The suitability of the ‘emergency’ foot-and-mouth disease antigens held by the International...

7/28/2019 The suitability of the ‘emergency’ foot-and-mouth disease antigens held by the International Vaccine Bank within a global context

http://slidepdf.com/reader/full/the-suitability-of-the-emergency-foot-and-mouth-disease-antigens-held 1/11

Vaccine 19 (2001) 2107–2117

The suitability of the ‘emergency’ foot-and-mouth disease antigensheld by the International Vaccine Bank within a global context

P.V. Barnett *, A.R. Samuel, R.J. Statham

Institute for Animal Health, Pirbright Laboratory, Ash Road , Pirbright, Woking , Surrey GU 24 0 NF , UK

Received 17 May 2000; received in revised form 28 September 2000; accepted 13 October 2000

Abstract

The International Vaccine Bank (IVB) based at the Institute of Animal Health (IAH) in Pirbright, United Kingdom (UK),

routinely monitors the suitability of the currently held strains of foot-and-mouth disease (FMD) vaccine virus, in anticipation that

vaccine may be required to control FMD outbreaks that pose a threat to member countries. Using primarily the two-dimensional

micro-neutralisation test (VNT), bovine polyclonal sera raised against each of the seven current ‘emergency’ antigens were utilised

to measure the relationship of IVB stocks to selected field isolates. The ‘O’ serotypes, Manisa and Lausanne, exhibited adequate

levels of cross-protection against most of the type ‘O’ field isolates examined. A22 Iraq 24/64 showed the broadest spectrum of

reactivity against the type ‘A’ field isolates examined and was supplemented by A15 Thailand 1/60. Some type ‘Asia1’ field isolates,

particularly those from South East Asia, showed antigenic difference to the Asia1 India 8/79 vaccine strain by VNT, but in-vivo

testing in the guinea pig model indicated this to be insignificant. The only ‘C’ serotype representative, C1 Oberbayern, may be one

of the least antigenically diverse of the current portfolio of bank antigens. Comparison of the serological and sequence data shows

that despite significant genetic variation between the field isolates examined the antigens held by the IVB should still proveefficacious in the field. © 2001 Elsevier Science Ltd. All rights reserved.

Keywords: Foot-and-mouth disease viruses; Vaccine; Antigenic relationships

www.elsevier.com/locate/vaccine

1. Introduction

Foot-and-mouth disease virus (FMDV) is exotic to

the member countries of the IVB, but the sporadic

occurrence of disease in Eastern Europe and in the

countries of trading partners poses a continuing threat.

Traditionally, control in the UK relied upon surveil-lance, rapid diagnosis, slaughter and movement restric-

tions. Future outbreaks may require a different

approach, including the use of ‘emergency’ vaccination,

due, in part, to the problem of disposing of large

numbers of slaughtered animals, together with the asso-

ciated welfare and environmental issues. To meet this

possible eventuality, the International Vaccine Bank

(IVB), established in 1985, maintains a bank of concen-

trated FMDV antigens stored over liquid nitrogen.

These can be rapidly formulated into vaccine as re-

quired by either the UK authority (The Ministry of

Agriculture, Fisheries and Food (MAFF)), or any of

the other IVB member countries (Australia, New

Zealand, Ireland, Norway, Sweden, Finland and the

associate member, Malta). The facility operates accord-

ing to the principles of Good Manufacturing Practice

(GMP) and currently holds licences from the UK De-partment of Health and the UK Veterinary Medicines

Directorate (VMD), for the production of FMD vac-

cines. Presently the IVB portfolio consists of 0.5 million

doses of each of seven strains of highly potent inacti-

vated FMD antigens, specifically O1 Lausanne, O1

Manisa, A22 Iraq, A24 Cruzeiro, A15 Thailand, C1 Ober-

bayern and Asia1 India, each strain having been se-

lected because of its wide antigenic spectrum. However,

the ever-changing global epidemiology requires the con-

tinued monitoring of contemporary viruses. Should an

outbreak threaten any one of the member countries, itwould therefore be possible to advise concerning the

appropriateness of the currently held antigens or the

need to acquire further strain(s).* Corresponding author. Tel.: +44-1483-232441; fax: +44-1483-

232448.

PII: S 0 2 6 4 - 4 1 0 X ( 0 0 ) 0 0 3 9 9 - 6

P.V . Barnett et al . / Vaccine 19 (2001) 2107–2117 2108

Little has changed in terms of the world distribution

of foot-and-mouth disease since the mid 60s, and read-

ers are referred to the recent detailed review by Kitch-

ing [1]. FMD is still endemic in many areas of the

world including Africa, Asia, the Middle East, India,

and South America.

Prophylactic vaccination against FMD ceased in Eu-

rope during 1990–1991 to allow the implementation of a harmonised policy and with it greater movement of

livestock and their products within the single market

(Directive 90/423/EEC). Since that time sporadic out-

breaks in countries in or bordering southern Europe

have maintained the threat of large outbreaks particu-

larly in the high density domestic livestock of mainland

Europe and the consequential risk to the UK [3]. FMD

remains endemic in Asiatic Turkey, and there have also

been viruses of FMD serotype ‘O’ in Bulgaria (1991,

1993 and 1996), Italy (1993), and Greece (1994 and

1996), and outbreaks due to serotype ‘A’ in the Former

Yugoslav Republic of Macedonia, and Albania (1996).

