Immune Monitoring

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Presentation by Jill Gilmour, IAVI.

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Immune Monitoring

  1. 1. Immune Monitoring in Vaccine Trials Jill Gilmour Exec Director IAVI HIL Imperial College, London Symposium on Innovations in Vaccine R+D Wellcome Trust , London Sept 5th 2012 1
  2. 2. Immune Monitoring in Vaccine Trials: Outline1. Introduction and background2. Context for immune monitoring on clinical trials3. Available assays4. Challenges 2
  3. 3. HIV Variability: The problem has a solution……. A globally effective HIV vaccine will likely need to elicit: • Broadly Neutralizing Antibodies: Prevent infection of any HIV strain • Broadly Reactive T cells (CMI): Control Infection of any HIV strain Neutralizing Abs Cell Mediated Immunity (CMI)AIM to elicit both in a final productPhase IIB testing of vaccine candidates that elicit either broad cellular or broadly neutralizingantibodies will yield valuable information for field re-distinct immunological spaces. 3
  4. 4. An Introduction The Human Immunology Laboratory (HIL) MissionDeliver high quality clinical trial data to prioritize and develop HIVvaccine candidates for IAVI and the fieldCoordinate and support IAVI’s global network of clinicallaboratoriesInform the next generation of HIV vaccine design and assessmentthrough translational clinical research on the African epidemic 4
  5. 5. IAVI R&D Network Global outlook IAVI Development Design and IAVI Human Development Lab Immunology Lab IAVI NAC at Scripps Innovation Fund grants IAVI India Lab ImmuneNeutralizing Vectors correlates/Antibody Consortium LACConsortium Clinical research centers IAVI collaborations with CAVD, HVTN, MVI and UK-HVC. 5
  6. 6. Immune Monitoring in Clinical Trials Context• Regulatory environment – In EU member states, laboratories performing testing on clinical trial samples should be accredited e.g. GCLP, and are subject to audit by regulatory bodies – Quality systems to ensure integrity of data – Maybe a requirement to validate assays• Standardization and method validation – Need to pool data from multiple clinical centers and compare data across products, regimens, sites etc – Ensures studies are powered to meet primary objectives• Go/ No-Go must be based on robust and scientifically sound data 6
  7. 7. Immune Monitoring in Clinical TrialsContext Cont’dStage of product development• Phase I/II Trials: – Primary assays: Accurately assess response rate, kinetics, magnitude to optimize dose and regimen. Hypothesis testing. – Research assays: Characterize immunogenicity and test/generate additional hypothesis• Efficacy Trials – Primary assays similar to phase I/II with some additional validation and streamlining – Test a proportion of volunteers. Important for manufacturing e.g. ensure potency between lots. – Research assays to assess correlate or mechanism of efficacy. – Implications for vaccine design and manufacturing e.g. optimizing dose, monitoring vaccine potency as product released to market etc 7
  8. 8. Correlates of protection after vaccination;defined quantitative measures. S. Plotkin, ClinInf Dis. 2008For many vaccines we don’t know exact correlates ofprotection; HIV, TB, Malaria 8
  9. 9. What have we learned from HIV vaccine efficacy studies to date?• Vaxgen gp 120 (No efficacy) – Induced Env-specific non-neutralizing antibodies and tier 1 neuts• STEP Merck Ad5 gag-pol-nef (No efficacy) – Induced “poly-functional” CD8 T cells – Limited CD8 breadth – Evidence of protection against vaccine matched viral strains in vaccine recipients in vivo and in vitro• RV144 Canarypox + gp 120 (31% reduction of HIV-1 acquisition with no viral load effect) – Induced Env-specific non-neutralizing antibodies and tier 1 neuts – Predominantly CD4 Env-specific T cells – Correlates analysis (V2 antibody binding)• DNA prime + Ad5 boost: gag-pol-nef, env A, B and C – Data expected 3-4Q 2013 – Induces “polyfunctional” T cells – Antibody and CD4 env responses 9
