clwlera Eds. D. Barua & w. Burrou¡s. I-ondon: lV'B' Saunders' pp' 23525:¿.

Hisatsune K, Kondo s., I(awata T & Kishimoto Y' onÐ Fatty acid composition of

lipopoþsaccha¡ides olVíbrìo cholqae35A3 (Inaba), NIH90 (ogaura) and 4715 (NAG)'

I. Bactøíol- 13E: 288-290'

Hohn B. & coilins J. (1gs0) A small coomid for efücient cloning of large DNA fragmenrs

Gqre Ll: Ðl-?9.8.

Holmgren J. (1gs1) Actions of cholera toxin and the prevention and t¡eatment of cholera

Naatre. 2922 413-417.

Holmgren J., Clemens J., Sack D.A, & svennerholm A'-M' (1989) Nerr cholera vacci¡es'

Vaccine. Tz 94'96.

Holmgren J. & l,önnroth I. (1976) Cholera toxin and the adenylate cyclase-activating signal

I. InfecLDi* 1i]3: Só4-S74'

Holmgren J., l_õnmoth I., Mansson J.-B & svennerholm L (1975a) Interaction of cholera

toxinandmembraneGMlgangtosideofthesmallintestine.hoc.NatLAcdSci.T2z

25m-2524.

Holmgren J. & rycke N. (1gs6) Immune mechanisms in entericinfections. hz Developmant

of vrccinæ øtd dntp again¡ diærhea Eds' J' Holmgren, A' Lindberg & R' Möllb5''

llth Nobel Conf., Stockholm 19s5. Sveden: Studentliterahtr. pp'9'ZZ

Holmgren J. & $ennerholm A.-M. (1gs5) Vaccine detelopment for the control of cholera

and related toxin-induced diarrhoeal diseases'

diseæe. L.ondon: Pitman PP' ?A2'?5,6'

In: Miqobiat tox¡n and diæthoeal

Ho}ngren J., Svennerholm rÁ\.-M., I-önnroth I', Fall-Persson M" Markman B' & Lundbeck H'

$nT Development of improved cholera vaccine based on subunit toxoid' Næute

269l. 6U2-604-

162

Holmgren J., Svennerholm À-M., OuchterlonY O', AnderssonA" Wallerström G' & lVeste¡-

berg.Berntssonu.(195b)Antitoxicimmunityinorperimentalcholeraprotection

and serum and local antibody responses in rabbits after enteric and parenteral im-

munizati,ot Infect Immun t2z 133l-1340'

HondaT & FinkelsteinR.A. (lg7g) selection andcharacteristics of.al/ìbrb eholaæ mutant

lacking the A (ADp-ribosytatinÐ portion of the cholera enterotoxin . Proe NatL Acad'

Sci U.SA- 76; Ð52-?Jùl56'

Hood M.A, & Ness G.E, (1gs2) suwival or,vibrío chorqæ andgscherüehía corí in estuarine

watersandsedimells.AppLfu'virØ.Miqobío|43:578-584.

Hornick R.8., Music S.I., Wenzel R., Cash R., Libonati J'P', Synder M'J' & lvood\ñ'ard TE

(191) The Broad St¡eet Pump revisited: resPome of volunteers to ingested cholera

vibrios. ButL N-Y. Acad- Med 47: 1181-1191'

Ilraniuþ I(W., Mulholland ,¿\., La¡son ,{D., Eubanks ER' & Hart LT (1980) charac-

terization of a flagellar sheath protein or'wbrio cholqæ' Infecr Im¡¡un Tl: 597ó03'

HuS R. (1%4) The proposed conservation of the generic name l/ibrío Pacini 1854 and

designation of the neotyPe strain ol Víbrúo cholqøe Pacini 1854' ht BulL Bacrqiol

Nomer Tax^ L4z 87-101'

Husband .ÀJ. & Gou,ans J.L (lgs) The origin and antþendependent distribution of

IgA-containingcellsintheintestine.r.Ery.Med.t4E:1146-1160.

Huq 4., Huq S.,{., Grimes DJ., O',Bfien lvL, chu trLH., C-ap]1tzzoJ'M' & C-otwell R'R' (1986)

c-olonization of tìe gut of the blue (.xab (cattineaes sryià/s)w wbtio cholcræ' AppL

Environ MbobbL 52: 586-588'

Ibrahim A,E.A, (19M) rmmunity to experimental cholera in the rabbit ph.D. thesis, utrecht

the Netherlands: The University of Ut¡echt

t63

Inman LR, Cantey J.R. & Formar s.B. (1gs6) Colonization, virulencæ and mucooal interac-

tion of an enteropathogenic,Erh qich¡ocoli (str. RDEC- 1) e:pressin g shiplla somatic

antþen in rabbit inæstine' I' InfectDrs' 154: 742-751'

International Study Group. (rg77) A positive effect on the nutrition of Phitippine children of

anoralgtucoeeælectroþæsolutiongivenathomeforthetreatmentofdianheaB¿ll

Wld- Htttl Org- 55: 87-94'

Jann 8., Jann trL & Beyert G.O. (193) 2-amino-2,6-dideory-D-gtucose (Dquinovosamine):

a constifirent of the lipopoþaccharide

531-534.

ol.Vtbrío clwlqae- European I' Bíochøl 372

Jansen lv.[L, Gieþn H, Riipkena s.G.T & Guinée Pfllvf (19SS) priming and boosting of

the rabbit intestinal immütr€ system with live and kitle4 smooth and rougþ V clwlcræ

ceÍs. MbobíatPatlogen 4: 2l-?ß'

Jertborn lvf,, Sennerholn A.-M. & Holmgren J' (1984) Gut mucooal' salivary and serum

anti_toxicandanti_bacterial antibody f€ßpons€s in svedes afterorarimmunizationwith

B subunit-whole cell choleravaccine. hrArc]æ'AltøgtAppL furntnol 75: 38-43'

Jertborn M., Svennerholm A,-M. & Holmgren J' (19Só) Saliva' breast milk and serum

antibody resPonses as indirect meiasufes of intestinal immunity afær oral'cholera

vaccintionornaturaldbease.I.ctínMigobiolAl?Ã3ffi.

Jones G.rv. (1gE0) The adhesive properties of wbrio chorsæ and other r4bì4j species-

RecqL Rerogtit- Set B' 6z 2Ð-?A9'

Jo¡es G.W., Abrams G.D. & Freter R (196) Adhesive p¡operties oî lörb c]øløaez

adhesion to isolated rabbit brush border membranes and hemagglutinating activity'

Infea- Im¡run. t4: 232-239'

tu

JonesG.w.&FreterR.(1976\Adhesivepropertiesor'Wbrioc}plerae:natureofthe

interaction with isolated rabbit brush border membranee and human erythrocytes'

Infect. Imman- t4z 2'40-?A5'

Jones G.w. & Rutter J .M. (rg72) Role of Kgg antigen in the pathogenesis of neonatardiarrhea

causedbyEschøichíøcotí|n,prdets.Infeclbnmun.6z9|8-127.

