1. Washington D.C., USA, 22-27 July 2012www.aids2012.org Statistical Design and Analysis for Immune Correlates Assessment: Basic Concepts and RV144 Illustration Yunda Huang 1, Holly Janes 1, 2, Peter Gilbert 1, 2 1 Vaccine and Infectious Disease Division Fred Hutchinson Cancer Research Center 2 Department of Biostatistics, University of Washington Seattle, Washington, USA IAS 2012 Correlates Workshop, Washington DC, USA

2. Washington D.C., USA, 22-27 July 2012www.aids2012.org Reasons to Be a Statistician … No one knows what we do so we are always right.

3. Washington D.C., USA, 22-27 July 2012www.aids2012.org Outline Definitions Correlates Study and Sampling Design Example: RV144 Immune Correlates Study Strategies to Evaluate Immune Correlates Summary

4. Washington D.C., USA, 22-27 July 2012www.aids2012.org In the context of preventive HIV vaccine clinical trials Rate of HIV infection: frequency of new HIV infections during a specified time frame – can be measured in vaccine (R v ) and placebo (R p ) groups separately Vaccine efficacy: proportion of infections prevented by vaccine relative to placebo (1- R v / R p ) – need to know the rate of infection in both vaccine and placebo groups Correlates of risk (CoR ): Immune markers statistically correlated with the rate of HIV infection in the vaccine group (Qin & Gilbert et al., JID, 2007) Correlates of protection (CoP): Immune markers statistically correlated with vaccine efficacy in the vaccine and placebo groups (Qin & Gilbert et al., JID, 2007; Plotkin & Gilbert, CID, 2012)

5. Washington D.C., USA, 22-27 July 2012www.aids2012.org Correlates of Risk (CoR) and Correlates of Protection (CoP) Vital for vaccine development –Choice of antigens included in vaccines –Bridge from previously collected protection data –Surrogate for efficacy evaluation –Population and individual level immunity measure Easily being confused and used inter-changeably Three facts: 1.A CoR may not be a CoP, but could be 2.A CoP must be a CoR 3.Not all CoRs or CoPs are created equal

6. Washington D.C., USA, 22-27 July 2012www.aids2012.org Fact #1a: A CoR may not be a CoP It is a CoR – because the levels of the immune response are correlated with the rate of infection in the vaccine group Is it a CoP? Immune Response Rate of HIV Infection

7. Washington D.C., USA, 22-27 July 2012www.aids2012.org Fact #1a: A CoR may not be a CoP It is a CoR It is not a CoP – if, in the same way, the rate of infection is correlated with the immune response (had it been measured) in the placebo as well Rate of HIV Infection Immune Response

8. Washington D.C., USA, 22-27 July 2012www.aids2012.org Fact #1a: A CoR may not be a CoP It is a CoR It is not a CoP The immune responses from this biomarker are not predictive of VE (≠ CoP), although overall VE=40% Vaccine Efficacy (%) Rate of HIV Infection Immune Response

9. Washington D.C., USA, 22-27 July 2012www.aids2012.org Fact #1a: A CoR may not be a CoP It is a CoR It is not a CoP Why: –Those individuals who could mount a strong immune response are better able to ward off infection “on their own” with no impact of the vaccine-induced immune responses of this marker –“On their own”: It may mark susceptibility to infection independent of Vaccination, e.g., risk behavior or host genetics Rate of HIV Infection Vaccine Efficacy (%) Immune Response

10. Washington D.C., USA, 22-27 July 2012www.aids2012.org Fact #1b: A CoR could be a (perfect) CoP It is a CoR Is it a CoP? Rate of HIV Infection Immune Response

11. Washington D.C., USA, 22-27 July 2012www.aids2012.org Fact #1b: A CoR could be a (perfect) CoP It is a CoR It is a CoP: those individuals who could mount a strong immune response are better able to remain uninfected, differently in vaccine and placebo recipients if assigned vaccine And, these immune responses (=CoR) are also predictive of vaccine efficacy (=CoP) Rate of HIV Infection Immune Response Vaccine Efficacy (%)

12. Washington D.C., USA, 22-27 July 2012www.aids2012.org Fact #2: A CoP must be a CoR It’s equivalent to show: If not a CoR, then not a CoP Immune responses from vaccinees are not predictive of rate of infection -- not a CoR Rate of HIV Infection Immune Response