  10. 10. Immunogenicity Go/No-go Criteria:Cellular and AntibodyGeneral Criteria (not product specific) Modeling suggests a public health benefit with a 50% partially effective vaccine Go > 60% response rate Demonstrate superiority either qualitatively and quantitatively to candidates which have been, or are in efficacy testing PLUSProduct Specific Criteria-test rationale Enhanced breadth, depth of coverage o Evidence of coverage in developing world Enhanced magnitude and quality of response o Anti-viral activity, proliferation, CD4 and CD8, avidity, affinity, B cell memory Induction of effective, boostable, memory response Mucosal targeting 10
  11. 11. Outline1. Introduction and background2. Context for immune monitoring on clinical trials3. Available assays4. Challenges for the future 11
  12. 12. Primary and secondary assays in common use to assess vaccine immunogenicity: Cellular Antibody Response rate ELISPOT ELISA1o Kinetics Magnitude Target antigens Flow cytometry Antibody class / subclass Phenotype CD8 CD4 Memory CD4 B Memory2o Function Viral Inhibition Assay Neutralisation Assay Proliferation, Cytokines…… Non-neutralising: ADCC, ADCVI, V2 Breadth ELISPOT: Epitope mapping Epitope mapping Viral Inhibition Assay Neutralization Assay3o Location GI for containment of viral reservoir GI for containment of viral reservoir (mucosal GU for containment of infection GU for containment of infection sampling)• Research assays: novel technologies e.g. Proteomics, genomics, DNA microarrays, RNA-sequencing, single cell multiplexing e.g. fluidym, nano-string,• Antibody gene sequencing 12
  13. 13. Example primary and secondary T cell immunogenictyassessment for an Ad35 GRIN-Env vaccine regimenIFN-g ELISPOT – Gag peptide pool >18 peptides mapped / 3000 2000 1000 Map Gag 8 unique regions in 8 SFC/106 PBMC 500 400 Epitopes individuals 300 1-3 epitopes / vaccinee 200 100 0 Low Mid High GRIN Conserved vs variable epitopes Log Inhib Polychromatic Flow Cytometry 13
  14. 14. Limitations of Most T Cell Assays High concentrationsHigh concentrations Indirect measurementsofof exogenous peptide synthetic antigen (peptide) (e.g. cytokines, peforins) New methods of assessing CTL function need to be developed Limited to peripheral blood Irrelevant Targets Do not measure antiviral function (B-cell lines) (MHC tetramer stain) Adapted from Watkins 2008
  15. 15. Alternative Assay: Viral Inhibition Assay High concentrations Indirect measurements Whole HIV Direct Antiviral Effects of synthetic antigen (peptide) CD8 The CellThe in vivoAntigen New methods of assessing CTL e.g. VIA Assess mucosal responses The site CD4 HIV infected Autologous CD4 T Cells XCD4 Measure antiviral activity The outcomeThe in vivo target
  16. 16. Viral Inhibition Assay: the principle CD4 CD4 CD4 CD4 CD4 CD4 CD4 CD4 CD4 CD4 CD4 Infection p24 CD4+CD8PBMCs CD8 CD8 CD8 CD8 Days CD8 CD8 CD4 CD4 CD8 CD8 Expansion Purification Inhibition
  17. 17. CD8 mediated inhibition of and HIV-1IIIB correlates with in vivo virus control HIV-1Spentzou et al (2010) JID 201: 720-729 17
  18. 18. In VIA Volunteers vaccinated with MRK-AD5 gag-pol-nef inhibit only viruses matched to vaccine insert:As predicted by efficacy data Total vaccinees = 16. Efficient inhibition > 1.5 log10 inhibition. 18