Jonson G., Svennerholm A._M. & Holmgren J. (1gSg) wbúo clprsae expresses cell surface

antigens during intestinal infection which are not expressed during invitro culture'

Inþa. Immtn- 57: 1809-1815'

Joo I. OnÐ cholera vaccines. l¡¡: cholera Eds" D' Barua & w' Burror¡'s' London: w'B'

Saunders& Co. PP' 333-355'

Kabir s. (1gg0) composition and immunochemical properties of outer membrane proteins

of.Vîbrb cholqæ' f' Buds¡oL t44z 382-389'

Ikbirs. (1gs2) characrerization of rhe ripoporysaccharide fromwbrb dplcræ395 (Ogau'a)'

Infea. bnnan ß: l?ß3-lZlL

IGbir s. (1933) Immunochemical properties of the m4or outer membrane protein or'víbrío

c]ølqae- hfect Inmun 39' 452455'

IKamatAM.$n4)Theseventhpandemicofcholeraln:CholgaEds.D.Ba¡ua&w.

Burrow¡- I-ondon: W'B' Saunders pp' 1-14'

IGper J.B. (1989'¡ l4brio elplsaev,accines. Rcu. Infea.Drs' 11: s568-S573'

Kaper J.B., L,ock6aû H, Baldini M.M. & I-evine M'lvf, (19Saa) A recombinant live and

choleravaccine. Bioæúnologt' 2¡' 345-349'

KaperJ.B.,I-ockman H., Baldini M.M. & I-evine M.lYf" (19s4b) Recombinantnontoxinogenic

Vtbrbc.holøæstrahs as attentuatedcholeravaccinecandidates' Nafrne' 3lB: 655ó58'

IGllog D.S, Peacock w.L, Deacon 1V.8, Brown L & Pirkle ct ( 1963) Ne,sdø goørrheoea'

I. Virulence geneticalþ li¡ked to clonalvariation' I' Baøìol E5: ln4'lng'

165

I(elly J.T & parker cD. (1gs1) Identification and p,reliminary characterization of wbtio

clpbaeoutermemb¡aneproteins.I.Bøcteríol145:1018-1024.

Kenne L, Lindberg 8., Unger P., Holme T & Holmgren J. $n9\' Structural studies of the

Wbrío cltolqaeO-antþn ' Øohyd'2R¿s' 6& l4-L6'

Khan M.U. & Greenough w.B. (1gs5) Epidemiolog of diarrhear diseases in Bangþdesh. In:

Bacterial d¡ûrhcnl dìseases' Eds' Y'

Publications. PP. 37 -5z-

Thkeda & T Miwatani. Toþo: KTK Scientific

Khan M.U., Samadi A.R, Huq M.I., Yunus M. & Eusof '{' (1934) Simultaneous classical and

El Tor cholera in Bangþdee h' I' Die' De* Rec 2: 13-18'

Kingc,A,. &van Heyningen w.E" (1973) Deactivation of cholera toxinby asiaridase-resistant

monosialosyl gangtioside ' J' hfect' Dís L2ilz 639-il7'

Kobayishi T, Enomotu s, sakazaki R. & Kuwahara s' (1%3) A new selective isolation

medium for the vifuiogroup: on a modified Nakanishi's medium (rcBS agar medium).

laP. I- BactqiaL LE¡' 387 -3lZ

Korhonen TIC, Nurmiaho E-L, Ranta H. & Edén c$ (1980) Nen'method for isolation of

immunologcally pure pili from E¡c]ærichia coli' Infea' Im¡n'utt' 272 569-575'

I-aBrec BH., Sprinz IL, Schneider H. & Formal sB' (1%Ð l-ocâlizåiton of vibrios in

experimental choleræ a fluorescent antibody study in guinea-pigs' Proc choL Rcc

Swp(Horclulu).U.S.PubLHlth.Serv.Pub.No.1328.pp,Z72.Z76.

I-aicy.,MendezE &changD. (ry76) Chemistryofcholeratoxin: treeubunitstructure'

I.InfecLDtu 133: S23-S30'

Lankford c.E & Legsomburana IJ. (1%5) Virulence factors of choleragenic vibrios. In:

ProcChot.M.Swp(Horcfuht).U.s.PubtHlth.Sew.Pub.No.1328.pp.109-120.

Lång H*4, & Paþa ET (19S?) A maior outer membrane protein or' lürío úolqæ ß

maltooe-inducible' Mioobiat Pæhogøt' 3z 143-147'

t6

r-ãtt^T (1331-1332) Malig'ant cholera: documents communicated by the central Board of

Health,I-ondon, rel¡ativetothe treatnentof cholerabythe copiousiniectionof aqueous

and saline fluids into theveins I'arcet' 2" 274-177'

r-efrrer H. & Svanborg_Edén c (1gg6) Gþcolipids as receptors rot Esdtqúùia corí lectins

or adhesins. In: Miqobiat lætitts md, øgþtkit's' Ed' D' Mirelman' New York: John

WileY & Sons. PP' 83-111'

I-er¡ine M.M. (1gg0) Immunity to cholera as evaluated in vorunteers. In: Chorerø mdreløed

dianheas. Bls. o. ouchterlony & J. Holmgren' 43rd Nobel $ropqiun' stockholm

1978. Basel S' Karger' pp' 195-203'

Irrrine M.M., Bl,ack R.F^, Clements M.L, Cisneros L, Nalin D'R & Young CR' (1981a)

Duration of infectionderived immunity to cholera I' Infø'Dfs' 143: 818{20'

I-eirine lvf.lvL, Btack R.E, Clements M.L, Cisneroa L, Saah A" Nalin D'R' Gill D'M" Cratg

J.P., YoungcR. & Ristaino P (1932) The pathogenecity of non+¡terotoxþenic wb¡o

chotøæserogfouPolbiotypeElTorisolatedfromseulagewaterinBrazilJ.Infecl

Dús. 145: 296-D9.

I-evine lvLM., BlackR.E , Clemenß M.L, Nalin D'R, Cbneros L & Finkelstein R'A' (1981b)

volunteer studies in development of vaccines egeinst cholera and enterotoxþenic

E*hqicttia cori: arerriew. rrz Aante enteñc hfeßtíons h chitùan- Ne*' prospecß fø

teøtmenl md præention FII& T Holme, J. Holmgren, M.H. Merson & R Mölby.