13. Washington D.C., USA, 22-27 July 2012www.aids2012.org Fact #2: A CoP must be a CoR It’s equivalent to show: If not a CoR, then not a CoP Immune responses from placebos will not be predictive of rate of infection Rate of HIV Infection Immune Response

14. Washington D.C., USA, 22-27 July 2012www.aids2012.org Fact #2: A CoP must be a CoR It’s equivalent to show: If not a CoR, then not a CoP Immune responses will not be predictive of vaccine efficacy Rate of HIV Infection Immune Response Vaccine Efficacy (%)

15. Washington D.C., USA, 22-27 July 2012www.aids2012.org Fact #3a: Not all CoRs are created equal Immune Response Rate of HIV Infection

16. Washington D.C., USA, 22-27 July 2012www.aids2012.org Fact #3a: Not all CoRs are created equal Immune Response Rate of HIV Infection

17. Washington D.C., USA, 22-27 July 2012www.aids2012.org Fact #3b: Not all CoPs are created equal Immune Response Vaccine Efficacy (%)

18. Washington D.C., USA, 22-27 July 2012www.aids2012.org Fact #3b: Not all CoPs are created equal Immune Response Vaccine Efficacy (%)

19. Washington D.C., USA, 22-27 July 2012www.aids2012.org Fact #3b: Not all CoPs are created equal Immune Response Vaccine Efficacy (%)

20. Washington D.C., USA, 22-27 July 2012www.aids2012.org Outline Definitions 1.A CoR may not be a CoP, but could be 2.A CoP must be a CoR 3.Not all CoRs or CoPs are created equal Correlates Study and Sampling design Example: RV144 Immune Correlates study Strategies to Evaluate Immune Correlates Summary

21. Washington D.C., USA, 22-27 July 2012www.aids2012.org Time-dependent and Time-independent CoR Time-independent immune correlates analysis: discover correlates at a specific time point –e.g. immune responses 2 weeks after the last vaccination –Peak immune response time point close to baseline –Informative and practical Time-dependent immune correlates analysis: discover correlates whose levels may change over time –e.g., most recent immune responses before diagnosis of infection –Immune response near the time of exposure with the acute risk of infection –Informative about the mechanism of protection

22. Washington D.C., USA, 22-27 July 2012www.aids2012.org CoR Study Design HIV vaccine-induced immune responses are only assessed in vaccinees Statistical power of an immune correlates study is driven by the number of HIV infections among vaccinees For a given total, the number of vaccinee infections depend on the vaccine efficacy: the higher the VE, the smaller number of infections from the vaccine group The smaller # infections is, the stronger the correlation between the immune response and the rate of infections needs to be, in order to have the same statistical power for CoR detection

23. Washington D.C., USA, 22-27 July 2012www.aids2012.org CoR Study Sampling design With unlimited resources, we could measure the post- vaccination immune responses from every vaccinees Several cohort study designs have been developed to save resources with minimal loss of power after adjusting for the sampling design Case-cohort (traditional): controls are sampled without regard to infection time as part of a subcohort Case-control: controls are sampled after ascertainments of cases

24. Washington D.C., USA, 22-27 July 2012www.aids2012.org CoR Study Sampling Design: Case-cohort Traditionally, controls are sampled without regard to failure times as part of a subcohort –Sampling can be done a priori without regard to case status or time –All cases are included whether they occur in the subcohort or not; controls are included only if in the subcohort –Estimate population level immune responses –Could select controls for multiple outcomes Lately, some case-cohort designs are also outcome-dependent

25. Washington D.C., USA, 22-27 July 2012www.aids2012.org CoR Study Sampling Design: Case-control Controls are sampled after ascertainments of cases Individual matching, frequency matching or stratification to sample appropriate controls for cases Matching addresses issues of confounding in the DESIGN stage of a study as opposed to the ANALYSIS phase, providing a more efficient analysis (reduction in standard errors of estimates) Matching on non-confounders may lose efficiency compared to the non-matched case-control approach

26. Washington D.C., USA, 22-27 July 2012www.aids2012.org Analysis Method Standard Cox or logistic regression models if data on the full cohort were available Modified Cox or logistic regression models if sub- sampling is done to account for the sampling design –Breslow and Holubkov (1997, Biometrika) –Borgan et al. estimator II (2000, Lifetime Data Analysis)