Since the breakdown of the Soviet Union into Inde-

pendent States, FMD has spread north from Iran,

Afghanistan and Turkey particularly into the trans-

Caucasian countries of Azerbaijan, Armenia and Geor-

gia and into Kazakhstan and neighbouring countries

constituting a significant threat to the borders of Eu-

rope. One of the FMD viruses isolated from the out-

breaks in Turkey, which had spread from neighbouring

Iran, was genetically and antigenically distinct and

prompted the development of a new vaccine from the

strain ‘A’ Iran ‘96. This vaccine was subsequently usedin Thrace to create a buffer zone and protect south-east

Europe. Genetically and antigenically distinct viruses

do occasionally arise emphasising the importance of

surveillance. Remarkably, three years later another

antigenically new strain of type A was identified in

Iran, termed A Iran ‘99, and this has also spread to

Turkey, again threatening Europe.

In eastern Asia, FMD was reintroduced into

Malaysia and there have been outbreaks due to virus

types ‘O,’ ‘A’ and ‘Asia1’; serotype O virus was intro-

duced into Taiwan Province of China (POC) in 1997

which resulted in around 3.8 million pigs being slaugh-

tered and more than 20 million doses of vaccine being

used to control the disease.

In 1999, FMD, due to type O, was declared in

Algeria. The disease rapidly spread both east and west

affecting Morocco and Tunisia. Although less extensive

than the previous epidemic of 1989–92 [4], the number

of outbreaks still reached 173 (160 in Algeria, 11 in

Morocco and two in Tunisia) involving almost exclu-

sively cattle [5]. Sequence studies and comparison to

recent isolates from West Africa suggested that the

virus was exotic to North Africa but related to strainsin West Africa and that it entered Algeria via illegal

movement of animals [5].

The latter part of 1999 saw the occurrence of out-

breaks of Asia 1 FMDV in Malaysia and Turkey.

Outbreaks caused by this serotype have been frequent

in Malaysia during the last decade. However, the out-

break of Asia1 in Turkey is of particular concern to the

To examine the antigenic relationships between iso-

lates from FMDV epidemics, of particular concern toIVB member countries, and the currently stored vaccine

strains we have primarily applied the well-established

two-dimensional virus neutralisation assay [6]. The epi-

demiological and genetic relationships of these isolates

have been examined by the analysis of nucleotide se-

quence data using phylogenetic techniques. The work

described here summarises these studies and evaluates

the relevance of the current portfolio of ‘emergency’

vaccine strains.

2. Methods

2 .1. Viruses

All the strains of FMDV were obtained from the

OIE/FAO World Reference Laboratory for FMD and

were grown on either baby hamster kidney (BHK-21)

cells or swine kidney cells (IB-RS2). The virus stocks

used for the micro-neutralisation tests were stored as

clarified tissue culture harvest material at −20°C in

50% glycerol.

2 .2 . Bo6ine polyclonal serum

Reference sera were derived from IVB cattle potency

tests using 21 day post-vaccination samples taken from

individually immunized animals receiving a half bovine

dose of antigen formulated as an aqueous aluminium

hydroxide/saponin vaccine. For each antigen, a pool of

sera from eight individual animals was used in the

serological tests.

2 .3 . Oligonucleotide primers

Oligonucleotide primer pNK72 labelled with a Cy5

amidite fluorescent dye for use with the ALFexpress™

automated sequencer was purchased from Pharmacia

Biotech. The sequences of primers used in this study are

detailed in Table 1.

2 .4 . RT -PCR

Reverse transcription-polymerase chain reaction

(RT-PCR) was performed using the primer set L463Fand NK61 essentially as described by Knowles and

Samuel [7].

2 .5 . Cycle sequencing

A fmol™ DNA sequencing kit (Promega, UK) which

utilises the method described by Murray [8] was used

according to the manufacturer’s protocol with the fol-

lowing amendments. Approximately eighty femto-moles

of cDNA template was used in the reactions and 1.5

pmol of the Cy5 amidite labelled pNK72 primer. Thereactions were heated to 94°C for 2 min and then

subjected to 30 cycles of the following programme on a

thermal heating block (Hybaid Ominigene, Hybaid

UK): 94°C for 1 min, 55°C for 1 min and 72°C for 1.5

min. The reactions were terminated by adding 4 ml of

Cy5 sequencing stop solution (Pharmacia Biotech. Swe-

den) and cooled to 4°C. The reactions were heated to

95°C for 3 min prior to loading on a ALF express™

DNA sequencer (Pharmacia Biotech, Sweden).

2 .6 . Analysis of sequencing data

The software AM V3.01 (Pharmacia Biotech, Swe-

den) was used to process the data which was then

exported as an ASCII text file onto an IBM compatible

personal computer where alignments were made manu-

ally in Wordperfect 6.1 before analysis using the soft-

ware EpiSeq v2.0 suite of computer programs written

by N.J. Knowles (IAH, Pirbright).

All pairwise comparisons were performed by giving

each base substitution equal statistical weight (ambigui-

ties were ignored). Phylogenetic trees were constructed

using the computer programme NEIGHBOR and theunpaired group mean averaging (UPGMA) method.