Amsærdam: ElsevierNorthHolland Biomedical Press pp 443459'

Irvine lvLM., Black RB, Clements M.L, I¡¡ata c, seals s', Honda T', Young cR' &

FinkelsteinR.A.(19s4)Evaluationinhumansofattenuatedwfuúoc}plgæElTor

ogawa strain Texas Star-sR as a live oral vaccin e' Infæt In¡runn 432 515-52

t67

I-evine M.M, Kaper J.8., Black R.E., & clements lvf'L (1933) New knou'ledge on

pathogeneoisofbacterialentericinfectionsasappliedtovaccinedevelopment.

Miqobiol Reu- 472 510-550'

I-evine M.M., Kaper J.8., Herrington D., Ketley J., I-osonsky G', Dcket co.' Dll B' e Ûyz

S. (19SSa) Safety, immunosgnicity a¡d efücacy of recombinant live oral cholera

vaccines, CDV 103 and CDV 103-Hg[.' Intø' 2: 46f'470'

I-evine M.M., KaperJ.B., Herringbn D., tosonsþG" Morris J'G.' Clements M'L' BlackRE '

Tall B & Hau R (19SSb) Volunteer studies of deletion mutants ofVîbrío clwlqae OL

prepared þ recombinanttechniqu æ' hfect' Innrun 56¡' 16l-167'

I-errine M.M., Nalin D.R., Craig J.P., Hoover D., Bergquist E J', Watenn¿rn D" Holley H'P"

HornickR.B.,PierceN.E&ubonatiJ.P.(19?9)Immunitytocholerainman:relative

role of antibacterialversus antitoxic immunity. Trans-R. Soc- Trop-Med Hyg' 732 3-9'

Levine lvf.ìá., Young cR., Hugþes TP., O'fbnnell S.' Black RB, Clements lvf,L' Robins-

Brou¡ne R. & Lim Y.-L (1981c) Du¡ation of serum antitoxin response follc,'Frfo¡gl4brfø.

chotsæ infection in North Americans:

EpËdmbL 114: 348-354'

relevance for seroepidemiologt' Am' J'

tin EY.C, Morris J.G. Jr., IbPer J.8., Gross T, Michalski J., Monison c' Libonati J'P' &

IsraelF^ (1ggó) persistenceof cholerainlheunitedstates: isolation or.víbríocholcræ

01 from a patient with diarrhea in Marylan d" I. ctin Mísobíol 232 6?A6?:6'

Lindberg¡, Lund 8., Johansso¡ L & Normark s (19sÐ I-ocalizåtion of the receptor-binding

p,rotein adhesin at the tip of the bacterial pilus Nøaue' 32E: 84-87'

ündh E. (1925) Increased resistâûce of immunogtobulin A dimers to proteolytic degradation

aftefbindingofsecretorycomponent.J.ÚnmwolL|422u.286^

168

I indholm L, Holmgren J., lVitst¡6m M., trkrlsson IJ., Andersson K & þcke N' (1983)

Monoclonal antibodies to cholera toxin with special reference to croes-reactions with

Eæhs¡IttiT æli heat-labile enterotoxin. Infed- bnmun' 4u 570-576'

Lonnroth I. & Hotmgren J. (rg3) subunit sürcture of cholera toxin. I. Gøt- MíqobbL 762

[fl4n.I-olvry O.H., Rooebrougþ N.J., Farr A.L & Randall RJ' (1951) Protein measureme¡twith

theFolinphenolreagenLl.BíoLC\eml!,322ß5.275.

Lospalluto J.J. & Finkelstein R.A. (Lg72) Chemical and physical propertiee of cholera

exoænterotoxin (choleragen) and its spontaneoueþ formed toxoid (choleragenoid)'

Bíochsn. Biophys- Acta 2572 158-166'

I-ugtenberg 8., Meiiers J., Peters R, van der Hoek P. & van Alphen L (195) Electropherctic

resolution of the lmaior outer menbrane protein" o1 Erchsichia colí KlZ into four

baûds. FønS l*fr- 5E; 254-258'

Þcke N., Eriben L & Holmgren J. (19S7a) Protection against cholera toxin after oral

immunization is thymus-dependent and associated with intestinal production of

neutralizinglgAantitoxin.ScødI.Invnwol25:413-419.

Þcke N., Hellstrom U. & Holmgren J. (1987b) Circulating cholera antitoxin memory cells

in the blood one year after oral choleravaccination in humans . Scand' I' ImmunoL 262

n7-21L.

I-y"k" N. & Holmgren J. (1987) Long-term cholera antitoxin memofy in the gut can be

triggered to antibody formation associated with protection within hours of an oral

challengeimmunizalto¡-Scørlú'I'ImtrutnolÉ:1A74lZ

þcke N. & Holmgren J. ( lggg) Adoptive transfer of gut mucoeal antitoxin memory þ isoLaþd

B cells l year afær oral immunization with cholera toxin . Infect - Imnu¿n 57 2 ll37 -ll4l'

t69

IyckeN.,IindþolmL&HolmgrenJ.(1985)Choleraantibodyproductionhvitroby

peripheralbloodþmphocytesfollowingq¡¿timmrrnizationofhumansandmice.dih

BE.Immu¡øL 62t 3947'

þcke N., Svennerholm.{.M. & Holmgren J. (198ó) strongbiotype andserotype cross-Protec-

tive antibacteriat and antitoxic immunity in rabbie after cholera infection. Mbobial

Paùogat L: 361-371'

Magnusson KE & stiernstrom I. (1gs2) Mucooar barrier mechanism¡. Interplay between

secretory IgA GIgA), IgG and mucins on the surface properties and association of

sabtonellacwith intestine and granulocytes . Immanotog¡t' ßz B9'?A8'

Mahalanabis D. (1gs1) Rehydration therapy in diarrhea. r¡z Aq¿tc entqíc ínfectíøs h

chilùan.Eds.THolme,J.Holmgren,M.H.Merson&RMollþ.Amsterdam:

Elsevier. PP. 303-318'

Mahalanabis D. & Merson rvr" (lgsó) Deveropement of an improrred formul,ation of orar

rehydration salts (oRs) with antidiarrheal and nutritional properties: a"su¡rer oRsn'

|nzDevebpttaúofVrcchæaúDntpøgahstDiarrheaEds.J.Holmgren,A"Iindbery

& R Moltby. llth Nobel conf., stockholm, 1935' $veden: studentlitæratur' Pp'

2n-2s6.

Mandara M.p. & Mhalu F.S (1gs0) cholera cont¡or in an inacceesible district in Dnzania:

importanceoftemporaryruralcenters.Med-I.Zmúia.15:10.13.

