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Laboratory of Immunobiochemistry Research review. Jay E. Slater, MD OVRR/DBPAP 18 March 2009. LIB Research Program. Projects Publications Support. Projects. Rabin Characterization of innate immune responses to respiratory syncytial virus Slater Endotoxin in mite extracts

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laboratory of immunobiochemistry research review

Laboratory of ImmunobiochemistryResearch review

Jay E. Slater, MD

OVRR/DBPAP

18 March 2009

lib research program
LIB Research Program
  • Projects
  • Publications
  • Support
projects
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

Bacterial endotoxin and DNA in house dust mite cultures and extracts

Cherry Valerio

Larry Arlian, PhD

Patrick Murray, PhD

Bhavini Trivedi, MD

initial studies
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.

can we detect bacterial dna in live mite cultures
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
dna from mites

D. farinae D. pteronyssinus

EcoR1 digests

undigested DNA

DNA from mites
slide9

Df

TM = 42

Dp

16s rrna sequences recovered
Bartonella species

B. henselae

B. quintana

B. vinsonii

B. elizabethae

E. coli

Pseudomonas species

Acinetobacter species

Uncharacterized

a-proteobacteria

endosymbionts from

Ixodes scapularis

Vestimentiferan tubeworms

Brevipalpus phoenicis

Metaseiulus occidentalis

Aspidiotus nerii

16S rRNA sequences recovered

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.

bartonella organisms
Gram-negative rods

0.6 by 1.0 mm

facultative

intracellular

fastidious

Harbored by

Lice

Fleas

Ticks

Hippoboscidae flies

(house dust mites)

Bartonella organisms
bartonella associated diseases
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.

conclusions 1
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
next questions
Next questions
  • Are the bacterial DNA sequences detectable in commercial allergen extracts?
  • Are the bacterial DNA sequences detectable wild mite species?
are the bacterial dna sequences detectable in commercial allergen extracts methods
Are the bacterial DNA sequences detectable in commercial allergen extracts?Methods
  • DNA isolation by QIAamp
  • PCR
  • Sequence
are the bacterial dna sequences detectable wild mite species
Are the bacterial DNA sequences detectable wild mite species?
  • Chortoglyphusarcuatus
  • Lepidoglyphusdestructor
  • Euroglyphusmaynei
  • Acarus siro
  • Tyrophagusputrescentiae
are the bacterial dna sequences detectable wild mite species18
Are the bacterial DNA sequences detectable wild mite species?
  • Extract genomic DNA (DNAzol) from fresh, washed mites
  • Amplify with 16S rRNA sequences
  • Sequence after high fidelity amplification (pfx)
  • Identify predominant organisms (BLAST)
conclusions
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)
next steps
Next steps
  • Population analyses
  • Bartonella culture
  • Endotoxin analyses
slide22

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

how do we measure potency
How do we measure potency?
  • Total protein (hymenoptera)
  • Overall allergen (grasses, mites)
    • Pooled human antibody
  • Specific allergen (cat, ragweed)
    • Sheep antibody
specific loss of a single allergen
Specific loss of a single allergen

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.

the dilemma of these potency measures
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
two possible solutions
Two possible solutions:
  • Divide the signal by
    • Separating the allergens, or
    • Separating the antibodies
an assay that will do both
An assay that will do both

Identify currently known allergens

And recognize potentially important allergens

yet to be identified

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

slide28
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
slide29
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
slide30

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

slide31

1

7

8

9

1.5

*

*

*

1

*

OD 450 nm

0.5

0

6

7

8

9

2

1

3

4

5

10

11

12

control

clone number

ragweed

cat hair

scFvs are then screened at both protein and DNA level

PCR and restriction

digest of select clones

Amb a 1 clones vs ragweed and cat hair extract

recombinant antibodies recognize specific allergens

2.5

Amb a 1

2

ragweed

1.5

Fel d 1

1

cat hair

0.5

0

A 113

A 121

A8

A9

A23

A 24

A 55

A 107

A 119

2.5

Fel d 1

2

cat hair

1.5

Amb a 1

1

ragweed

0.5

0

F10

F11

F46

Recombinant antibodies recognize specific allergens

OD 450 (nm)

Anti-Amb a 1 clone number

3.0

OD 450 (nm)

F38

F 7

F 17

F118

F124

Anti-Fel d 1 clone number

slide33
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
multiplex microbead technology
Multiplex microbead technology

O-

O

0

O

0

O

O

S

NH2

C

N

O

c

C

O

o

O

  • 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.

scFv

EDC +

Sulfo NHS

Carboxy labeled bead

Sulfo-NHS esther

scFv attached via amide bond

multiplex microbead technology35
Multiplex microbead technology

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

www.bio-rad.com

multiplex microbead technology36
Multiplex microbead technology
  • Up to 100 different bead types can be combined into a single well of a 96-well plate
assay design
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 standard

Cat hair

Company A

Cat hair

multiplex array technology
Multiplex array technology
  • 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 ratio
    • of 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.

635 nm

532 nm

Luminex 200, Luminex Corp.

analyzing dose response curves

30000

20000

10000

0

-5

-4

-3

-2

-1

0

Analyzing dose response curves

maximum

slope

MFI

EC50

minimum

Allergen extract (log dilution)

relative potencies

35000

Standard cat hair

30000

Sample cat hair

25000

20000

15000

10000

Log EC50

5000

0

-5

-4

-3

-2

-1

0

Relative potencies

Relative potency = EC50 standard / EC50 sample

standard sample

LOGEC50 -2.46 -2.74

EC50 0.0034 0.0018

rp = .0034/.0018 = 1.8

slide41
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
summary of anti amb a 1 data
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.
summary of anti fel d 1 data
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.
slide46
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
slide47

Summary of the work performed by Millegen

  • 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

Data from Millegen Labege, France

future experiments
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
publications rabin
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.
publications rabin50
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.
publications rabin reviews
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.
publications slater
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.
publications slater53
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.
publications slater reviews
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.
laboratory of immunobiochemistry site visits

Laboratory of Immunobiochemistry site visits

January 2002

June 2006

[June 2010]

lib research program57
LIB Research Program
  • Projects
  • Publications
  • Support