27. Washington D.C., USA, 22-27 July 2012www.aids2012.org Outline Definitions Correlates Study and sampling design –Number of infections drives the power of the study –Sampling designs with corresponding analysis methods Example: RV144 immune correlates study Strategies to evaluate different immune correlates Summary

28. Washington D.C., USA, 22-27 July 2012www.aids2012.org RV144 Thai Trial Primary Results 28

29. Washington D.C., USA, 22-27 July 2012www.aids2012.org 29 RV144 Thai Trial

30. Washington D.C., USA, 22-27 July 2012www.aids2012.org Impetus for the Correlates Study: Evidence for Partial Vaccine Efficacy Objective: To carry out an immune correlates analysis to begin to identify how the vaccine might work 0.00.51.01.52.02.53.03.5 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 Years Probability of HIV Infection (%) Placebo Vaccine C. Modified Intention-to-Treat Analysis* *N=16,395 assessed; 51 Vaccine, 74 Placebo HIV-1 infected Estimated VE = 31% [95% CI 1−51%], p=0.04 30

31. Washington D.C., USA, 22-27 July 2012www.aids2012.org RV144 Correlates of Risk Results 31

32. Washington D.C., USA, 22-27 July 2012www.aids2012.org What the RV144 Correlates Study Assessed The analysis sought to discover Correlates of Risk (CoR): Immune response variables that predict whether vaccinees become HIV-1 infected Thus, the study is designed to generate hypotheses that certain immune responses are Correlates of Protection (CoP) that would need validation in future research 32

33. Washington D.C., USA, 22-27 July 2012www.aids2012.org RV144: Two Tiers of Studies Pilot immunogenicity studies –Multiple immunology labs to perform assays on sample-sets from HIV uninfected RV144 participants –Conducted standardized comparative analyses of all candidate assays, to down-select the best performing assays and to optimize the immune variables to study as correlates Case-control study –Assessed the selected immune variables as correlates of infection risk 33

34. Washington D.C., USA, 22-27 July 2012www.aids2012.org RV144 Example Pilot Data: gp70-V1V2 Binding Antibodies (ELISA) 34

35. Washington D.C., USA, 22-27 July 2012www.aids2012.org RV144: Criteria for Advancing Assays to the Case-Control Study 35 Criterion 1. Represents a niche in immunological space (not highly correlated with other assays)  2. Low false positive rate (judged in placebo recipients and Week 0 responses of vaccinees)  3. Vaccine-induced responses with broad variability  4. Relatively low noise (e.g., high reproducibility on replicate samples)  5. Relatively low specimen volume requirement  6. Previously supported as a correlate of infection in the North American VaxGen trial of AIDSVAX 

36. Washington D.C., USA, 22-27 July 2012www.aids2012.org RV144 Case-Control Analysis: Two Tiers Primary Analysis: 6 priority immune response variables Secondary Analysis: All other immune response variables that passed pilot study criteria This division maximizes statistical power for the priority immune variables while allowing a broader exploratory analysis 36

37. Washington D.C., USA, 22-27 July 2012www.aids2012.org Down-Selected Primary Immune Variables Primary VariablePrincipal Investigator Plasma IgA Binding (14 envelope panel)Georgia Tomaras IgG avidity score to A244 gp120Munir Alam Antibody-dependent cellular cytotoxicity-AE- 92TH023. HIV infected CD4 T cells David Evans Michael Alpert Neutralization of Tier 1 viruses (6 envelope panel) David Montefiori Rungpeung Sutthent Chitraporn Karnasutra IgG binding to scaffolded gp70-V1V2Susan Zolla-Pazner CD4 T cell intracytoplasmic cytokines (IFN , IL-2, TNF , CD154) stimulated by AE-92TH023 peptides Julie McElrath Nicole Frahm 37

38. Washington D.C., USA, 22-27 July 2012www.aids2012.org RV144 Case-Control Study Time-independent CoR: What are the immunologic measurements at a fixed time-point (wk26) in vaccinees that predict HIV-1 infection over a 3 year follow-up? Sampling design: Balanced stratified random sampling for vaccinees− 5:1 (control:case) ratio within each of the following covariate strata Gender × Number of vaccinations × Per-protocol status –41 infected vaccinees (all available) –205 uninfected vaccinees (5:1 stratified random sample) –40 placebo recipients (simple random sample) Outcome-dependent 2-phase sampling case-cohort study 38