Manbtis T, Fritsch EF. & Sambrook J.c (1932) Motcotb ctonhg: a lúortury mannl

C-old Spring Harbor laboratory' C-old SpringHarbor' N'Y'

Manning P.,{., Heuzenrrceder lvL\\t, Yeadon J., Læavesle'y D'I.' Ree'ves P'R' & RO¡/ley D'

(19Só) Molecular cloning and eryression in Fscnq¡a¡a cotiKlzof the o antigens of

thel¡abaandogwaserotyPesofthewbríocholqæollþpoþsaccharirlesandtheir

poæntialforvaccinedorelopmeltlnfectlmmutlsSzTl2-T17.

170

Manning P.4., Imbesi F' & Ha¡rnes D'R. (1932) Cell envelope proteins inWbtúo cholqæ'

FEI¿IS Mbobiol l-en Lilz 159-166.'

Marrs CE, Ruehl lv.w., Schoolnik G.K & Falkow S. (19Es) Pitin gsne phase variation of

It[oræclhbovisiscausedbyaninversionofthepilinçnes.I.BadqíoLflÙ.3f/32-3['39.

Martin .{.R, Mqley w.H., Sau 8.8., Ahmad s. & Huq I' (1J}69) Epidemiologic ana$nis of

endemic cholera in urban EastPakista¡1964-lg6' Am'f' Epktcm¡ot Spt 572-582'

Mazia D., Schatten G. & sale w. (1g7s) Adhesion of cells to surfaces coatedu'ith poly-lysine'

l. CclI BioL 6z 198-200'

McCormack w.M., Mæley \il.H., Mohammed E & Benenson A'S (1J)69) Endemic cholera

in rural Fast Pakis ta¡' Am' I' Epídmiol' SÈ 393-404'

McDermottM.R.&BienenstockJ.Qng,)Evidenceforacommonmucosalimmunologic

syÉteD I. Migration of B immunoblasts into intestinal' respiratory and genital tissues'

J- In¡nnol L22: t8l2-1898'

MclntyreR.C,TiraT,FtoodT&BlakeP.A"(1979)Modesoft¡ansmissionofcholeraina

nady infected poprlation on an atoll: implications for control meaf¡ufe8' LmceÍ' lz

3ll-314.

Mekalanæ J.J. (1gs5) Cholera toxin: genetic anaþsis, regulation and role in pathogenesis'

Gut Top MisobioL bnmutPl 11E: 97-118'

Mestecþ J. (1gS7) The oommon mucosal immune system and current strategier for i¡duction

of immune resPonses i¡ efernalsecretions' I' ClÍn In¡nanol 7z ?ß5'T16'

Mestecþ J. & Mcchee J.R. (1987) Immunogtobulin A (Ig[): molecular and cellular

inte¡actions involved in IgA bioeynthesis and immune response' Adu' hwìtutpL 4Ù'

ts3-245.

t7l

Mhalu F,S., Mmafi P.lv. & Iiumba J. (199) Rapid emergence of El Tot lnbrb choleræ

¡çsi8tanttoantimicrobialagentsduringfirstsixmonthsofthefourthcholeraepidenic

in li¡nzania Larc*' lz 345.347'

Mharu ES., Mt^ngo ED.E & Msengj A,E. (1gg4) Hoopital outbreaks of cholera t¡ansmitted

througþclogepereon-to-Pefsoûco¡tactl-/,nc¿t.2282.84'.

Migasena S., Pitisuttitha- P., Suntharasamai P', haynra-HongB" Supanaranond lV" Desakorn

v.&BlackRE(19s9)Comparisonofthereactivitiesandimmunogenicitiesof

procholeragenoid and the B subunit of cholera toxin in Thai voluntee$' Infæt' Irunwt'

572 1942-1945.

Miller J.H OnÐ epqimørt hmoteailarganeÍícs. coldspringHarborLaboratory,cold

SpringHa¡bor, N'Y'

Miller vL & Mekeranos JJ. (lgss) A novel suicide vector and it use in construction of

insertionmutations:osmoregulationofoutermembraneproteinsandvinrlencedeter-

minants irl4brio ehobærequires tüR. J- BrctqioL L7Ù' 2575-2583'

Miller cJ., Feachem RG. & Drasar B.S. (1gs5) Cholera epidemiologl in developed and

derrelopingcountries: ne$, thougþs on transmission, seasonality and cont¡oL LanceL

l| ?ßl-2ßZ

Moch T, Hoechützky H., Hacker J', K¡öncke KD. & Jann trC (19ST) Isolation and charac-

terizationof thecr+iaþl-ß-1,3-garactoøyr-specificadhe¡in fromfrmbnarBscl.-.íchiacolí

hac NdLAcad- Sci U-SA U: 3462-%6'

Möller G. (1989) Lynphocyte homing Immunolog'Rsv' No' 108' Denmark: Munlsga^fi

International Publishers'

Monsur KA. (1%3) Bacteriotogical diagnoois of cholera under ñeld conditions. fulL 144d,

Htttt. Ory. 2E: 387-389.

172

Morgan R.L,Isaacson RB, Moon H.lv., Brinton cc & To C-C (19S) Immunization of

suckling pigs againet enterctoxigenic Fsenq¡A¡a coli-induced dianhe¿l disease þ

vaccinating dams with purified 987 or K99 pili: protection correlates with pilus homol-

ogt ofvaccine and challangs' Inþa' Imtrunt 222 771-777'

Morona R., Fonest 8., Attridge S.R., Tackett CO', Herrington D" Morris G" I-aBrooy J"

I.er¡ineM.M.&RowleyD.(198s)ConstnrctionandvaccineefficacyofEX64S,a

bivalentcholera-typhoidvaccine,Abstracßof24iht'L.S.-IøpøtC.ooperdíveMedicøl

Sciøtcehogrøtt, ChoteraandrelatedDíarrheatDúæasesPøteL bkyo, Japan' tr 58'

Morris J.G. & Btack RE (1gs5) Chorera a¡d other vibriooes in the united States. New Fng

I. Med StLz 343350.

Morris J.G., lvest G.R, Holck s.E, Blake P.r{., Echeverria P'D' & Karaulnik M' (1982)

Cholera amongrefugees in Rangsit, Thaitanô I. hrer|DÍs. 145: l3l.l3/¡,

Moeley lv.H. (1969) The role ef immunity in cholera A re'ien' of epidemiological and

serological studies' Tæ' FeP' BìoL n'Ied' 2ilz EI CAL

Moeley lv.fl, Benenson A-S. & Barui R. (1963) A serological suwey for cholera antibodies

in rural Fast pakistaû L The distribution of antibody in the cont¡ol population of a

cholera-vaccine ñeld-trial area and the reLation of antibody tit¡e to the pattern of

endemic cholera Bu& Wtd- HblL Otg' 3t¡' 377-33/.'

