ProImmune Antigen Characterization Summit Sanja Selak

10-Jan-2011 Vaccine antigen selection: lessons from infected hosts Sanja Selak

Vaccine discovery at Intercell ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Vaccine discovery at Intercell ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],1 st contact 2 nd contact ACUTE CONVALESCENT

Vaccine discovery at Intercell ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],SERUM CONTROLS FOR ANTIGEN SELECTION

Selection of human sera for antigen identification ,[object Object],[object Object],Serum dil. 4000x Lysate amount: 35µg/lane Patients who overcame infection: Acute/Convalescent serum pairs Chronic disease Healthy individuals

Identification and selection of vaccine antigens ,[object Object],>2000 Pathogen proteome Etz et al. (2001) J. Bacteriol. 183, 6924-6935. Etz et al. (2002) PNAS 99, 6573-6578. Henics et al. (2003) BioTechniques 35, 196-202. Nagy et al. (2004) Genomics, Proteomics & Vaccines, ed. Grandi Meinke et al. (2004) Curr. Opin. Microbiol., 7, 318. Meinke et al. (2005) Vaccine, 23, 3025. In vitro validation In vivo validation Antigen Identification <200 Antigenome Gene conservation Peptide serology Surface location Functional activity <50 Recombinant proteins Protection in mice Donor and serum selection Antibodies Immunoblot In vitro assays ELISA Exposed humans Genomic library screening Sequence analysis Clone selection Genome Antigen screen Surface display <5 Protective antigens

Vaccine discovery at Intercell ,[object Object],[object Object],[object Object],[object Object]

Principle of candidate antigen and epitope selection by ELISA using human sera Patients (group A) Patients (group B) Healthy individuals Peptide screening with human Abs (ELISA) Gene accession number: ORF# X ORF X -01 ORF X -02 ORF X -03 ORF X -04 ORF X -05 ORF X -06 ORF X -07 ORF X -08 An example of the “candidate antigen” selected by genomic library screening Epitope – the antibody binding site on an antigen (5-30 aa)

Examples of reactivity with selected peptides by ELISA ,[object Object],[object Object],Recurrent episodes Seroconversion: acute/convalescent Other clinical manifestation ELISA UNITS: (OD 405nm – BLANK) x 1000 PEPTIDE X-01 X-02 X-03 X-04 X-05 Y-01 Y-02 Y-03 Y-04 Y-05 Y-06 Y-07 Z-01 Z-02 Z-03 Z-04

Evaluation of functional activity of antibodies Y Y Y Ag-specific antibodies Y Y Flow Cytometry (FACS) Y Y ,[object Object],[object Object],[object Object],[object Object],ANTIGEN SELECTION CRITERIA BASED ON in vitro ASSAYS Western Blot Y Y Y Y Y Y Y Y Y Y Cross-protection

Challenges associated with the studies of vaccine protection against human pathogens ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Pathogen : host interactions ,[object Object],CDC/Division of bacterial and mycotic diseases Encapsulated bacteria ( S.pneumoniae ) Neutrophil Nat Rev Microbiol 2: 442 S. pyogenes

Strategies promoting antigen expression in vitro

Strategies for inducing antigen expression in vitro Microbiol Mol Biol Rev, Mar. 2004, p.154–171 BACTERIAL IRON ACQUISITION FROM INFECTED HOST

Strategies for inducing antigen expression in vitro SURFACE STAINING OF BACTERIA GROWN UNDER IRON DEPLETED CONDITIONS ,[object Object],[object Object],ANTIGEN X ANTIGEN Y 95.6 100 HI antigenX 129.9 50 HI antigenX 17 0 HI antigenX 0 0 Desferal [µM] 6.6 Preimmune 4.1 2nd Ab MEDIAN SAMPLE 127 100 HI antigenY 164 50 HI antigenY 148 0 HI antigenY 0 0 Desferal [µM] 6.7 Preimmune 4.1 2nd Ab MEDIAN SAMPLE