39. Washington D.C., USA, 22-27 July 2012www.aids2012.org Why 5:1 Sampling? Only a Small Power Loss Moving from 5:1 to 10:1 39

40. Washington D.C., USA, 22-27 July 2012www.aids2012.org RV144 Immune Correlates Study Main Result* Variable Relative risk per sd P-valueQ-value** IgA Binding to Envelope Panel1.540.0270.08 IgG Avidity A244 gp1200.810.370.56 ADCC AE.HIV-1 Infected CD4 Cells0.920.68 Tier 1 Neutralizing Antibodies1.370.220.45 IgG Binding to gp70-V1V20.570.0150.08 CD4+ T Cell Intracellular Cytokines1.090.610.68 *Multivariate logistic regression (quantitative variables) adjusted for gender, baseline behavioral risk (low, medium, high) **1-Qvalue ≈ estimated prob. that the immune variable correlates with infection rate All 6 variables together in multivariate analysis: p=0.08 The 2 correlates in multivariate analysis: p=0.01 40

41. Washington D.C., USA, 22-27 July 2012www.aids2012.org V1V2-gp70 Scaffold ELISA Medium High Low 41

42. Washington D.C., USA, 22-27 July 2012www.aids2012.org Cumulative Infection Rates with V1V2-gp70 Scaffold Assay Estimated Relative Risk High vs. Low = 0.29 HighV1V2 Low/Medium V1V2 42

43. Washington D.C., USA, 22-27 July 2012www.aids2012.org Plasma IgA Binding To Envelope Panel Medium High Low 43

44. Washington D.C., USA, 22-27 July 2012www.aids2012.org Cumulative Infection Rates with IgA Env Binding Assay Estimated Relative Risk High vs. Low = 1.89 High Env IgA Low/Medium Env IgA 44

45. Washington D.C., USA, 22-27 July 2012www.aids2012.org Sieve Analysis is an Integral Part of Immune Correlates Assessment The correlates analysis showed V1V2 Abs predicted infection in the vaccine group only Sieve analysis examines evidence for a difference in the sequences of viruses infecting vaccinees versus placebo recipients –Observed differences can be attributed to the vaccine in a randomized trial –Detection of a ‘sieve effect’ may suggest that the vaccine blocks infection with some types of exposing HIVs If certain epitope-specific Ab responses are protective, then would expect to see a relative absence of these specific epitopes in sequences of infected vaccinees compared to infected placebo recipients Found additional evidence for vaccine pressure on the V2 mid-loop region 45

46. Washington D.C., USA, 22-27 July 2012www.aids2012.org The gp70-V1V2 antibody CoR would be most useful for vaccine development if it strongly predicted VE (i.e., was a good CoP) What it Could Mean (Most Useful for Vaccine Development) 46

47. Washington D.C., USA, 22-27 July 2012www.aids2012.org The gp70-V1V2 antibody CoR does not predicted VE (≠CoP) But, It Could Also Mean 47

48. Washington D.C., USA, 22-27 July 2012www.aids2012.org Outline Definitions Correlates Study and Sampling design Example: RV144 Immune Correlates Study –Case-control study –Evidence for two correlates of infection risk in vaccinees –IgG antibodies that bind to scaffolded-V1V2 recombinant protein correlated inversely with infection rate –Plasma IgA antibodies correlated directly with infection rate Strategies to evaluate different immune correlates Summary

49. Washington D.C., USA, 22-27 July 2012www.aids2012.org Collect the requisite data for correcting the CoR analysis for potential exposure confounding (e.g., risk behavior, host genetics) Collect the requisite data for directly assessing the ability of a CoR to predict VE (more on next few slides) Conduct sieve analysis of HIV sequences to assess whether the vaccine applied pressure on the HIV Env target(s) specific to the immune correlate Collaborate with other groups (e.g, CHAVI, CAVD, VRC) conducting experiments (e.g., in non-human primates) testing hypotheses about the CoRs 49 Strategies to Assess CoRs as VE-Predictors (CoPs) and as Mechanisms of Protection