Mooley lV.H., McCormack w.lvL, Ahmed A-, Chowdhury A'KM'A" & Banri R'lL (1f)ó9)

Report of the 19ól.-1967 choleravaccine field trial in nrral Fast Pakistaû' II' ResultB

of the serological sufveys in the study population - the relationship of case rate to

antibody titre and an estimate of the inapparent infection ntewil\l/ibrio cholsæ' BulL

Wd- Httlv Org. 4u 187 -t97'

Mooley w.H., Woodward lv.E, Aaz KlvLA" Rahman A'S'M'M., Chowdhury A"trLM-A"

Ahmed A, & Feeþ J.c: (1g0) The lf}68-lf}6g cholera-vaccine field trial in rural East

173

pakistan. Effectiveness of monovalent ogawa and Inaba vaccines and a puriñed Inaba

antrgeq with comparative results of serological "¡¿

¿¡imal protection tests' I' Infed

Dú'c t;Zlz S1-S9.

Mootov K.E, Friedtander M. & Blohell G. (19&4) The receptor for transepithelial transport

of IgA and lgþl contains multiple immunogtobulin-like domains Naare' 3lE: 3743'

Mukeriee s. (1%3) The bacteriophage+usceptibility test in difre¡entiating whrío cholcræ

a¡ôVihriøEl Tor. ButL WId- HÃfrL Org' 8z 333-336'

Muir LL, Stnrgnell R.,{. & Davies J.K (19SS) hoæins that aPPear to be associatedwith pili

u.Ne/ssvlír ' hfect'hwruut- 56;1743-1747'

Nag 8., Moon H.w. & Isaacson R.E (|g77> Colonization of porcine intestine þ

enterotoxi genrc4sc)øichíacoJi: eelection of piliated forms in rauo, adhesion of piliated

forms to epithelial ce¡s h vítro, and incidence of a pilus antþn among porcine

enteropathogenic.&ch qict,¡4 colL Infect. Immun t6z 344-352

Nas¡ 8., Moon H.lv., Isaacson R.B, To CC & Brinton cc. (198) Immunization of suckling

pig against enteric enterotoxþe úc Eschøichi¿coJi infection þ vaccinating d¡ms with

purified PtlL hfea- Innun 2t 269-274'

Nakashima Y., Napiorkonski P., Schafer D.E & Konigsberg W'H' (1976) Primary structure

oftheBsubunitofcholeraentefotoxin.FEB9l.ú.6ft¡2n5.n8.

Nakasone¡.¿þtvanagalvL (19SS) Piliof genusWbri¿r.' Abwøasof24thU'S'-IqmCnopaa-

tíve Mdicat Scíance hogron, Choba oú rebted Dúû,,,e41 Dúseaæs Pøe]^ ftþo,

JaPan. P- 49.

NalinD.R.(1976)Choleracopepodsandchitinaæ'I-øtú'2:958'

Nalin D.R. & Cash R.^d Qnq oral therapy for cholera l!¡z clplcra E&' D' Barua & w'

Burrows.I.ondon:}V.B.Saunders&Co.pp.256.2ß1.

t74

Natin D.R., Daya V, Reid.au, Ladne M.M. & Cisneroe L (199) Adsorption and growth of

Víbrio clwlqaeon chitin' Infect' Im¡nun' 25: 768-770'

Nelson E T, Clemens J.D. & Finkelstein R..{. $nq wbrûo crøtsøeadherence and coloniza-

tion in experimental cholera: Elect¡on microscopic studies' Infecf' bnmwt' 14" 5n'

547

Neoh s.H. & Rowley D. (rg0) The antigens of wbrio chorqae involved in tre vibriocidal

actionofantibodyandcomplementl.InfeclDís.L212505-513.

NeohS.H.&RowþD.(1972)hotectionofinfantmiceagainstcholerabyantibodiesto

threeantigensolVibrbcholqael.InfecLDi's.1:2624147.

Nishibuchi M., Seidler RJ., Rollins D.M. & Joneph J'W' (1983) Vibrio factors ca.use rapid

fluid accumuliation in sucklingmice. Infect l¡nmwt 4û 1083-1091'

Noriki H. QnT Evaluation of toxoid field trial in the Philippines' Proc' of the 12th It hnr

Cholera(U.S..IapøttØopøativeMedic4lscíqt.cehqramLpp.32.3|.

Normarks, Båg¡ M., Göransson M., lindbergEP" I-'nd B.' Norgren M' & u-hlin B'E (1986)

Genetics and biogenesis of Eschabhiacoll adhesins. lliz Microbial lectins md ag-

glutinbt. Ed D. Mirelman, Nerr,York: John Wiley & Sons. pp. 113.143.

Norris H.T & Maino G. (1g6s) on the role of the ilear epitherium in the pathogenesis of

e4perimental cholera' Am' J' PatlL 532 ?ß3-279'

o,Brien AD., Chen M.F-, Holmes R.K, Kaper J. & Levine lYLM' (1984) Environmental and

human isolates oî. V cholcræ arrd V pøahaanotytiars produce a Shigella dysøtaiae I

(Shþ)-like cytotoxin' Iarcet' Lz 77 -78'

ohtomoN. (1972) safetyandpoæncytestsofchoreratoxoidl-otllinanimalsandvolunteers'

Proc. l2tlt Ioinf C-onføencc ø cholera' (tlS-Iapørt Cæpøative Medicgl Sciøce ho

gûn),úøPan PP' ?ß6-?96'

175

oppenheimer J.R, Ahmad IvLG., Huq d, Haque KA" Alam A.KM'A" Aziz Klvf's.' Ali s'

& Haque A,s.M. (1978) Limnological shrdies of three ponds in Dacca, Bangladesh' J'

Físhqics t l-?3-

o,shaugþnessy w.B. (1S31-1832) Proposal of a neu',method of treating the blue epidemic

cholera by the iniection of higþþ-o:rygenized salts into the venous syst'en LowÍ' lz

ffi-371.

orren IL L ( 1933) And noç, pathophysiolog of M cells - good neu's and bad ¡eu's from Peyer's

patches GaÑroantøotogt t5: 468-470'

onen RL, Pierce N.E, Apple R.T & Cray l\¡.CJr. (198ó) M cell transport of wbrío cholcræ

from the intestinal lumen into Peyer's patches: a mechanism for antigen sampling and

for microbial transepithelial migration. J- Infæt'Dís' 153: 1108-1118'

orren RL & Jones LL Qna) Epithelial cell specialization within humnn Peyer's patches:

an ultrastnrcûrral study o¡¡þsrinal þhoid follicles. Gøstoqúqobgt' lÍ¡t' 189-m3'

peterson IcM. & Mekalanos JJ. (198s) cha¡acterization of the lftrío e-Iølqæ ToxR regulon:

identification of novel ggnes invotved in intestinal colonizatiot Infe6' Immwt 562

28n 28Ð.

