Laboratory of Immunobiochemistry Research review Jay E. Slater, MD OVRR/DBPAP 18 March 2009.

Laboratory of Immunobiochemistry

Research review

Jay E. Slater, MDOVRR/DBPAP

18 March 2009

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LIB Research Program

Projects Publications Support

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Projects

Rabin Characterization of innate immune

responses to respiratory syncytial virus

Slater Endotoxin in mite extracts Multiplex allergen extract potency

assay

Bacterial endotoxin and DNA in house dust mite cultures and extracts

Cherry ValerioLarry Arlian, PhD

Patrick Murray, PhDBhavini Trivedi, MD

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Initial studies Endotoxins are present in many

standardized allergen extracts Cat and mite > pollens Cat pelt > cat hair D. farinae >> D. pteronyssinus Next step:

Investigate differences between D. farinae and D. pteronyssinus using live mite cultures

Trivedi B, Valerio C, Slater JE. Endotoxin content of standardized allergen vaccines. J Allergy Clin Immunol 2003; 111:777-783.

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Can we detect bacterial DNA in live mite cultures?

Extract genomic DNA from fresh, washed mites

Amplify with 16S rRNA sequences Quantify using internal standards Sequence after high fidelity

amplification Identify predominant organisms

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DNA from mites D. farinae D. pteronyssinus

EcoR1 digests

undigested DNA

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0

20

40

60

80

100

120

0.1 1 10 100DNA (ng)

Ba

nd

de

ns

ity

Df

Dp

TM = 42

Dp Df

slope 0.02 0.02

int 0.57 -0.55

r^2 0.97 0.98

TM equiv (ng) 19.57 1.45

TM number (copies) 1500 1500

copies/ng 77 1033

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16S rRNA sequences recovered

Bartonella species B. henselae B. quintana B. vinsonii B. elizabethae

E. coli Pseudomonas species Acinetobacter species

Uncharacterized -proteobacteria endosymbionts from

Ixodes scapularis Vestimentiferan

tubeworms Brevipalpus

phoenicis Metaseiulus

occidentalis Aspidiotus nerii

Valerio CR, Murray P, Arlian LG, Slater JE. Bacterial 16S ribosomal DNA in house dust mite cultures. J Allergy Clin Immunol. 2005; 116:1296-300.

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Bartonella organisms Gram-negative

rods 0.6 by 1.0 m facultative intracellular fastidious

Harbored by Lice Fleas Ticks Hippoboscidae

flies (house dust mites)

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Bartonella-associated diseases Zoonotic: cat-scratch disease (B. henselae) Louse-borne (Pediculus humanus) (B. quintana)

trench fever urban trench fever

Sandfly-borne (Phlebotomus) (B. bacilliformis) Oroya fever (Carrion’s disease) verruga peruana

Uncertain transmission (B. henselae and B. quintana)

bacillary angiomatosis bacillary peliosis culture-negative endocarditis

Emerg Infect Dis 1995; 1(1):16-21.N Engl J Med 1997; 337(26):1916-7.

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Conclusions (1) D. farinae allergen extracts contain more

endotoxin than D. pteronyssinus extracts No evidence of adverse events associated with

endotoxin in allergen extracts Culture data uninformative Analysis of amplified mite DNA suggests

the presence of about 10-fold more bacterial DNA in D. farinae than in D. pteronyssinus

Sequence analysis of recovered bacterial DNA indicates the presence of Bartonella species as well as other Gram-negative organisms No evidence of iatrogenic infection

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Next questions

Are the bacterial DNA sequences detectable in commercial allergen extracts?

Are the bacterial DNA sequences detectable wild mite species?

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Are the bacterial DNA sequences detectable in commercial allergen extracts? Methods

DNA isolation by QIAamp

PCR

Sequence

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Are the bacterial DNA sequences detectable in commercial allergen extracts?

DNA present? fD1 fD2D farinae (13) 12/13 5/13 6/13D pteronyssinus (14) 12/14 0/14 0/14Cat hair (2) 0/2 0/2 0/2Cat pelt (2) 0/2 0/2 0/2German roach (3) 2/3 1/3 1/3Honeybee venom 0 0 0Bermuda grass 0 0 0Ryegrass 0 0 0

PCR

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Chortoglyphus arcuatus Lepidoglyphus destructor Euroglyphus maynei Acarus siro Tyrophagus putrescentiae

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Extract genomic DNA (DNAzol) from fresh, washed mites

Amplify with 16S rRNA sequences Sequence after high fidelity

amplification (pfx) Identify predominant organisms

(BLAST)

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16S rRNA sequences recovered

Forward Reverse Bartonella henselae strain M40SHD Bartonella henselae strain M40SHD

A. siro Bartonella henselae strain 882_ANT5 Bartonella henselae strain 882_ANT5 Bartonella henselae strain 882_ANT5 Bartonella henselae strain Houston-1 Bartonella australis strain Aust/NH1 Bartonella sp.