Dendrograms were plotted with the program DRAW-

GRAM. These programmes were from the PHYLIP 3.5c

phylogeny package ([9]). The UPGMA method con-

structs a tree by successive (agglomerative) clustering

using an average-linkage method of clustering. UPGMA

assumes a clock but the branch lengths are not opti-

mized by the least squares criterion. This makes the

method very fast and thus able to handle large data

2 .7 . Neutralisation assay

A two-dimensional micro-neutralisation technique

similar to that described by [6] was used. Briefly, dou-

bling dilutions of antibody were reacted with 0.5 log10

dilutions of virus for 1 h at room temperature, BHK-21

or IB-RS2 cells were added as indicators of residual

infectivity and the test incubated at 37°C for 3 daysprior to fixing and staining. Antibody titres were calcu-

lated from regression data as the log10 reciprocal anti-

body dilution required for 50% neutralisation of 100

tissue culture infective units of virus (log10 SN50/

100 TCID50). The antigenic relationship of viruses

based on their neutralisation by antibodies is given by

the ratio: ‘r’=neutralisation antibody titre against the

heterologous virus/neutralisation antibody titre against

the homologous virus. The significance of differences in

the values of ‘r’ obtained by the polyclonal antiserum

was evaluated according to the criteria of [10]. Gener-

ally, serological relationships between two viruses in the

range ‘r’=0.3–1.0 are indicative of reasonable levels of

cross protection whereas values less than 0.3 indicate

very dissimilar strains and the need to acquire or de-

velop a new vaccine strain.

2 .8 . In 6i 6o analysis

The potency (PD50) of the IVB strain Asia India 8/79

against field isolates Asia Thailand 4/95 and Asia Nepal

28/90 was determined in guinea pigs according to the

procedure described by Barnett and Statham [11] andcompared with that observed against the homologous

strain.

3. Results

3 .1. Serological relationships

3 .1.1. O1 Manisa and O1 Lausanne 6ersus O type field

isolates

According to the two-dimensional test neutralisation

results, adequate levels of cross-protection should be

attained by the high potency O1 Manisa vaccine

(PD50]112) against the field isolates examined, includ-

ing those from Greece in 1994, Philippines in 1995 and

the more recent Taiwan POC and Vietnam outbreaks

(Table 2).

A comparable study with O1 Lausanne (Table 2) also

indicates that adequate levels of cross-protection should

be attained from this vaccine strain against most of the

viruses examined. However, direct comparison of the

serological results and ‘r ’ values, indicate that the Lau-

sanne vaccine strain is unlikely to protect against cer-tain isolates, such as O Philippines 11/94 (‘r’=0.23**)

or O Algeria 2/99 (‘r’=0.16**).

Table 1

Oligonucleotide primers used for the RT-PCR molecular epidemiol-

ogy sequencing studies

Sense GeneVirusPrimer Primer sequence

(5% –3%)

Universal NegativeNK61 P2B GACATGTCCTCC

TGCATCTG

NK72 Universal Negative GAAGGGCCCAGGP2A

GTTGGACTC

O universal L ACCTCCRACGGGL463F PositiveTGGTACGC

Table 2

Serological relationships (r) between recent O FMDV field isolates

determined in 2D microneutralization tests using IVB bovine 21 day

post vaccinal O1 Manisa (lot 0163) and O1 Lausanne (lot 6.0.142C)

Virus Bovine antisera

O1 Manisa O1 Lausanne

1.00† – O1 Lausanne

1.00O1 Manisa –

0.5O Bulgaria 1/93 \1.00, 0.93

O Cambodia 3/92 \1.00 0.59

0.45O Cambodia 1/98 \1.00

\1.00, 0.79O Greece 4/94 \1.00

O Greece 38/94

0.21* – O Hong Kong 1/92

– 0.69 O Hong Kong 4/95

0.54 – O Hong Kong 5/95

O Italy 1/93 0.16, 0.12 0.05**

0.89O Morocco 1/91 –

0.81 0.28* O Philippines 2/95

0.45\1.00

O Philippines 9/95

– O Taiwan 10/97 0.85

0.43O Thailand 1/92 \1.00

0.150.35 O Turkey 19/91

\1.00O Vietnam 7/97 0.56

0.66O Algeria 2/99 0.16

0.28O Philippines 1/99 0.95

a, indicative of reasonable levels of cross protection; , indica-

tive of very dissimilar strains. Bold data represents a repeated series

of tests.† The (r) values shown represent the neutralization titre against

heterologous virus/neutralization titre against homologous virus.

* r significantly less than 1.0 (P=0.05).

** r significantly less than 1.0 (P=0.01).

Turkish isolates such as Turkey 8/99 and 10/99 with ‘r’

values of 0.11* and 0.17*, respectively (Table 3). These

viruses, together with those from epidemics from the

end of the last decade and earlier part of this decade,

which also exhibited a dissimilarity to the vaccine strain

(data not shown), warranted further investigation. Due

to the historical high potency of the Asia1 India vaccine

(PD50=61), it was assumed this strain would still beefficacious against these distantly related viruses. To

examine this, guinea pig adapted viruses of two serolog-

ically dissimilar field viruses, namely Nepal 28/90 and

Thailand 4/95 were tested by challenge against the IVB

Asia1 India vaccine strain (Table 4). As indicated, the

vaccine strain held by the IVB afforded acceptable

potency figures against these selected strains, suggesting

the vaccine could be expected to provide immunity

against these field isolates and that a further Asia 1

strain was not required by the IVB.