Peterson J.Sl., LoSpalluto JJ. & Finkebtein ILA" çnzìt Localization of cholera toxin hvövo'

I- Infect Dis- L2'62 617{128'

pierce N.E, (1g3) Differential inhibitory effects of cholera toxoids and gangliooide on the

enreroroxins of l4brb clpbræ atd&srhqúchia cotí. I. W- Me¿ 1i]7: 10(Þ-l0B.

Pierce N.E, Banwell J.G., Sack RB, Mitra R.C & Mondal A" (1970) Magnitude and duration

of antitoxic response to human infection with, l4brìo dtolqae. I- hfeá' D¡s' L2lz

s31-S35.

,t

È

*,

H

l,

176

Pierce N.E, Cray Jr. lv.c, trbper J.B. & Mekalanaos JJ. (1938) Determinants of im-

nunogenicity and mechanisms of protection by virulent and mutantwb¡o cholqæ'

Infee. Immun- 562 L42-148'

pierce N.E, Cray w.cJr. & Sircar BIC (1g7g) Induction of a mucosal antitoxin response and

its role in immunity to e:perimental canine cholera Infecl bnmun 21: 185-193'

Pierce N.E, Greenougþ \il.[ & Carpenter CCJ. (19?1) Wb¡o dpbæ enterotoxin and i¡g

modesof action. BacíRq' 35" t-13'

Pohlner J., Meyer TE, Jalaiakumari M.B. & M'nning P'A" (19Sóa) Nucleotide sequence of ii

ompv, the ggne for a m4 ot wbrio cttobæ oulr'I membrane protein' Moler' Gq^ GeneÍ'

Ð5: 494-500.

Pohlner J., Meyer TE & Manning P.,4,. (19S6b) Serological properties and processing in

E&hsíchío coti Kl2of OmpV fusion proteins of'Vîbrb úobæ' Mokt' Gq' Genet

ã)5: 501-506.

Pollitzer R. (1959) Chole¡a Monograph series No. 431\¡.H.O. Gener¡a'

Rabbanic.H. (198ó) Cholera Clh.Gastoqttqol LSz 507-528'

Rahanan M.M., A^ZKNLS., patwari y. & Munshi lvf"fl. QnÐ Diarrheal mortality in two

Baniladeshi vill4ges with and without community based oral rehydration therapy'

I-øcet.2: 8'09S12

Rappaport RS., Bonde G., McCann T, Rubin BA & Tint H. $nÐ Development of a

purified cholera toxoid" II. heparation of a stable, antþenic toxoid þ reaction of

purified toxinwith glutaraldehyde. Infect Immun u' 304-317.

Redmond J.!V. (1g7g) The structure of the o-antigenic side chain of the lipopoþsaccharide

olVíbrb clptqæ5698 (tnab"). B¡oehct L Bioplryx Acta. 5E/,: 346-352^

Re€dLJ.&MuenchH.A, (1g3S) Asimplemethodof estimatingfiftypercentendpoinß. Am

I. HYg. 272 493497.

I'

177

i¡I'i

Þ-

Richardson S.H. & Noftle trL,{. (1970) Purification and properties of permeability fac- i

tor/choleraenterotoxinfromcomplexandsyntheticmedia J.InfectDi^* L2lt s73-s?9' It

Riipkema s.G.T, Jansen .lV.H., Gielen H. & Guinée P.A'M. (1937) tmmunogtobulins in bile i

and serum of the rabbit associated with protection after Wbtúo eholsæinfection and i

vaccination Miøobiat Pælngan' 3: 365'375'

Rogers R.C, Cuffe R.G.CJ., Cossins Y.M, Murphy D.M. & Bourke A"TC (1980) The

eueensliand cholera incide¡t or.l977.II. The epidemiological investigation. BulI wld'

Hlth. Org. 5E: 6ó5-6ó9.

Rosenbergc.E (1%2) The chobøyeøt. chicago: university of chicago Press'

RutterJ.M.&JonesG.l\t(1973)ProtectionaginstentericdiseasecawdJ-byMærúchiacoli

- a model forvaccinationwith avirulence determinant? Nøture. A2z 531-532

Rutter J.M., Jones G.W., Brou¡n G.TH., Burrows M.R & Luther P'D' (1976) Antibacterial

activity in coloetrum and milk associated u'ith protection of piglets apinst enteric

disease caused by KSS-poe iwe Eç,clwíchia coli. hfecl Imtnt¿n t:3z 667 47 6'

Sack G.H., Pierce N.E, Henness€y KN., Mitra RC, Sack R'B' & Mazumdâf D'N'G ' (1972)

Gast¡ic acidity in cholera and non-cholera diarrhoea BulL Wld- HttlL otg' 472 3l'36'

sak^zaki R- & Shimada T (1y77') Serovars o|.Víbrb chobae. Jøp. J. Med sci BíoL 3lÌ

n9-28L

Salmaso S., Greco D., Bonfigtio 8., Castellani-Pastoris M., de Felip G', Bracciotti A"' Sitzia G"

congn 4., Piu G., Angion G., Barra L ,TamprenA" & Baine w'B' (1980) Recurrence

of pelec5rpod-aseociated cholera in Safdini¿ I'ûucd' 2t ll?A-llZ7'

samadi A,R, shahid N., Eusof 4., Yunus M., Huq lvLl, I(han M'U.' Rahman Às'M'lvf' &

Faruque A.SG. (1933) Classical lürio choleræ biotype displaces El Tor in

Banþdesh- I-anca t 805Sü/'

t78

sattler J., lViegandt H., Staerk J., Kranz T, Ronnebergef HJ., Schmidôerger R & zilgH'

(1975) Studies of the subunit stmcürre of choleragen' hropeot I BiochøL 572

309-316.

sanyal s.G, Alam K, Neogi P.KB., Huq M.I. & Al-Mahmud KA" (1983) A nerü cholera toxin'

I-Øcet 1z 1337.

Satterwhite TIC, Dupont H.L, Evans D.G. & Evans D'J' (1978) Role of Eschsichía colí

colonization factor antigen in acute diarrhea. Løtcet 2: 181-1M'

Schneider D.R. & parker c.D. (1g7g). Isolation and characterization of proteasedeficient

mutants o|'lhbrb cholqae t' InfæLDir lÍlt: 143-151'

schrank G.D. & verwey s/.E, (1976) Distribution of cholera orsaniôms in e:rperimeDltzlvíbrúo

chobæinfections: proposedmechanisms of pathogeneeis and antibacterial immunity'

Infect. hnmw- L3:' 195â3'

sciortino cv & Finkelstein RA. (1983) wbío clpbae exPresses iron-regulated outer

membraneproteinsinvívo.InfecLImmun42:99o-96.