C. arcuatus Bartonella clarridgeiae strain M9HN-SHQ Bartonella clarridgeiae strain M9HN-SHQ Bartonella sp. Bartonella rattiaustraliensis strain AUST/NH14 Bartonella henselae strain M40SHD Bartonella henselae strain M40SHD

E. maynei Bartonella henselae strain 882_ANT5 Bartonella henselae strain 882_ANT5 Bartonella henselae strain Houston-1 Bartonella henselae strain Houston-1 Bartonella henselae strain M40SHD Bartonella henselae strain M40SHD

L. destructor Bartonella henselae strain 882_ANT5 Bartonella henselae strain 882_ANT5 Bartonella henselae strain Houston-1 Bartonella henselae strain Houston-1

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Conclusions

D farinae endotoxin content is high, and associated with the presence of Bartonella DNA

Confirmed in Mites Mite extracts One wild mite species (C arcuatus)

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Next steps

Population analyses Bartonella culture Endotoxin analyses

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An Antibody-Based Multiplex Bead Assay to Determine the Potency and Composition of Allergen Extracts

Nicolette deVore, PhD

Jonny Finlay, PhD

Susan Huynh

Ekaterina Dobrovolskaia

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How do we measure potency?

Total protein (hymenoptera) Overall allergen (grasses, mites)

Pooled human antibody Specific allergen (cat, ragweed)

Sheep antibody

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Specific loss of a single allergen

0

0.1

0.2

0.3

0.4

0.5

-6-4-20

log dilution

resp

onse

(a

bsor

banc

e)

Soldatova LN, Paupore EJ, Burk SH, Pastor RW, Slater JE. The stability of house dust mite allergens in glycerinated extracts, J Allergy Clin Immunol 2000;105:482-488.

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The dilemma of these potency measures:

In order to measure specific allergens, we need to know which allergens are relevant

If we measure overall allergenicity, we are unable to detect the absence of specific (and potentially important) allergens

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Two possible solutions:

Divide the signal by

Separating the allergens, or

Separating the antibodies

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An assay that will do both

Identify currently known allergens And recognize potentially important allergens yet to be identified

?

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??

? ?

?

?

?

??

??

??

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Aims To develop an multiplex antibody-

based method for profiling complex allergen mixture Antibodies Assay development Apply to cat and ragweed

Apply this technique to German cockroach allergen standardization

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To produce recombinant antibodies

Step 1. Inject chicken with allergen mixture of interest.

Step 2. Once a strong immune response is detected, Remove bone marrow and spleen and purify total RNA

Step 3. PCR is performed to amplify antibody repertoire.

Step 4: PCR products are digested with Sfi I and ligated into a vector.

Step 5: Plasmid containing antibody library is then electroporated into F´ E. coli along with helper phage. The scFv is then expressed on the PIII coat protein attached to the phage

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scFvs are then screened at both protein and DNA level

1 7 8 9

Amb a 1 clones vs ragweed and cat hair extract

**

*

*

0

0.5

1

1.5

1 2 3 4 5 6 7 8 9 10 11 12

cont

rol

clone number

OD

450

nm

ragweed

cat hair

PCR and restriction digest of select clones

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Recombinant antibodies recognize specific allergens

F10 F11 F46 F118 F124

Anti-Fel d 1 clone number

Amb a 1

ragweed

Fel d 1

cat hair

0

0.5

1

1.5

2

2.5

F38 F 7 F 17

OD

450

(n

m)

3.0

Anti-Amb a 1 clone number

0

0.5

1

1.5

2

2.5

A8 A9 A23 A 24 A 55 A 107 A 119 A 113 A 121

OD

450

(n

m)

Amb a 1

ragweed

Fel d 1

cat hair

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The surface of each bead is coated with carboxylic acid groups.

Using EDC and sulfo-NHS, recombinant antibodies can be covalently bound to the bead surface via an amide bond.

Multiplex microbead technology

o

0

c N

O

OO

S

O

O

O-

C

O

C

O

NH2

0

EDC +Sulfo NHS

Sulfo-NHS estherCarboxy labeled bead scFv attached via amide bond

scFv

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Each bead type can be bound to recombinant antibodies with different specificities.


www.bio-rad.com

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Up to 100 different bead types can be combined into a single well of a 96-well plate


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Assay design

•Each well contains the same mixture of six different beads bound to six different anti-Feld 1 recombinant antibodies

•12 2-fold dilutions of each extract are added to each well of each row

Extract dilutions Streptavidin – RPE

Anti-rabbit biotin

Fel d 1 specific rabbit sera

Fel d 1 in allergenic extract

scFv bound to

Carboxy labeled bead

E4 standardCat hair

Company ACat hair

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The beads are drawn up single file into the detection chamber

Here the sample is hit with two lasers:

• A 635nm laser excites the dyes within the bead.