Table 3

Serological relationships (r) between recent Asia1 FMDV field iso-

lates determined in 2D microneutralization tests using IVB bovine 21

day post vaccinal Asia1 India 8/79 (735 lot L244) seraa

Bovine antiseraVirus

Asia1 India 8/79

Asia1 India 8/79 1.00†

Asia Cambodia 2/91 0.32, 0.27

Asia India 5/89 0.34, 0.29

0.17**, 0.23 Asia Israel 3/89

Asia Malaysia 11/92 0.43, 0.26

0.16**, 0.31 Asia Nepal 28/90

Asia Saudi 13/92 0.63, 0.78

Asia Thailand 10/94 0.22, 0.15*

Asia Thailand 4/95 0.24, 0.13*

Asia Malaysia 9/99 0.43

Asia Turkey 8/99 0.11*

0.17* Asia Turkey 10/99

tive of very dissimilar strains. Bold data represents repeated tests

using new high titre pool of bovine polyclonal sera from small scale

cattle potency test (18/8/97).† The (r) values shown represent the neutralization titre against

heterologous virus/neutralization titre against homologous virus.* r significantly less than 1.0 (P=0.05).

** r significantly less than 1.0 (P=0.01)

3 .1.2 . Asia1 India 6ersus Asia type field isolates

The IVB vaccine strain Asia1 India 8/79 may not

induce protection against some of the Asia1 field

viruses, in particular the isolates from South East Asia,here represented by Thailand 10/94 and 4/95, ‘r’ values

of 0.15** and 0.13**, respectively, and the more recent

Table 4

Guinea pig potency test results following vaccination with Asia1 India 8/79 and challenge with heterologous Asia1 field isolates

Potency*Challenge viruses Vaccine dilution

1/3 1/9 1/271/1 1/81 Controls

5/5 5/5 4/5 3/5 0/2 72.5Asia India 8/79 5/5†

4/5 4/5 3/55/5 3/5Asia Thailand 4/95 0/2 37.5

5/5Asia Nepal 28/90 5/5 3/5 4/5 3/5 0/2 46.7

† Number of animals protected/ number of animals per group.

* PD50 value as determined by the Karber method.

3 .1.3 . A15 Thailand , A22 Iraq and A24 Cruzeiro 6ersus

A type field isolates

The A22 Iraq vaccine strain held by the IVB was

shown serologically to have a wide antigenic spectrum

when studied against a number of diverse field isolates

(Table 5). The only exceptions being the more recent

isolates, Brazil 2/95, Malaysia 38/95 and Turkey 1/98

with ‘r’ values of 0.15**, 0.13** and 0.08**,respectively.

The serological cross-specificity indicated by the A15

Thailand strain generally appeared to meet the needs of

the member countries (Table 5), particularly in respect

to the isolates Brazil 2/95 and Malaysia 38/95, which

were antigenically distant from A22 Iraq. The only

major concern were the new variants of type ‘A’ FMD

viruses originating from Iran in 1996 which spread to

Turkey in 1997 and 1998, and the more recent Turkey

and Iran 1999 isolates. These were not antigenically

covered well by the current IVB type ‘A’ vaccine strains(micro-neutralization ‘r’ values of 0.08*, 0.13* and

0.10* for A Turkey 1/98, and 0.13*, 0.21 and 0.25 for A

Iran 22/99, against A22 Iraq, A24 Cruzeiro and A15

Thailand, respectively).

Another A variant was also isolated in Malaysia and

Thailand in 1997, however, samples proved difficult to

grow in tissue culture and were therefore not analysed

by the virus neutralisation assay. An alternative test,

the liquid phase blocking ELISA [12] indicated that

none of the IVB vaccine strains were likely to confer

protection (data not shown) and that another commer-

cially available vaccine strain, A Iran 6/94, may bemore applicable.

A more recent acquisition to the IVB’s portfolio, A24

Cruzeiro, which replaced the first batch of antigen of

the same strain acquired by the Vaccine Bank in 1985,

displayed a minimal relationship to all the field isolates

examined (Table 5). This antigen is currently consid-

ered to be one of the least important vaccine strains,

and was not examined further, since the A22 Iraq and

A15 Thailand strains appear to offer adequate cover.

3 .1.4 . C 1 Oberbayern 6ersus C type field isolatesC1 Oberbayern may be one of the least antigenically

diverse of all the current vaccine strains held by the

IVB. All four isolates examined by VNT appear to be

serologically dissimilar to the vaccine strain (Table 6).

However, the number studied reflect the C’s received by

the World Reference Laboratory in the last few years.

These same field isolates were examined by liquid phase

blocking ELISA (data not shown) and, in particular,

confirmed the dissimilarity of Bangladesh 1/92 and

Bhutan 2/91. Therefore, like the Asia1 India study,there is a need to examine the efficacy of this vaccine

strain and three of these isolates namely, Bangladesh

1/92, Nepal 1/94 and Philippines 4/90 will undergo

in-vivo protection studies.

3 .2 . Sequence analysis

3 .2 .1. O1 serotypes

Compared to O1 Manisa (Fig. 1), the O Greece 4/94

virus demonstrated a 9% difference in the sequence

between nucleotides 475–639 (representing amino acids158–213 of VP1) and similar levels of difference were

observed with Bulgaria 1/93, Morocco 1/91, Italy 1/93

and Turkey 19/91. The differences in sequences exhib-

ited by the Taiwan, Philippine and Vietnam isolates

were considerably greater (18%). However, it should be

noted that although a small percentage of nucleotide

differences are an indication of the similarity of an

isolate to the vaccine strain, a high percentage of

sequence difference does not necessarily equate to an

inappropriateness of the vaccine strain being compared.