50. Washington D.C., USA, 22-27 July 2012www.aids2012.org Once a positive CoR is discovered in vaccinees Collect the requisite data for directly assessing the ability of a CoR to predict VE To assess the relationship between VE and an immune marker (i.e., CoP), we need to know the level of the immune marker for both vaccine and placebo recipients – fill in all the blanks GroupOutcome Immune response levels 1234 Vaccine Uninfected Infected Placebo Uninfected Infected

51. Washington D.C., USA, 22-27 July 2012www.aids2012.org Once a positive CoR is discovered in vaccinees Collect the requisite data for directly assessing the ability of a CoR to predict VE To assess the relationship between VE and an immune marker (i.e., CoP), we need to know the level of the immune marker for both vaccine and placebo recipients -- fill in all the blanks GroupOutcome Immune response levels Total 1234 Vaccine Uninfected Infected Total Placebo Uninfected ? ? ? ? Infected ? ? ? ? Total~~~~ CoR study Clinical Trial

52. Washington D.C., USA, 22-27 July 2012www.aids2012.org Predicting the potential HIV specific immune response (X) to HIV vaccination for placebo recipients (Follmann, Biometrics, 2006) BIP approach: baseline immunogenicity predictor (W) –W is correlated with X –At baseline, measure W in both vaccine and placebo recipients, e.g., immune responses to a non-HIV vaccine –Randomization ensures W same in both vaccine and placebo groups –Build statistical models between W and X based on vaccinees’ data –Use placebo subjects’ W to impute X and pretend X is what we would have seen, had the placebo subjects received the HIV vaccine CPV approach: close-out placebo vaccination –At the end of the trial, inoculate placebo uninfecteds with HIV vaccine –Measure immune response on the same schedule as was measured for vaccinees –Pretend that is what we would have seen, had we inoculated at baseline

53. Washington D.C., USA, 22-27 July 2012www.aids2012.org Remarks Correlates of Risk (CoR) and Correlates of Protection (CoP) are different but both important Impact of a positive CoR –Vaccine development (other speakers) –HVTN go/no go guidelines based on RV144 immune correlates finding CoR assessment in vaccine efficacy trials –Future HVTN efficacy trials will use two-stage sequential designs* with important secondary objectives to examine immune correlates BIP and/or CPV approach for the assessment of CoP * References: Gilbert P, Grove D, Gabriel E, Huang Y, Gray G, Hammer S, Buchbinder S, Kublin J, Corey L, Self S (2011, Statistical Communications in Infectious Diseases) Corey L, Nabel GJ, Dieffenbach C, Gilbert PB, Haynes BF, Johnston M, Kublin J, Lane HC, Pantaleo G, Picker LJ, Fauci AS (2011, Science Translational Medicine)

54. Washington D.C., USA, 22-27 July 2012www.aids2012.org Acknowledgement Research supported by –NIAID, NIH –USMHRP –Bill and Melinda Gates Foundation –Henry Jackson Foundation Statistical Center for HIV/AIDS research and Prevention (SCHARP) –Youyi Fong –Allan DeCamp –Ying Huang –Paul Edlefsen –Steve Self RV144 Immune Correlates Study –Leadership: Bart Haynes, Peter Gilbert, Jerome Kim, Nelson Michael, Julie McElrath –Host of laboratory scientists from several institutions Duke University led the antibody work (Bart Haynes, Georgia Tomaras, David Montefiori) FHCRC led the T cell work (Julie McElrath)

55. Washington D.C., USA, 22-27 July 2012www.aids2012.org Appendix

56. Washington D.C., USA, 22-27 July 2012www.aids2012.org Statistical Approach to Sieve Analysis Local sieve analysis (high-dimensional) –Assess Env amino acid (AA) sites as ‘signature sites’ Signature = site with different distribution of residues vaccine vs. placebo relative to an insert-residue* –Assess sets of AA sites as ‘signature sets’ E.g., 9-mers potentially constituting T cell epitopes E.g., clusters of sites potentially constituting antibody epitopes Global sieve analysis (low-dimensional) –Assess if and how vaccine efficacy depends on the distance of the exposing virus to an insert-sequence –Global = summarize a breakthrough HIV by one or a few numbers quantifying distance *3 vaccine-inserts: ALVAC-AE.92TH023, rgp120-AE.CM244, rgp120-B.MN