  19. 19. VIA activity correlates with viral control in HIVand vaccinated NHP controlling SIV challenge■ 1st generation VIA correlates to in vivo virus control and excellent specificity in HIV vaccine trials (Spentzou et al. JID. 2010)■ Higher inhibition in the VIA correlates to lower viral loads in vivo. (Julg et al. JVI 2010)■ VIA inhibition correlates with viral load dynamics over time (CHAVI 001) (Freel et al. JVI 2012)■ VIA in DNA-Ad5 vaccinated NHP correlate with lowered peak and set point viral loads (Yamamoto et al. J.Virol. 2012)■ VIA like assay (ICS) results correlate with in vivo control (LTNP) (Migueles et al. PLoS Path. 2011)■ VIA in LN cells correlates with protection against live-attenuated SIV (Picker et al. unpublished data) 19
  20. 20. Location, location, location. S. Plotkin, Clin Inf Dis. 2008 HIV – Mucosal sampling is critical Over 50% of all T cells reside in the gut The gut is the initial site of High-level HIV replication Massive CD4T cell depletion --occurs within the first two weeks after infection --observed after intravenous, intra-rectal, oral and vaginal challenges Limiting the initial viral replication in the gut could be crucial for an AIDS vaccine 20
  21. 21. Mucosal Assay Development• Assess the feasibility of various mucosal sampling methods in clinical trial setting• Develop clinical sampling methods and assays to assess vaccine induced mucosal immune responses in UK and Africa, focused on two assays (a) cellular responses in the gut (b) antibody responses in genital secretionsApply into IAVI sponsored/collaborative clinical trials 21
  22. 22. CD8 T cell responses to gag in blood compared with gutDifferences in phenotype and function between blood and gut,and between colon and duodenum also observed
  23. 23. KAVI developing as a Centre of Excellence in Mucosal Immunology in East Africa Supported by and IAVI and HIL, KAVI has embarked on a number of studies on mucosal immunology: - 2009 – initiated study to develop mucosal sampling methods and assays to assess mucosal immune responses in GU tract - 2011 – initiated pilot study to assess cellular responses in GALT (colon) using IAVI-HIL methodologies - 2011 – initiated mucosal sampling in vaccine trialsHaas et al Nature 2010 Mar 11; 464(7286):217-23 23
  24. 24. Mucosal Specimen Collection and Assay MethodsSampling Method AssayMerocel sponge Antibody (Ab)(rectal and cervical)Digene cytobrush MMC(rectal and cervical)Semen MMC and AbRectal biopsies MMCSoftCup MMC and AbAspirator AbSaliva (active and passive) AbOro and naso-pharyngeal swabs Ab 24
  25. 25. Other assays for assessment of T cells For HIV – Makedonas & Betts 2010 TB, Malaria & other diseases plethora of possible responses TCR avidity, affinity, clonotype ‘The degree of polyfunctionality of CD8+ T cells correlates to the number of functions for which are assayed.’Novel technologies: e.g. Proteomics, genomics, DNA/RNA microarrays,RNA-sequencing, single cell multiplexing e.g. fluidigm, nano-string, 25
  26. 26. Gene expression profiles induced by YF-17D vaccination in the European and Ugandan populationA. YF-17D vaccination induces different gene expressions in Lausanne and Entebbe. (A) Number of genes at 5% FDR with a threshold of 2 or 3 fold change. The magnitude of gene expression changes was higher in Lausanne compared to Entebbe. (B) Fold changes of the top 100 genes in Lausanne and Entebbe at 3, 7, 14, 56 and 84 days after a first vaccination. The expression changes were observed at days 3 and 7 after YF-17D vaccination. The expression changes of individuals genes was different in VGTI (Rafick Sekaly), UVRI and IAVI Lausanne and Entebbe.B.
