ProImmune Antigen Characterization Summit Sanja Selak


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Sanja Selak of Intercell AG, Vienna, Austria, presents at the ProImmune Antigen Characterization and Biomarker Discovery Summit, January 2011.
Intercell develops vaccines for the prevention and treatment of infectious diseases

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ProImmune Antigen Characterization Summit Sanja Selak

  1. 1. 10-Jan-2011 Vaccine antigen selection: lessons from infected hosts Sanja Selak
  2. 2. Vaccine discovery at Intercell <ul><li>LEARNING FROM THE HOST IMMUNE SYSTEM </li></ul><ul><ul><li>Antigen selection by relying on the current knowledge of basic attributes of protective antigens: </li></ul></ul><ul><ul><ul><li>(1) expression during human disease, </li></ul></ul></ul><ul><ul><ul><li>(2) accessibility to functional antibodies or effector immune cells, </li></ul></ul></ul><ul><ul><ul><li>(3) conservation among clinical strains, </li></ul></ul></ul><ul><ul><ul><li>(4) importance of contribution to in vivo survival in order to avoid counter selection, </li></ul></ul></ul><ul><ul><ul><li>(5) protection in animal models mimicking human disease </li></ul></ul></ul>
  3. 3. Vaccine discovery at Intercell <ul><li>ANTIGEN SELECTION USING PATIENTS SERA </li></ul><ul><ul><li>Acute and convalescent serum pairs from the patients who successfully recovered from infection </li></ul></ul><ul><ul><ul><li>Increase in antibody titers against bacterial antigens in the course of infection (seroconversion) suggests that: </li></ul></ul></ul><ul><ul><ul><ul><li>the antigen was expressed during the infection in human host </li></ul></ul></ul></ul><ul><ul><ul><ul><li>the antigen was immunogenic </li></ul></ul></ul></ul><ul><ul><ul><ul><li>the antibodies may have contributed to the bacterial clearance </li></ul></ul></ul></ul>1 st contact 2 nd contact ACUTE CONVALESCENT
  4. 4. Vaccine discovery at Intercell <ul><ul><li>Sera from healthy individuals: </li></ul></ul><ul><ul><ul><li>who were colonized without developing disease </li></ul></ul></ul><ul><ul><ul><li>who were exposed to bacterium without getting infected </li></ul></ul></ul><ul><ul><ul><ul><li>Protective or neutralizing antibodies? </li></ul></ul></ul></ul><ul><ul><li>From the patients who experienced recurrent disease episodes: </li></ul></ul><ul><ul><ul><li>Control for non-protective antibodies (antigens) </li></ul></ul></ul><ul><ul><ul><li>Control for non-protective antibody titers </li></ul></ul></ul>SERUM CONTROLS FOR ANTIGEN SELECTION
  5. 5. Selection of human sera for antigen identification <ul><li>RECOGNITION OF PROTEINS IN PATHOGEN CELL LYSATE </li></ul><ul><ul><li>Sera with significant increase in antibody titers in the convalescent disease phase are indicated with asterisk (*) </li></ul></ul>Serum dil. 4000x Lysate amount: 35µg/lane Patients who overcame infection: Acute/Convalescent serum pairs Chronic disease Healthy individuals
  6. 6. Identification and selection of vaccine antigens <ul><li>ANTIGEN IDENTIFICATION TECHNOLOGY (AIP) </li></ul>>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
  7. 7. Vaccine discovery at Intercell <ul><li>ANTIGEN EPITOPE SELECTION </li></ul><ul><ul><li>Human sera containing „protective“ antibodies are used for screening antigen peptide maps and truncated antigen versions </li></ul></ul><ul><ul><ul><li>This strategy further defines most immunogenic regions on bacterial proteins </li></ul></ul></ul><ul><ul><ul><ul><li>Especially useful for large and membrane embedded antigens which are difficult to express as full length ORF </li></ul></ul></ul></ul>
  8. 8. 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)
  9. 9. Examples of reactivity with selected peptides by ELISA <ul><ul><li>High seroconversion values on sequential peptides belonging to the same ORF </li></ul></ul><ul><ul><li>Correlation between seroconversion values in individuals who overcame infection and antibody titers in the sera from healthy individuals </li></ul></ul>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