• The dyes emit distinct photons. • Photons are detected and the

ratioof photon wavelengths emitted is calculated to determine the bead

type.• A 532 nm laser detects the RPE

bound to the bead.• Output consists of median

fluorescence index (MFI) of each bead-type in each well.

Multiplex array technology

Luminex 200, Luminex Corp.

635 nm

532 nm

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Analyzing dose response curves

-5 -4 -3 -2 -1 0

0

10000

20000

30000

maximum

minimum

EC50

slope

MF

I

Allergen extract (log dilution)

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Relative potencies

Relative potency = EC50 standard / EC50 sample

Log EC50

-5 -4 -3 -2 -1 00

5000

10000

15000

20000

25000

30000

35000Standard cat hairSample cat hair

standard sample LOGEC50 -2.46 -2.74 EC500.0034 0.0018

rp = .0034/.0018 = 1.8

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Summary of anti-Amb a 1 data

The average calculated potencies of ragweed extract vary greatly when anti-Amb a 1 scFvs are used alone or in groups

The potency of some ragweed extracts can be accurately computed from extracts with known potencies using the microbead method.

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Potencies of ragweed extracts obtained using bead assay are consistent with manufacturer data

Extract RID Microbead assay

I 111 28 109 20

II 205 51 248 9

III 209 52 152 12

IV 107 27 100 1

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Summary of anti-Fel d 1 data

When anti-Fel d 1 scFvs are used alone or in groups the average calculated potencies of cat hair extracts are similar

Potency of cat hair extracts can be accurately computed from extracts with known potencies using the microbead method.

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Comparing microbead data to RID data for cat hair extracts

Extract RID Microbead assay

V 4 1 2 0

VI 6 2 6 0

VII 18 5 16 2

VIII 7 2 5 1

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test new anti-cockroach scFvs

0.000

0.500

1.000

1.500

2.000

2.500

6A1

6A2

6A3

6B11

6B12

6G2

4B7

6E1

2A1

1E11

1D9

clone number

OD

450

(n

m)

positive

negative

•Selection of 250 positive clones•DNA sequencing of 250 clones: 150 unique clones •Select 85 clones to express in a soluble form and analyze by ELISA•Select 50 best clones to purify

Summary of the work performed by Millegen

Data from Millegen Labege, France

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Future experiments

Binding of soluble scFv’s to bead-bound known allergens

Inhibition assays using known allergens Analysis of scFv binding patterns in

Western blots Identification of scFv-recognized

antigens by N-terminal sequencing

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Publications: Rabin Le Nouën C, Munir S, Losq S, Winter CC, McCarty T, Stephany DA,

Holmes KL,Bukreyev A, Rabin RL, Collins PL, Buchholz UJ. Infection and maturation of monocyte-derived human dendritic cells by human respiratory syncytial virus, human metapneumovirus, and human parainfluenza virus type 3. Virology. 2009 Mar 1;385(1):169-82.

Mane VP, Heuer MA, Hillyer P, Navarro MB, Rabin RL. Systematic method for determining an ideal housekeeping gene for real-time PCR analysis.J Biomol Tech. 2008 Dec;19(5):342-7.

Chi B, Dickensheets HL, Spann KM, Alston MA, Luongo C, Dumoutier L, Huang J, Renauld JC, Kotenko SV, Roederer M, Beeler JA, Donnelly RP, Collins PL, Rabin RL. Alpha and lambda interferon together mediate suppression of CD4 T cells induced by respiratory syncytial virus. J Virol. 2006 May;80(10):5032-40.

Zhang M, Drenkow J, Lankford CS, Frucht DM, Rabin RL, Gingeras TR, Venkateshan C, Schwartzkopff F, Clouse KA, Dayton AI. HIV regulation of the IL-7R: a viral mechanism for enhancing HIV-1 replication in human macrophages in vitro. J Leukoc Biol. 2006 Jun;79(6):1328-38.

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Publications: Rabin Zhang J, Alston MA, Huang H, Rabin RL. Human T cell cytokine

responses are dependent on multidrug resistance protein-1. Int Immunol. 2006 Mar;18(3):485-93.