Strategies for inducing antigen expression in vitro ,[object Object],[object Object],[object Object],[object Object],SURFACE STAINING OF P.AERUGINOSA GROWN UNDER THE CONDITIONS MMIMICKING HOST ENVIRONMENT D1 mAb D5 mAb BHI medium 50% human plasma BHI medium RPMI + 10% FBS

Functional immune assays in vitro

Strategies for predicting antigen protection in human host ,[object Object],[object Object],[object Object],T. Areschoug et al. Vaccine 22S (2004) S9–S14 IMMUNE EVASION IN HUMAN AND ANIMAL HOST Animal model Human system

[object Object],Strategies for predicting antigen protection in human host 50x 150x 450x 1350x ,[object Object],[object Object],YYZ BBA SERUM DILUTION Rabbit complement (12.5%) 0 20 40 60 80 100 anti-lysate M2 M3 M1 M3 % killing Inactive C‘ Human complement (12.5%) anti-lysate M2 M3 M1 M3 Inactive C‘ YYZ BBA

Strategies for predicting antigen protection in human host ,[object Object],[object Object],[object Object],Fc Ab-binding protein Antibodies (Immunoglobulins) Complement inhibitors-binding protein C‘ pathway inhibitors Additional human protein-binding molecules Fibrinogen Complement S.PYOGENES IMMUNE EVASION MECHANISMS Antigen Ag Bacteria Complement inhibition C‘ pathway inhibitors Complement Fibrinogen binding Fc binding unstained bacteria preimmune 91% Hyperimmune #1 25µg : 85% 10 0 10 1 10 2 10 3 10 4 0 30 60 90 120

Selection of non-immune human plasma for S. pyogenes in vitro assays ,[object Object],[object Object],SURVIVAL IN PLASMA FROM DIFFERENT DONORS http://www.innov-research.com/innovative/index.php 0 50 100 150 200 250 300 350 400 T=0 11- 36765 11- 36766 11- 36761 11- 36757 11- 36860 11- 36754 23- 10942 23- 10963 23- 10982 23- 10987 Assay controls Donor plasma actC‘ inactC‘ Donor IC138 CFU average

Reactivity of mouse sera with S.pyogenes in the presence of human plasma ,[object Object],[object Object],[object Object],[object Object],2% BSA FL2 Fluorescence unstained bacteria preimmune 5% Hyperimmune #1 25µg 33% Mouse preimmune and HI serum samples 50x 100x 50x 100x 50x 100x No 2nd Ab 1% 12% 3% 3% 4% 3% No primary Ab (buffer) 4% 11% 5% 3% 4% 7% anti- lysate preimmune (-ctr) 73% 76% 11% 6% 20% 12% anti-lysate hyperimmune (+ctr) 83% 79% 41% 26% 59% 42% IS2846 group 1 preimmune 84% 77% 7% 6% 13% 9% ANTIGEN #1 50µg 88% 86% 51% 51% 41% 46% IS2846 group 2 preimmune 87% 78% 7% 5% 14% 9% ANTIGEN #1 25µg 91% 85% 48% 33% 45% 41% IS3099 group 2 preimmune 84% 75% 7% 5% 9% 10% ANTIGEN #1 10 µg 91% 86% 17% 9% 31% 15% IS3099 group 3 preimmune 86% 79% 7% 6% 12% 6% ANTIGEN #1 10µg 89% 87% 21% 10% 30% 18% Without blocking 60% human plasma 30% human plasma % positive counts unstained bacteria preimmune 91% Hyperimmune #1 25µg : 85% 10 0 10 1 10 2 10 3 10 4 0 30 60 90 120 2% BSA