Sears S.D., Richardson IG, YoungC, Parker CD. & Levine M'M' (1984) Evaluation of the

human immune responõe to outer memb¡ane proteins of lftrb cholqae Infecl

Irntnutt. 4: 439 444'

shahidN.S.,SamediA.R.,KhanM.U.&HuqM.I.(1984)Classicalvs'ElTorcholera:a

prospective famity study of a concurrent ouöreak I' Díû'Ds' R¿s' 2t 73-78'

Shandera \ryJC, Hefkin 8., Martin D.L, Taylor J'P', Maserang D'R' Wells J'G" Kelbt M.'

GhandhitrL,KaperJ.B.,[-eeJ.V&BlakePá'(1983)Persisænceofcholerainthe

United States. Am- I- Tlop' Med IIyg' 322 8l2Al7'

simon R, hiefer v & pührer,A. (1gs3) A broad hoet range mobilization qrctem lot h viw

genetic engineering transposon mutagenesis in gram negative bacteria Biotechnologt'

i

tz 784-791.

t79

singþton EL, Attwell Rl\t, Jang M.S. & Coì*'elt RR. (1982å) Influence of salinity and

nutrient concentration on survival and grcwrth oLl1brio dtobøein aquatic nüc¡ocosrns'

AppL Fnvirm- M¡qobíoL 43: 1081-1085'

Sing[eton F.L, Attwell R.W., Jangi M.S. & Cotwetl R.R' (1982b) Effects of temperature and

salinity ot vîbrío c]ølerægrosrth. Appt. hvírot. Miqobíol 44: 1O47-1058'

SminiaT, Janse E M. & lVilde¡s M.M. (1932) Antigen-trappingcells in Peyer',s patches of the

¡alu Scand I- immunol 16: 481-485'

smith H.L (199) Serotypingof non*,holeravibrios. L Clh. Micrabbl 10: 85-90'

smith H.W. & linggood M-a (19?1) Observations on tle pathogenic properties of the K88,

Hþ and Ent plasmids of Escl, erúchíacoli with particular reference to porcine diarrhea

I. Med Mísobiol 4; 467485'

sneller M.C. & Suobe¡ lv. (1936) M celts and host defense. I- InfæLDís' 154: 737:741'

Snou, J. (1S5Ð On ihc mofu of cønmtniætíot of choba M editbn London: John

Churchill

snyder J.D., Allega D.T, I-evine M.M., Craig J.P., Feeley J.c, Delvitt !v'E & Blake P'A'

(1931) Serologic shrdies of naturalþ acquired infectio¡ with'Víbrb e]øl¿rø serogrcuP

Ol in the Uniæd States. I- Infed'Dfu' 143: 182-187'

sprra \v.lvl, Sack RB. & Froehlich J.L (1981) Simple adult rabbit model lor víbrb choleræ

and enterotoxigenic Bc]1øi1hi4 coli dizrùea hfecr" hntnun- 32t 739-747'

Sæele F-J., chaicumpa rv. & Ronley D. (rg74) Isolation and biological properries of three

classes of rabbit antibodies to víbrb chotsæ. J. InfæLDts 13lh 93-103'

Stevenson G., Leavesly D.L, I-agnado C:4., Heuzenroeder M'W' & Mandng P'A (1985)

Purification of the 25 kDavibrb cholerae maior outer-membrane protein a¡d the

molecular cloningof is gene: ompv. European I. B¡ochstt 1¿ft: 385-390'

180

stocker B.A,D. (1g4g) Measurements of rate of mutation of flagellar antigenic phase in

Sabnotætla tphímühnt' J' Hye 472 398413'

st¡eeterP.R, BergF^L, Rouse B.TN., Batgalz.eR.E & Butcher E C (1983) Atissue-specific

endothelial cell molecule involved in þmphocyte homing Natute 33tz 4l'46'

svennerholm A.-M. (19?5) Experimental studies on cholera immunization. [v. The antibody

respo$e to formalized Wbrb cholqaeand purified endotoxinwith special reference to

protectivecapacity.Int.Archs.AtløgtApplhnmunoL4g:434452

sennerhorm A._M. (1gs0) The nahrre of protective immunity in cholera - r¡ crtorqa and

relared diûïtteAs E(ls. o. ouchterlony & J. Holmgren 43rd Nobel symposium'

Stockholm 1978. Basel: S' I(arger' pp' 171-184'

Sennerholm ,{ -M., Gothefors L, Sack D.,{., Bardhan P.IC & Holmgren J. ( 19S4a) I-ocal and

qntemic antibody responses and immunological memory in humans ¿fte¡ immunization

withcholeraBsubunitþdifferentroutes.Bulll44d'HlûLOrg'6'229Ú-918'

ñennerholm ^A.-M. & Holmgren J. $nq Synergistic protective effect in rabbits of im-

munization $t\wfuìo clplcræ lipopoþaccharide and toxinltoxoid" htfeã. I¡rvnun

t3z 735-740.

ñen¡erholm A--M., Jertborn MT Gothefors L, Iørim A"M'M'M', Sack D'À & Holmgren J'

(1gs4b) Mucosal antitoxic and antibacterial immunity after cholera disease and after

immunization with a combined B ¡ubunit-whole cell vaccine' l' hfeá' Dis' L49 :'

884S93.

TabtiengR., Wattanasri s., Echeverria P., seriwatana J., Bodhidaüâ L, Chatkaeomorakot '4"

& Rowe B. (lgsg) An epidemi c oi.víbrío chotsæ El lbr Inaba resistant to several

antibiotics with a coniugative Group C plasmid coding for Type II dihydrofolate

reductase in Thailan ð" AnL L TIop' Med Hyg' 41: 680ó86'

181

Tamaru T & Brou,n lV.R. (1985) IgA antibodies in rat bile inhibit cholera toxin-i¡duced

secretion in ilealtoops ¡n síûL Imrunologt 55: 579-583.

Þuxe R.V., Holmberg s.D., Dodin,{., wells J.G. & Blake P.A" (19SS) Epidemic cholera in

Mali: higþmortalityandmultiplercutesof transmissioninafamine arca. Epídem'Inf'

LW n9-2ß9.