  27. 27. Single Cell Genomics  Just like flow cytometry, this technology provides us with two independent pieces of information: – How many cells express a gene? – How much do these cells express?  Standard (bulk) analysis confounds these two measurements to generate an average  Single cell analysis allows us then to answer another question: – What is the co-expression of genes?Slide courtesy Mario Roederer and CAVD VIMC-T cell consortium
  28. 28. Fluidigm TechnologyDispense cDNA into Microfluidics Sample cDNAChipsample vesselsPrimers & probes intoreagent vessels Primers & ProbesMicrofluidics mixes allcombinations innanoliter-sizedchambers40 Cycle RT-PCRMonitor fluorescencefrom each chamber 28
  29. 29. Why is Single Cell Important? BLIMP1 CD84 (SLAMF5) TNFR-1 BLIMP1 CD84 (SLAMF5) TNFR-1Single cell analysis reveals a completely different picture ofregulation of these genes!
  30. 30. Fluidigm Analysis Summary• On Single Cells: – Distribution of gene expression – Coordinate regulation of genes – Reveals further heterogeneity (subsets) – potential correlates for vaccine or disease analysis• On Bulk Populations (~100 cells) – Remarkable precision & sensitivity (RT-PCR) – Economical, directed micro-array-like analysis• Needs enormous bioinformatics support! 30
  31. 31. Assays to Monitor Humoral Responses• Primary Assays (Validated) – Binding ELISA – Neutralizing antibody • TZM-b1 tier 1 and tier2 • A3R5• Secondary (Qualified) – Antibody isoptype – Non-neutralizing activity e.g. ADCC / ADCVI, virus capture – Avidity and affinity – Epitope mapping 31
  32. 32. Assays to Monitor the Humoral ResponseResearch Assays• B Cells – B cell Elispot (memory B cells) – B cell phenotyping• Antibody – Deep sequencing/moAb isolation to characterize engagement & mutation of Ab genes. – Isolation and characterization of Env-specific monoclonal antibodies (IgG and IgA) from B memory cells and plasmablasts sorted according to homing receptors in the systemic and mucosal compartments – Use antibodies to explore anti-viral functions e.g. aggregation, inhibition of trancytosis, mucus inhibition – Epitope mapping 32
  33. 33. Challenges• Need a pipeline of immunogens for clinical testing• Lack of human challenge models and efficacy trial and data – E.g. Need to define what an effective broadly cross- reactive antibody or anti-viral T cell response• Bio-informatics and data management are not keeping up with technology and data bases not tranlsational• Define targets-functional or structural constraints• Standardization and qualification in vaccine trials 33
  34. 34. Developing Assays Defining Immunogenicity Go/No-go Criteria •Viral control is possible •Effective immune responses HIV •Target immunogens Pathogenesis •Transmission NHP •Host and viral genetics Efficacy• Protection is possible •Optimize dose, regimen, e.g. passive antibodies Clinical Trial •Safety, experience with product• Viral control possible Data •Immunogenicity e.g response rate, e.g. CMV, Live attenuated, kinetics, function, breadth Ad26/MVA mosaic, •Test strategy e.g. Increased breadth, DNA/IL12/Ep+Ad5 function, location• Immune correlates e.g. LAV-VIA in lymph-node, CMV broad TEM, DNA+Ad5 VIA Efficacy Trials Correlates of protection Mechanism of protection 35
  35. 35. Assay Validation and Qualification• Primary Immunogenicity Assays – Validate and document fit for purpose – Hypothesis testing – Good precision, accuracy, rigorous and robust – Ideally low cost and high throughput – Critical as progress to large scale manufacturing• Secondary – Qualified – Characterize immunogenicity, test secondary hypothesis, generate new hypothesis• Exploratory – R+D 36
  36. 36. Immune Monitoring in Clinical Trials Context Cont’d• Data from the field – e.g. Step trial and RV144 – Advancements in systems biology, deep sequencing, and other technologies enable us to look at immune responses in exquisite detail• Post RV144 and STEP – Tendency to assess every cellular and humoral immune response possible –may not feasible for every trial• Partnerships, collaborations, multiple clinical centers, study populations and funders / sponsors required – Complex operations, reporting, coordination, compromise, 37

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