OPK assay using human granulocytes (HL60) Opsonization (40min, RT) Human HL60 cell line differentiated into granulocytes Incubation 20min at 37C = uptake of bacteria Continue incubation for additional time points at 37C to determine intracellular killing rate (30min, 1hr, 2hr and 3hr) Lysis after 20min incubation with HL60: plated out released bacteria to determine the uptake Lysis and plating of the released bacteria STEP 1: STEP 2: S.pyogenes + + Complement (4% baby rabbit serum) ASSAY SETUP WITH RABBIT COMPLEMENT Antibiotic killing 40min at 4C ( 2% or 20 µg/ml Pen/Streptomycine) Antibodies

OPK assay using human granulocytes (HL60) and baby rabbit serum (complement) UPTAKE AND KILLING KINETICS ,[object Object],Preimmune Hyperimmune Bacterial lysate specific serum CFUave 0 200 400 600 800 1000 ANTIGEN #1 specific sera Preimmune Hyperimmune M2 Hyperimmune M7 Hyperimmune M9 0 CFUave 200 400 600 800 1000 20 min + 1hr + 2hr + 3hr + 4hr UPTAKE KILLING CFUave 0 200 400 600 800 1000 HBSS buffer +active RabC´ +inactive RabC´ Assay controls:

OPK assay using mouse macrophages TESTING UPTAKE AND KILLING KINETICS PAGE Non-specific (pre-existing) Ab S.pyogenes-specific Ab S.pyogenes + Mouse antibodies OPSONIZATION INCUBATION 1hr , 37C =uptake of bacteria Continued incubation for additional 6hr at 37C to determine intracellular killing Lysis and plating released bacteria. UPTAKE: KILLING: Antibiotic killing of extracellular bacteria 40min at 4C (Pen/Streptomycine) Lysis after 1hr incubation with MFs: plating out released bacteria to determine the uptake.

S. pyogenes OPK assay with mouse macrophages (RAW264.7) ,[object Object],[object Object],[object Object],[object Object],OPK IN THE ABSENCE OF HUMAN PLASMA PAGE 24-NOV-2009 pre= preimmune serum HI= hyperimmune serum No Ab Pre HI Pre HI m.6 HI m.7 1hr: phagocytic uptake CFUave 0 400 800 1200 1600 2000 6hr: killing after uptake No Ab pre HI pre HI m.6 HI m.7 CFUave Candidate antigen Bacterial lysate 0 400 800 1200 1600 2000 0 20 40 60 80 100 % killing pre HI pre HI m.6 HI m.7 Candidate antigen Bacterial lysate % OPK (preimmune) % OPK (uptake)

OPK assay using mouse macrophages TESTING EFFECT OF HUMAN PLASMA ON UPTAKE AND KILLING OF S. PYOGENES PAGE Non-specific (pre-existing) Ab S.pyogenes-specific Ab Pre-incubation with human plasma Without human plasma + Mouse antibodies OPSONIZATION *binding of both specific and non-specific Abs S.pyogenes Non-immune human plasma + Y Y Bacteria coated with human plasma proteins OPSONIZATION Mouse Abs bound in the correct orientation S.pyogenes + Mouse antibodies UPTAKE KILLING

OPK assay using mouse macrophages ,[object Object],[object Object],OPK IN THE PRESENCE OR ABSENCE OF HUMAN PLASMA PAGE 24-NOV-2009 pre= preimmune serum HI= hyperimmune serum 6hr: killing after uptake No Ab No Ab pre HI pre HI m.6 HI m.7 pre HI pre HI m.6 HI m.7 CFUave Bacterial Lysate 1hr: phagocytic uptake 0 400 800 1200 1600 2000 CFUave Without human plasma With human plasma 0 400 800 1200 1600 2000 Candidate antigen Bacterial Lysate Candidate antigen

OPK assay using mouse macrophages ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],PAGE 24-NOV-2009

[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Conclusions

Serology and Immune Assays (SIA)

For more information be invited to: www.intercell.com

ProImmune Antigen Characterization Summit Sanja Selak

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