Song K, Rabin RL, Hill BJ, De Rosa SC, Perfetto SP, Zhang HH, Foley JF, Reiner JS, Liu J, Mattapallil JJ, Douek DC, Roederer M, Farber JM.Characterization of subsets of CD4+ memory T cells reveals early branchedpathways of T cell differentiation in humans. Proc Natl Acad Sci U S A. 2005 May 31;102(22):7916-21.

Gupta N, Arthos J, Khazanie P, Steenbeke TD, Censoplano NM, Chung EA, Cruz CC, Chaikin MA, Daucher M, Kottilil S, Mavilio D, Schuck P, Sun PD, Rabin RL, Radaev S, Van Ryk D, Cicala C, Fauci AS. Targeted lysis of HIV-infected cells by natural killer cells armed and triggered by a recombinant immunoglobulin fusion protein: implications for immunotherapy. Virology. 2005 Feb 20;332(2):491-7.

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Publications: Rabin (reviews) Rabin RL, Levinson AI. The nexus between atopic disease and

autoimmunity: a review of the epidemiological and mechanistic literature. Clin Exp Immunol. 2008 Jul;153(1):19-30.

Rabin RL. Recombinant and modified allergens: the U.S. perspective.Arb Paul Ehrlich Inst Bundesamt Sera Impfstoffe Frankf A M. 2006;(95):191-3;discussion 193-4.

Rabin RL. Regulation of allergenic products in the United States: The promise and problem of adjuvants in allergen immunotherapy. Arb Paul Ehrlich Inst Bundesamt Sera Impfstoffe Frankf A M. 2009, in press.

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Publications: Slater Slater JE, James R, Pongracic JA, Liu AH, Sarpong S, Sampson HA,

Satinover SM, Woodfolk JA, Mitchell HE, Gergen PJ, Eggleston PA.Biological potency of German cockroach allergen extracts determined in an innercity population. Clin Exp Allergy. 2007 Jul;37(7):1033-9.

Soldatova LN, Tsai C, Dobrovolskaia E, Marković-Housley Z, Slater JE.Characterization of the N-glycans of recombinant bee venom hyaluronidase (Api m 2) expressed in insect cells. Allergy Asthma Proc. 2007 Mar-Apr;28(2):210-5.

Padavattan S, Schirmer T, Schmidt M, Akdis C, Valenta R, Mittermann I,Soldatova L, Slater J, Mueller U, Markovic-Housley Z. Identification of a B-cell epitope of hyaluronidase, a major bee venom allergen, from its crystal structure in complex with a specific Fab. J Mol Biol. 2007 May 4;368(3):742-52.

Finkelman MA, Lempitski SJ, Slater JE. beta-Glucans in standardized allergen extracts. J Endotoxin Res. 2006;12(4):241-5.

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Publications: Slater Dobrovolskaia E, Gam A, Slater JE. Competition enzyme-linked

immunosorbant assay (ELISA) can be a sensitive method for the specific detection of small quantities of allergen in a complex mixture. Clin Exp Allergy. 2006 Apr;36(4):525-30.

Valerio CR, Murray P, Arlian LG, Slater JE. Bacterial 16S ribosomal DNA in house dust mite cultures. J Allergy Clin Immunol. 2005 Dec;116(6):1296-300.

Finlay WJ, deVore NC, Dobrovolskaia EN, Gam A, Goodyear CS, Slater JE.Exploiting the avian immunoglobulin system to simplify the generation of recombinant antibodies to allergenic proteins. Clin Exp Allergy. 2005 Aug;35(8):1040-8.

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Publications: Slater (reviews) Slater JE. Standardized allergen vaccines in the United

States.Clin Allergy Immunol. 2008;21:273-81.

James R, Mitchell H, Gergen PJ, Eggleston PA, Slater JE. Analyzing of ID50EAL data for the standardization of German cockroach allergen extracts in the U.S.Arb Paul Ehrlich Inst Bundesamt Sera Impfstoffe Frankf A M. 2006;(95):117-27;discussion 127, 155.

Slater JE. Characterization of allergen extracts. Dev Biol (Basel). 2005;122:145-52.

Slater JE. A global view of allergenic product potency. Arb Paul Ehrlich Inst Bundesamt Sera Impfstoffe Frankf A M. 2009; in press.

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LIB research support

FY source

2008 2009

Intramural $211,000 $275,000

Critical Path $178,000 $90,000

Extramural $290,000

Laboratory of Immunobiochemistry

site visits

January 2002

June 2006

[June 2010]

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LIB Research Program

Projects Publications Support

Laboratory of Immunobiochemistry Research review Jay E. Slater, MD OVRR/DBPAP 18 March 2009.

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Transcript of Laboratory of Immunobiochemistry Research review Jay E. Slater, MD OVRR/DBPAP 18 March 2009.