A case in point is the O1 Manisa strain, still effective at

protecting against the Taiwanese outbreak despite an18% difference in the compared sequences. Sequence

comparison of the Algerian 2/99 with the more serolog-

ically related O1 strain Manisa recorded a 15% differ-

ence between nucleotides 475– 639 of VP1. In many

cases the sequence relationships between the same se-

lected field isolates and O1 Lausanne [2] (Fig. 1) showed

even greater differences (]18%) than those observed

with O1 Manisa.

3 .2 .2 . Asia1 serotypes

Sequence comparison of Asia isolates, such as Thai-land 10/94 and 4/95 and the vaccine strain Asia1 India

also revealed nucleotide differences of around 14%

(Fig. 2). In contrast, the sequence examined from Saudi

13/92 was identical to the Asia1 vaccine strain and it is

therefore a probability that this outbreak was caused by

the vaccine. The three years between the occurrence of

the Indian strain, which has been used as a vaccine, and

the 1992 outbreak in Saudi Arabia could have been

expected to show, at a conservative estimate, 3% differ-

ence in nucleotide homology. The recent Malaysian

isolate (May 9/99) belongs to a Far Eastern genetic

lineage. However, the Turkish isolates (Tur 8/99 and

10/99) show close sequence homology to strains iso-

Table 6

Serological relationships (r) between recent C FMDV field isolates

post vaccinal C1 Oberbayern (batch 320) seraa

Bovine antiseraVirus

C1 Oberbayern

C1 Oberbayern 1.00†

0.13**C Bangladesh 1/92

C Bhutan 2/91 0.20**

C Nepal 1/94 0.16**

0.21**C Philippines 4/90

tive of very dissimilar strains.†

The (r) values shown represent the neutralization titre againstheterologous virus/neutralization titre against homologous virus.

Table 5

Serological relationships (r) between recent A FMDV field isolates

post vaccinal A22 Iraq 24/64 (lot 661), A24 Cruzeiro (lot 4219) and

A15 Thailand 1/60 (lot 4220) seraa

Virus Bovine antisera

A22 Iraq A24 Cruzeiro A15 Thailand

1/6024/64

A22 Iraq 1.00† – –

– A24 Cruzeiro 1.00 –

A15 Thailand – – 1.00

0.20A Albania – 0.26*

A Brazil 3/93 0.11* 0.11* –

0.12* 0.32 A Brazil 2/95 0.15*

0.14* 0.36 0.37A Iran 6/94

0.370.15*A Malaysia 0.13*

A Saudi 0.12** 0.13* 0.55

0.25A Saudi 0.09* 0.12**

– – 0.70 A Thailand

0.48A Thailand 0.10* 0.22

0.47 0.14* – A Turkey1/92

0.28 0.12**A Turkey 0.18*

0.08** A Turkey 0.10*0.13*

A Zambia 90 0.05**0.11*0.29

0.13*A Iran 22/99 0.25 0.21

tive of very dissimilar strains.† The (r) values shown represent the neutralization titre against

heterologous virus/neutralization titre against homologous virus.* r significantly less than 1.0 (P=0.05).

lated in the Indian sub-continent, and to those found in

Iran (#2.5%) during 1999.

3 .2 .3 . A serotypes

Sequence comparison of all these field isolates with

A24 Cruzeiro [21], A22 Iraq or A15 Thailand showed

differences in many cases of \14% (Fig. 3), and even

Fig. 1. Dendrogram depicting genetic relationships between selectedfield isolates of FMD virus serotype O and IVB antigen strains O1

Lausanne and O1 Manisa (bold).

Fig. 2. Dendrogram depicting genetic relationships between selected field isolates of FMD virus serotype Asia1 and the IVB antigen strain Asia1

India (bold).

as high as 24% for one isolate, Zambia/90. The closest

sequence comparisons, 8% against A22 Iraq, were the

isolates from Albania (1/96) and Saudi Arabia (16/95)which suggests that both of these outbreaks were

caused by a Middle East strain of type A. The new

variant of FMD virus type A, first isolated from sam-

ples originating from Iran in 1996, was confirmed to be

genotypically similar to isolates from later outbreaks in

Turkey in 1997 and 1998. However, the more recent A

isolates found in Iran and Turkey in 1999, which are

now appearing in Iraq, are also antigenically and geno-

typically unique and very different from the 1996 vari-

ant. Current evidence suggests that both these variants

are co-existing in the susceptible population and like

the A Iran ‘96 the development of another A vaccine

strain to control this outbreak is conceivable. The

inadequacies of current vaccines and the need to de-

velop further A vaccine strains is a good indication of

the problems still caused by this serotype.

3 .2 .4 . C serotypes

Sequencing showed that all four isolates in the panel

exhibited 18% or greater nucleotide differences in the

region examined on comparison with C1 Oberbayern

vaccine strain (Fig. 4). Distinct differences between the

Asian isolates (Bangladesh 1/92, Bhutan 2/91 and CNepal 1/94) and the Far East isolate C Philippines 4/90

were also evident.

Fig. 3. Dendrogram depicting genetic relationships between selectedfield isolates of FMD virus serotype A and IVB antigen strains A24

Cruzeiro, A15 Thailand and A22 Iraq (bold).

Fig. 4. Dendrogram depicting genetic relationships between selected field isolates of FMD virus serotype C and the IVB antigen strain C1

Oberbayern (bold).

