Laboratory of Immunobiochemistry Research review

1. Laboratory of ImmunobiochemistryResearch review Jay E. Slater, MD OVRR/DBPAP 18 March 2009

2. LIB Research Program • Projects • Publications • Support

3. Projects • Rabin • Characterization of innate immune responses to respiratory syncytial virus • Slater • Endotoxin in mite extracts • Multiplex allergen extract potency assay

4. Bacterial endotoxin and DNA in house dust mite cultures and extracts Cherry Valerio Larry Arlian, PhD Patrick Murray, PhD Bhavini Trivedi, MD

5. 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.

6. 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

7. D. farinae D. pteronyssinus EcoR1 digests undigested DNA DNA from mites

9. Df TM = 42 Dp

10. 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.

11. Gram-negative rods 0.6 by 1.0 mm facultative intracellular fastidious Harbored by Lice Fleas Ticks Hippoboscidae flies (house dust mites) Bartonella organisms

12. 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.

13. 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

14. Next questions • Are the bacterial DNA sequences detectable in commercial allergen extracts? • Are the bacterial DNA sequences detectable wild mite species?

15. Are the bacterial DNA sequences detectable in commercial allergen extracts?Methods • DNA isolation by QIAamp • PCR • Sequence

16. Are the bacterial DNA sequences detectable in commercial allergen extracts?

17. Are the bacterial DNA sequences detectable wild mite species? • Chortoglyphusarcuatus • Lepidoglyphusdestructor • Euroglyphusmaynei • Acarus siro • Tyrophagusputrescentiae

18. 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)

19. 16S rRNA sequences recovered

20. 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)

21. Next steps • Population analyses • Bartonella culture • Endotoxin analyses

22. 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

23. How do we measure potency? • Total protein (hymenoptera) • Overall allergen (grasses, mites) • Pooled human antibody • Specific allergen (cat, ragweed) • Sheep antibody

24. 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.

25. 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

26. Two possible solutions: • Divide the signal by • Separating the allergens, or • Separating the antibodies

27. An assay that will do both Identify currently known allergens And recognize potentially important allergens yet to be identified ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?

28. 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

29. 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

30. 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

31. 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

32. 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

33. 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

34. 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

35. Multiplex microbead technology Each bead type can be bound to recombinant antibodies with different specificities. www.bio-rad.com

36. Multiplex microbead technology • Up to 100 different bead types can be combined into a single well of a 96-well plate

37. 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

38. 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.

39. 30000 20000 10000 0 -5 -4 -3 -2 -1 0 Analyzing dose response curves maximum slope MFI EC50 minimum Allergen extract (log dilution)

40. 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

41. 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

42. 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.

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

44. 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.

45. Comparing microbead data to RID data for cat hair extracts

46. 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

47. 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

48. 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

49. 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.

50. 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.