Tøylor R.K, Miller vL, Furlong D.B. & Mekalanoo JJ. (19SÐ Use olphoA gene fusions to

identi$ a pilus colonization factor coordinateþ regulatedwith cholera toxin. hoc- NaL

Acod. Scû^ U-SA- Uz 7ß33-?ß37'

Taylor R., Shau, c, Peterson trL, Spears P & Mekalianoo J. (19SSa) Safe, live wbrto choleræ

vaccines? Vrccine 6: 151-154'

?ylor R.K Shaw cE, Sun D., Rhine J..A,., Peterson KM. & Mekalanos JJ' (1988b)

Molecular mechanisms or.Wbrío cholqaecolo¡ization and its prerrention Absrøcß of

24ih (,L1.-Iapø C-oopødive Mdicat Scíaæe hogrøn, Choba ød relatcd DiÛheal

Dúseaæs Pøtel Toþo, Japan' pp' 50-51'

Teppema J.S., Guinée PA.M., Ibrahim 4.4., Paques M. & Ruitenberg E J' (1937) In víw

adherence and colonization of Vibrb cløbæ strains that differ in hemagglutinating

activity and motility- Infecr.Immun 532 ÐI3-2IAZ

Tou¡bin H., staehelin T & Gordon J. (1979) Electrophoretic transfer of proteins from

poþacrylamide gels to nitrocelluloee sheets: procedure and some applicatio¡x hoc

NøtLAcd. ScLU-SA- 76: 435O-4354'

lamont E.C. & Boolego J.l\t (1935) Pilus vaccines' Væche' 3: 3-10'

Tveedy J.lvL, park Rlvá. & Hodgkiss w. (1%s) Evidence for the pfesence of fimbriae (pili)

on Vibrio species. J. GqL M¡súíoL 5t: B5'2M'

t82

uhlin B.E , Baga M., Goransson M., LindbergEP., Lund 8., Norgren M' & Normarks (198Ð

Genes determining adhesin formation in uropathogenic Escherichia coli Cun Top'

Microbíol hntnunol 1:1:&' 163-177'

{-[iiye ,4.., Nakatomi M., utsunomiya A., Mitsui K, sogne s., Iwanaga M' & Kobari K (1968)

Eryerimental cholera in mice. I. First report on the oral infection. Trop. med 1û

65-71.

underdown BJ. & Schiff M.J. (1936) Immunogtobulin A: strategic defense initiative at the

mucosal surface. AnnRev' bnmunoL 4z 3E94t7'

Vaerman J.-P., Deriik-I angendriês A, Rits M. & Delacroix D' (198Ð Neutralization of

cholera toxin þ ratbile secretory IgA antibodtç. Intrutnolo&t 54: 601603'

Van Heyningen s. (197) Choleratoxin. B¡oL Rct. 52: 509-549.

van Hgningen w.E, carpenter ccJ., Pierce N.E & Greenougþ w'B' (191) Deactivation

of cholera toxin þ gangtiooide. I.Infea. Dis- fi24z 415418.

vougþt llt (1s93) A Chrytø on cholqa ro, by reafurc. History swPø^+ hevøtiot and

Tleafinqt of thc Di,æase Phitadelphia: The EA. Davis company.

wanke cA, & Guerrant RL (1gS7) Small borvel colonization alone is a cauee of diarrhea

btfect. Imtru¿n. 552 197A-197;6'

watanabe Y. & Verwey lV.E (1%5) The preparation and properties of a purified mouse-

protective lþpoþsaccharide from the Ogawa subtype of the El Torvarie$ olVîbrío

chobæ. ln: hocetÅkgs ol fric Cholerø Raseodt Symposùnr' U'S' Deparment of

Health, Education and \trelfare publication SPHS 1328. \Vashington: Government

Printing Office. PP. 253-259'

lVata¡abe Y., Verwey W.H,, Guckian J.C, rili[iams H.R., Phillips PE & Rocha S'S' (1J]69)

Some of the properties of mouse protective antþns derived from Vibrio cholerae- T¿r'

ReP. BioL Med 272 n5-29E'

183

Watren R.H., Morgan EM, SonÉhla Y.N., Vanikiati B. & PhilliPs RA. (1959) Water a¡d

electrolyte sürdies in cholera r. cl¡r\^ hvest 3E: 1879-1889.

lVeissma¡ J.8., Dewiü W.E, Thompson J., Muchnick CN., Portnoy B.L, Feeley J.C &

Gangarosa F^J. (1974) A case of cholera in Texag ln3. Artt- I. Epífuníol. 10(}

487498.

lVestphal O. & Jann K (lf)65) Bacterial lipopolysaccharides. Extraction with phenol-water

andfurtherapplicationsof theprocedure. Metlødsíncøbolryùatechanístry- 5: 83-91.

Wilders lvf.M., Sminia T, Plesch B.E, Drexhage H..au, Welter¡reden F-E & Meuwissen S.G.

(19g3) Large mononuclear la-pooitiveveiled cells in Peyer's patchee. U. Localization

in ratPeyer's patches Intnwolog- 4Ez 461467'

Wolf J.L & Rye W.A" (1934) The membranous epithelial (M) cell and the mucooal immune

systen. Rev. Med 35: 95-112

\iloodruff JJ. & Clarke LM. (1987) Specific cell-adhesion mechanisms determining mi8fa-

tion pathways of recirculatinglymphocytes . Ann Rev. Immunol 5z Ðt'22'.

kid.y N.G. (1%S) The lattice spacingof cryrstalline catalase as an internal st^ndard of length

in elect¡on microscopy. J- Illtastrtrct- Res- A: 454'4&'

yamamoto T, tr(amano T, Uchimura M., Iwaaaga M. & Yokota T (19SS) Vbrb ùobae Ol

adherence to villi and þmphoid follicle epitheliumz in vìtro model using formalin-

treated human small intestine and correlation between adherence and cell-associated

hemaggtutinin lerrels' Infec| Dnmun 56z 32Al-3250'

yamamoto trC, Ichinoee Y., Nakasone N., Þnabe lvL, Nagahama M., Sakurai J. & Iwanaga M.

(1936) Identity of hemoþins produced by Vcholøæ non-Ol al'd V choløae o1,

biotype El Tor. Infeú- hnmwu SLz 9/7-93t'

184

yamemoto T & yokotaT (l9SS) Electron microecopicstuÛ ot.Víbrío e]øløaeOl adherence

to the mucus coat andvillus surface in the human small intestine. hfæt Imtrutn 56:'

n$-n5e.

yancey RJ., Willis D.L & Berry LJ. (199) Flagella-induced immunity against experimental

cholera in adult rabbits. Infæt bnmun 252 ffi'28'

ychiyama y. & Brou¡n W.R (1937) Specific antibodies to cholera toxin in rabbit milk are

pmotective egainst Wfu¡o choleræ-tnduced intc¡tinat secretion. Iwntnolog- 61: 543-

547.

185