4. Discussion

Emergency vaccination can play an important role in

the control of FMD outbreaks in countries normally

free of FMD. This was demonstrated in 1966 in Sweden

when the policy was used as an adjunct to stamping out

to control a single outbreak and eradicate the causal

virus. The storage of conventional formulated FMD

vaccines in a strategic reserve for such eventualities is

expensive as vaccine must be replaced every 18 months

due to limited shelf-life. An alternative to this costly

practice is the well-established method of indefinitestorage of concentrated, inactivated, FMD antigen at

ultra-low temperatures over liquid nitrogen. Cryogeni-

cally stored antigen can be rapidly formulated into

vaccine when required. This concept is not unique to

the IVB, and the recent establishment of a European

Community FMD antigen reserve and other examples

of individual countries establishing their own FMD

reserves, indicate the increasing popularity of this ap-

proach. However, the IVB is unique in that it also

houses its own dedicated manufacturing facility for the

emergency formulation of FMD vaccine, which can be

despatched within 2– 3 days of receiving a request.Other advantages of this system include choice of adju-

vant during formulation, and regular quality assurance.

FMD virus is antigenically very diverse, existing as

seven major serotypes with numerous sub-types, and

strains selected for incorporation into vaccines must be

antigenically similar to current field strains of FMDV,

to ensure an optimal immune response and protection.

Arguably the most important advantage of storing

concentrated antigens is the versatility to choose the

most applicable vaccine strain to deal with an epidemic

rather than be reliant on the strain/s already incorpo-rated in a pre-formulated vaccine. Vigilant global

surveillance of FMD viruses is of extreme importance

in order to advise on the protection afforded by a givenvaccine strain, the necessity for additional antigen

strains, and to maintain the ability to respond rapidly,

should an infection escalate and threaten any member

country of the IVB.

The seven strains currently held by the IVB were

accepted following the demonstration of a high potency

value in cattle (greater than 10 PD50) when formulated

with Al(OH)3 and saponin. Such high potency vaccine

is essential where the primary aim of the emergency

programme is to prevent the spread of FMDV as

rapidly as possible. In this respect, the authors havereported on the rate of development of protection in

cattle, sheep and pigs following vaccination with some

of these emergency vaccines and demonstrated protec-

tion against natural airborne infection by pig challenge

within 4 days of immunization [13–16]. All the antigens

produce vaccines of extremely high potency and offer

protection against a broad range of field isolates.

In this study, the authors have monitored the appli-

cability of all the IVB antigen strains against current

and past virus outbreaks by in vitro and, where neces-

sary, in vivo methods.

Serological results indicate that adequate levels of cross-protection should be attained against ‘O’ isolates

using O1 Manisa or O1 Lausanne and against ‘A’

isolates using either A22 Iraq or the more recently

acquired A15 Thailand. The only important exceptions

being the new type ‘A’ variants isolated from Iran and

Turkey in 1996 and 1999 and those from Thailand and

Malaysia in 1997. Indeed, none of the available vaccine

strains were likely to confer protection against the Iran

and Turkey strains which created the need for the

commercial sector to develop new ‘A’ type vaccine

strains. Acquisition of strains such as A Iran ‘96 by theIVB is a matter being considered by the Technical

Committee. In light of the risk to South East Europe

and thereby the European Union, as outlined at the

General Session of the European Commission for the

Control of Foot-and-Mouth Disease in FAO Rome,

this may be acquired at some future date. Both A22

Iraq and A15 Thailand compensate for the limited

antigenic spectrum displayed by the A24 Cruzeiro vac-

cine strain against the field isolates studied. The A24

Cruzeiro virus, originating from Brazil in 1955, is awell established and historically important antigen but

is considered to be of less relevance today. Improved

cross-specificity was observed from use of bivalent

A22/A24 vaccine tested against two unrelated strains,

Brazil 3/93 and Malaysia 38/95 (unpublished results).

In some instances, therefore, the mixing of two or

more vaccine strains has a synergistic effect and

improves efficacy against antigenically unrelated iso-

lates.

According to micro-neutralisation results, Asia1 In-

dia vaccine may not confer protection against someof the Asia1 field isolates, particularly those from

South East Asia. Repeat testing of this panel of

viruses using a new pool of bovine Asia1 anti-sera

substantiated some of the previous results, highlight-

ing Thailand 10/94 and 4/95 in particular as being

serologically different. However, the ‘r’ values for

many of the other isolates were also low, i.e. B0.3,

and therefore could also be considered as dissimilar.

The appropriation of an additional Asia1 vaccine

strain, specifically Asia1 Shamir, was considered, and

further in vivo tests were carried out using guinea pig

adapted isolates. The results of these challenge experi-

ments appeared to indicate Asia1 India vaccine would

provide adequate protection against the isolates

tested, namely Thailand 4/95 and Nepal 28/90. A

similar formulation of Asia1 Shamir vaccine was also

compared against these two isolates and the resulting

elevated PD50 values supported the wide spectrum of

serological protection observed (data not shown). The

latter part of 1999 saw the occurrence of outbreaks of

Asia 1 FMDV in Malaysia and Turkey. Outbreaks

caused by this serotype have been frequent in

Malaysia during the last decade. However, the out-break of Asia1 in Turkey is of particular concern to

the IVB. Although the serological results indicated

that Asia1 India 8/79 should be effective against the

Malaysian isolate its appropriateness for the Turkish

isolates was in doubt. For this reason the candidate

vaccine strain, Asia1 Shamir, was also examined sero-

logically against these Turkish isolates and showed

that it would be a better choice for field application.

Therefore, despite the indication from in vivo studies,

that Asia1 India should provide the adequate protec-

tion required by the member countries, more confi-dence could be placed on the Asia1 Shamir strain,

and therefore this remains a strong candidate for in-

clusion in the IVB portfolio, particularly if future

outbreak strains become increasingly dissimilar to the

current IVB vaccine strain.