  10. 10. Evaluation of functional activity of antibodies Y Y Y Ag-specific antibodies Y Y Flow Cytometry (FACS) Y Y <ul><li>Target Ag is expressed under in vitro culturing conditions </li></ul><ul><li>Localized on the cell surface, accessible to antibody binding </li></ul><ul><li>High-affinity antibodies capable of pathogen neutralization (functional activity) </li></ul><ul><li>Protective antigenic epitopes are conserved among different bacterial strains </li></ul>ANTIGEN SELECTION CRITERIA BASED ON in vitro ASSAYS Western Blot Y Y Y Y Y Y Y Y Y Y Cross-protection
  11. 11. Challenges associated with the studies of vaccine protection against human pathogens <ul><ul><li>Strictly human pathogens such as S.pyogenes, S.pneumoniae and P. aeruginosa have evolved mechanisms that facilitate propagation within human host: </li></ul></ul><ul><ul><ul><li>Expression of proteins that prevent recognition by the host immune system or facilitate invasion of host tissues </li></ul></ul></ul><ul><ul><ul><ul><li>Fibrinogen and plasminogen binding proteins </li></ul></ul></ul></ul><ul><ul><ul><li>Expression of proteins that interfere with immune response activation </li></ul></ul></ul><ul><ul><ul><ul><li>Complement inhibitor binding; prevents complement activation and opsonization </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Antibody Fc fragment binding; prevents recognition by phagocytes </li></ul></ul></ul></ul><ul><ul><ul><li>Expression of proteins which increase survival within host specialized tissues </li></ul></ul></ul><ul><ul><ul><ul><li>Acquisition of host-derived nutrients and minerals </li></ul></ul></ul></ul><ul><ul><li>Different pathogens employ different survival and virulence mechanisms: </li></ul></ul><ul><ul><ul><li>Experimental strategies have to be adapted to a specific bacterium </li></ul></ul></ul>
  12. 12. Pathogen : host interactions <ul><li>COLONIZATION AND INVASION OF HOST TISSUES </li></ul>CDC/Division of bacterial and mycotic diseases Encapsulated bacteria ( S.pneumoniae ) Neutrophil Nat Rev Microbiol 2: 442 S. pyogenes
  13. 13. Strategies promoting antigen expression in vitro
  14. 14. Strategies for inducing antigen expression in vitro Microbiol Mol Biol Rev, Mar. 2004, p.154–171 BACTERIAL IRON ACQUISITION FROM INFECTED HOST
  15. 15. Strategies for inducing antigen expression in vitro SURFACE STAINING OF BACTERIA GROWN UNDER IRON DEPLETED CONDITIONS <ul><ul><li>Significant increase in the surface expression of antigen X under iron limiting conditions </li></ul></ul><ul><ul><ul><li>Up-regulation of the proteins involved in iron acquisition </li></ul></ul></ul>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
  16. 16. Strategies for inducing antigen expression in vitro <ul><ul><li>Change in the antigen conformation due to growth in human plasma </li></ul></ul><ul><ul><ul><li>Increased accessibility of D5 epitope </li></ul></ul></ul><ul><ul><li>Phenotypic change during the growth in eukaryotic cell culture medium </li></ul></ul><ul><ul><ul><li>Fluorescence induction </li></ul></ul></ul>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
  17. 17. Functional immune assays in vitro
  18. 18. Strategies for predicting antigen protection in human host <ul><ul><li>Many bacterial pathogens express proteins which specifically bind human complement inhibitors, reducing the amount of complement activation </li></ul></ul><ul><ul><li>During the animal infection, complement activation proceeds without inhibition: </li></ul></ul><ul><ul><ul><li>It is important to test functional activity of Abs in the presence of human complement </li></ul></ul></ul>T. Areschoug et al. Vaccine 22S (2004) S9–S14 IMMUNE EVASION IN HUMAN AND ANIMAL HOST Animal model Human system
  19. 19. <ul><li>SBA ACTIVITY OF ANTIBODIES IN THE PRESENCE O F HUMAN COMPLEMENT </li></ul>Strategies for predicting antigen protection in human host 50x 150x 450x 1350x <ul><ul><li>Dramatic reduction in bactericidal activity of antibodies against YYZ antigen (M3): </li></ul></ul><ul><ul><ul><li>Not a good candidate for the protection in human host </li></ul></ul></ul>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
  20. 20. Strategies for predicting antigen protection in human host <ul><ul><li>Problems with the in vitro analysis of antibodies: </li></ul></ul><ul><ul><ul><li>Binding to bacterial Fc receptors </li></ul></ul></ul><ul><ul><ul><li>Animal complement sources not suitable, due to absence o f complement regulation </li></ul></ul></ul>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