The only type ‘C’ representative in the bank’s re-

serve, C1 Oberbayern, is possibly the least antigeni-

cally diverse of the current portfolio of IVB antigens.

Less importance has been placed on this particular

serotype since it has been absent throughout theworld in recent years as judged from the isolates re-

ceived by the WRL. This is reflected in the small

panel of four viruses examined by both neutralisation

and liquid phase blocking ELISA (data not shown)

which showed this antigen to have narrow specificity.

In a similar analysis to that involving the Asia1 India

vaccine strain, the authors are now examining the

efficacy of C1 Oberbayern by in vivo challenge

against selected field isolates.

Although the classical serotypes have been shown

to have good correlation with genetic groupings byphylogenetic analysis, and to some extent also the

subtypes [17], there is no direct link between percent-

age sequence homology and antigenic relationships. It

has been reported that quite distantly related isolates

may have similar antigenic characteristics due to

molecular mimicry at important antigenic sites [18,19].

Conversely, very close sequence homology may con-

ceal large antigenic differences. [20] described a mon-

oclonal antibody escape mutant which was isolated

by serial selection with a panel of five neutralizing

monoclonal antibodies. This mutant virus is able to

evade neutralization by polyclonal antisera elicited

against the parental virus (O1/Kaufbeuren/FRG/66).

However, if a comparison is made between the mu-

tant virus and the parent strain the percentage nucle-

otide difference is only 1.17%. This clearly

demonstrates the danger in making the assumption

that if two isolates are closely related genetically that

the same vaccine can be used. It is important there-

fore that, when a field isolate is studied, appropriate

techniques are used simultaneously to gain the re-

quired information and enable a well thought out dis-

ease control programme to be implemented. Theserological tests used in this study, are able to show

the likely efficacy of vaccines to control outbreaks.

The sequence data, on the other hand, by reference

to a large database of virus sequences provides more

detailed information on possible sources of the strain

causing an outbreak. This is particularly highlighted

by the Asia1 Saudi 13/92 isolate whose sequence

was identical to that of the Asia1 India vaccine strain

and strongly suggests that incompletely inactivated

vaccine may have been applied in the field. Phyloge-

netic analysis can pinpoint where animal movementsor importation may have been responsible. Decisions

to implement effective animal control measures

can then be taken on a more informed and rational

basis.

Another aspect of sequence studies are that if some

isolates are found to be genetically divergent to existing

isolates it may highlight the need to study these isolates

more fully with serological tests to gain further insight

into their antigenic properties. The recent appearance

of genetically and antigenically distinct outbreakviruses in Turkey in 1996 and 1999, that prompted the

development of new vaccine strains, appear to be excel-

lent candidates. In the future it would be advantageous

to be able to predict the likely antigenic consequences

of changes in sequence. With the continuing accumula-

tion of data and studies with monoclonal antibodies

this could ultimately be a distinct possibility.

All the antigens stored in the IVB are tested annually

for stability and efficacy in vivo and in vitro incorporat-

ing guinea pig potency tests, sucrose density gradient

and SDS– PAGE analysis. The antigens are therefore

unique in respect to the amount of quality control

testing undergone. In many cases these antigens have

also been examined experimentally with differing adju-

vant formulations and for the rapidity with which they

confer protection in the target host. This permits a high

degree of confidence in vaccine stability, potency and

efficacy. The only remaining area of doubt is antigenic

relevance to current and future FMD outbreaks. Retro-

spective studies have shown that the criteria used to

select the majority of the bank’s antigens though intu-

itively based were accurate. Techniques now available

allow the selection to be more scientifically based.Nevertheless, the appropriation of further strains re-

mains an important consideration particularly with re-

gard to the Asia1 and C serotypes, which are each only

represented by a single strain, and the newly developed

and antigenically unique A Iran ‘96 vaccine strain.

These observations underline the importance of contin-

ually surveying and characterising field isolates.

Acknowledgements

This work was supported financially by MAFF, UK(project number SE2805).

References

[1] Kitching RP. A recent history of foot-and-mouth disease. J

Comp Pathol 1998;118:89–108.

[2] Beck E, Strohmaier K. Subtyping of European foot-and-mouth

disease virus strains by nucleotide sequence determination. J

Virol 1987;61:1621– 9.

[3] Donaldson AI, Doel TR. Foot-and-mouth disease: the risk for

Great Britain after 1992. Vet Rec 1992;131:114–20.

[4] Samuel AR, Knowles NJ, Mackay DKJ. Genetic analysis of typeO viruses responsible for epidemics of foot-and-mouth disease in

North Africa. Epidemiol Infect 1999;122(3):529– 38.

[5] Mackay DKJ. Serological surveillance for FMD in North

Africa. Report, Closed Session of the Research Group of the

Standing Technical Committee of the European Commission for

the Control of Foot-and-Mouth Disease and the Foot-and-

Mouth Disease Subgroup of the Scientific Veterinary Committee

of the Commission of the European Community, Paris, 1999.

[6] Booth JC, Rweyemamu MM, Pay TWF. Dose response relation-

ships in a microneutralization test for foot-and-mouth disease

viruses. J Hyg 1978;80:31–42.

[7] Knowles NJ, Samuel AR. Polymerase chain reaction amplifica-

tion and cycle sequencing of the ID (VP1) gene of foot-and-

mouth disease viruses. Report, Closed Session of the Research

Group of the Standing Technical Committee of the European

Commission for the Control of Foot-and-Mouth Disease, Vi-

enna, Austria, Rome, Appendix 8, 1994: 45–53.

[8] Murray V. Improved double-stranded DNA sequencing using

the linear polymerase chain reaction. Nucleic Acids Res

1989;17:88–9.

[9] Felsenstein J. PHYLIP — Phylogeny inference package (version

3.2). Cladistics 1989;5:164– 6.

[10] Rweyemamu MM, Hingley PJ. Foot-and-mouth disease virus

strain differentiation: analysis of the serological data. J Biol

Stand 1984;12:323– 37.[11] Barnett PV, Statham RJ. Long term stability and potency of

antigen concentrates held by the International Vaccine Bank.

Report, Session of the Research Group of the Standing Techni-

cal Committee of the European Commission for the Control of

Foot-and-Mouth Disease and the Foot-and-Mouth Disease Sub-

group of the Scientific Veterinary Committee of the Commission

of the European Community, UK. Appendix 38, 1998: 272–5.

[12] Kitching RP, Rendle R, Ferris NP. Rapid correlation between

field isolates and vaccine strains of foot-and-mouth disease virus.

Vaccine 1988;6:403– 8.

[13] Doel TR, Williams L, Barnett PV. Emergency vaccination

against foot-and-mouth disease: rate of development of immu-

nity and its implications for the carrier state. Vaccine

1994;12(7):592–600.[14] Cox SJ, Barnett PV, Dani P, Salt JS. Emergency vaccination of

sheep against foot-and-mouth disease: protection against disease

and reduction in contact transmission. Vaccine 1999;17:1858–68.

[15] Salt JS, Williams L, Statham R, Barnett PV. Further studies on

the rate of development of protection in cattle given emergency

vaccination against FMD. Report Session of the Research

Group of the Standing Technical Committee of the European

Commission for the Control of Foot-and-Mouth Disease and the

Foot-and-Mouth Disease Subgroup of the Scientific Veterinary

Committee of the Commission of the European Community,

Moeldling, Vienna, Austria, Appendix 17, 1995: 90–7.

[16] Salt JS, Barnett PV, Dani P, Williams L. Emergency vaccination

of pigs against foot-and-mouth disease: protection against dis-

ease and reduction in contact transmission. Vaccine1998;16(7):746–54.

[17] Martinez MA, Dopazo J, Hernandez J, Mateu MG, Sobrino F,

Domingo E, Knowles NJ. Evolution of the capsid protein genes

of foot-and-mouth disease virus: antigenic variation without

accumulation of amino acid substitutions over six decades. J

Virol 1992;66:3557– 65.

[18] Samuel AR, Knowles NJ, Kitching RP. Serological and bio-

chemical analysis of some recent type A foot-and-mouth disease

virus isolates from the Middle East. Epidemiol Infect

1988;101:577–90.

[19] Hernandez J, Martinez MA, Rocha E, Domingo E, Mateu MG.

Generation of a subtype-specific neutralization epitope in foot-

and-mouth disease virus of a different subtype. J Gen Virol

1992;73:213–6.[20] Crowther JR, Farias S, Carpenter WC, Samuel AR. Identifica-

tion of a fifth neutralizable site on type O foot-and-mouth

disease virus following characterization of single and quintuple

monoclonal antibody escape mutants. J Gen Virol

1993;74:1547–53.

[21] Weddell GN, Yansura DG, Dowbenko DJ, Hoatlin ME, Grub-

man MJ, Moore DM, Kleid DG. Sequence variation in the gene

for the immunogenic capsid protein VP1 of foot-and-mouth

disease virus type A. Proc Natl Acad Sci USA 1986;82:2618–

The suitability of the ‘emergency’ foot-and-mouth disease antigens held by the International...

Documents

Transcript of The suitability of the ‘emergency’ foot-and-mouth disease antigens held by the International...

Practical Blood Bank Lab 1 ABO Grouping. ABO blood group antigens present on red blood cells (Natural Antigens)antigens IgM antibodies present in.

Antigens and antibodies interact…

Immunogens, Antigens, and Haptens

Antigens & Antibodies Teacher - elysciencecenter.comelysciencecenter.com/yahoo_site_admin/assets/docs/Antigens__Anti… · Antibodies Background Transfusion perrormea. Concepts Antigens

Nature of antigens

Characterization of Candida albicans Antigens with ... · Monoclonalantibody 15C9reactedwith antigens of87, 50, and34kDapresentin the germtube extract andwith antigens of92, 50, 34,

Antigens and Immunogens

Antigens - repository.limu.edu.ly

Lec6.Immunogenes or Antigens

BMLS 3 Antigens

3. Antigens

Cancer Antigens

4. Erythrocyte Antigens & Antibodies

Blood Group Antigens and Antibodies - New York … Group Antigens and Antibodies 2 Blood Group Antigens and Antibodies • Blood Group Immunology/ •Pre-transfusion Testing • ABO

5. Leukocyte Antigens

Efficient generation of monoclonal antibodies against ...€¦ · Monoclonal antibodies speciﬁc for foreign antigens, auto-antigens, allogeneic antigens and tumour neo-antigens

Immunogenes or Antigens

CHAPTER 3 RESEARCH METHODOLOGY - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/6106/8/08_chapter 3.pdf · CHAPTER 3 RESEARCH METHODOLOGY ... 2 Suitability for word of mouth

Antigens & immunogensF.ppt

Antibodies and antigens Antibodies = immunoglobulins Antibodies bind antigens.