  21. 21. Selection of non-immune human plasma for S. pyogenes in vitro assays <ul><ul><li>In total human plasma from 80 different donors was commercially obtained and tested for the ability to support the growth of various S. pyogenes strains </li></ul></ul><ul><ul><ul><li>One donor was identified without pre-existing opsonizing antibodies against one strain (serotype 4) </li></ul></ul></ul>SURVIVAL IN PLASMA FROM DIFFERENT DONORS 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
  22. 22. Reactivity of mouse sera with S.pyogenes in the presence of human plasma <ul><li>SURFACE STAINING USING CANDIDATE Ag-SPECIFIC SERA </li></ul><ul><ul><li>In human plasma non-specific, Fc binding of antibodies is prevented </li></ul></ul><ul><ul><ul><li>Only antigen specific antibodies bind bacterial surface </li></ul></ul></ul><ul><ul><ul><li>Increased surface binding of hyperimmune serum antibodies </li></ul></ul></ul>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
  23. 23. 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
  24. 24. OPK assay using human granulocytes (HL60) and baby rabbit serum (complement) UPTAKE AND KILLING KINETICS <ul><ul><li>OPK of S.pyogenes occurs equally in the presence or absence of specific antibodies </li></ul></ul>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:
  25. 25. 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.
  26. 26. S. pyogenes OPK assay with mouse macrophages (RAW264.7) <ul><ul><li>Bacterial killing is detectable only by hyperimmune sera </li></ul></ul><ul><ul><ul><li>Preimmune sera induced the same bacterial CFU# uptake, but no killing </li></ul></ul></ul><ul><ul><li>In the absence of antibody, only 50% of bacteria are taken up by phagocytes; </li></ul></ul><ul><ul><ul><li>Growth inside the cells (absence of killing) </li></ul></ul></ul>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)
  27. 27. 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
  28. 28. OPK assay using mouse macrophages <ul><ul><li>In the absence of human plasma, antigen-specific sera induce effective killing 6hr after internalization </li></ul></ul><ul><ul><li>No uptake or killing in the presence of human plasma </li></ul></ul>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
  29. 29. OPK assay using mouse macrophages <ul><li>CONCLUSIONS </li></ul><ul><ul><li>Macrophages are able to distinguish between “specific” and “non-specific” opsonization of bacteria! </li></ul></ul><ul><ul><ul><li>Sorting of bacteria into different intracellular compartments? </li></ul></ul></ul><ul><ul><ul><li>Opsonized bacteria are less efficient in escaping phagosomal killing? </li></ul></ul></ul><ul><ul><li>In the presence of human plasma, S.pyogenes uptake by phagocytic cells is dramatically reduced; and so the killing </li></ul></ul><ul><ul><ul><li>Additional conditions and factors are needed for more efficient uptake </li></ul></ul></ul><ul><ul><ul><ul><li>Longer incubation </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Inflammatory mediators (cytokines) to enhance macrophage OPK activity </li></ul></ul></ul></ul>PAGE 24-NOV-2009
  30. 30. <ul><li>CONSIDERATIONS FOR THE SELECTION OF BEST VACCINE CANDIDATES </li></ul><ul><ul><li>Human pathogens are specifically adapted to invade and overcome human host immune defenses: </li></ul></ul><ul><ul><ul><li>Vaccine protection studies in animal models are of limited value </li></ul></ul></ul><ul><ul><li>To ensure the selection of best vaccine material, experiments have to be designed under the conditions closely mimicking the situation during human infection: </li></ul></ul><ul><ul><ul><li>Use of differential bacterial growth medium </li></ul></ul></ul><ul><ul><ul><li>Co-culturing with human cells and tissues </li></ul></ul></ul><ul><ul><ul><li>Growth in human serum or plasma </li></ul></ul></ul><ul><ul><ul><li>Use of different phagocytic cells </li></ul></ul></ul><ul><ul><ul><li>Testing different complement sources: </li></ul></ul></ul><ul><ul><ul><ul><li>Animal serum (for correlating functional activity with in vivo protection) </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Human serum or plasma (for predicting bactericidal activity in humans) </li></ul></ul></ul></ul>Conclusions
  31. 31. Serology and Immune Assays (SIA)
  32. 32. For more information be invited to: