On Tuesday at the AIDS Vaccine 2011 conference in Bangkok, Barton Haynes described the initial results of a mammoth effort to uncover immune correlates of protection in the RV144 vaccine trial (the presentation is available online via the conference website). As reported in the New England Journal of Medicine in 2009, the trial demonstrated a very slight but statistically significant degree of efficacy in reducing HIV infection risk; there were 51 infections in the vaccine group compared to 74 in the placebo group, indicating the vaccine reduced HIV acquisition by 31.2% (95% confidence interval: 1.1 to 52.1; p=0.04). Of potential importance, the reduction in risk was greatest in the first year, during which there were 12 infections among vaccine recipients compared to 30 in the placebo group. Over the subsequent 2.5 years of follow up, there were an additional 39 infections in the former group and 45 in the latter (essentially no difference).
To investigate whether any vaccine-induced immune responses were associated with acquiring or resisting infection in the trial, Haynes led a large collaborative effort that prioritized six different immunological tests and then studied them in a case-control format, comparing samples from vaccine recipients who became infected to those who did not. Samples were taken at week 26, two weeks after the final booster immunization. Ten infections occurred among vaccine recipients prior to week 26, so the total number that could be included in the case control study was 41. The total number of uninfected vaccine recipients included for comparison was 205. The six immunological tests studied were:
- Binding of IgA antibodies in plasma to a panel of 14 HIV Env proteins.
- Avidity of IgG antibodies to one of the gp120 proteins included in the AIDSVAX vaccine used in the trial (derived from a Thai CRF01_AE HIV isolate codenamed A244).
- Antibody-dependent cellular cytotoxicity (ADCC) against cells infected with another a Thai CRF01_AE HIV isolate codenamed 92TH023 (the gp120 protein from this isolate is included in the ALVAC vector used in the trial).
- Neutralizing antibody responses against a panel of six “tier 1” (relatively easy to neutralize) HIV isolates.
- Binding IgG antibodies to the V1/V2 loops of HIV Env “scaffolded” onto a gp70 protein from murine leukemia virus (MLV). This assay was selected based on evidence that it identifies antibodies capable of binding to HIV Env in its natural conformation.
- CD4 T-cell responses, measured by the production of cytokines and cell surface markers in response to stimulation with peptides from the 92TH023 isolate.
Helpfully, two scientists involved in the work—Morgane Rolland and Peter Gilbert—explain the statistical methodology used to evaluate whether any of these measures correlated with HIV acquisition in a new paper (see abstract and link below; the journal has made the paper open access). The authors note that multiple comparisons are involved in the analyses, which is known to increase the possibility of obtaining significant results simply by chance. The standard statistical tool for addressing this possibility is called a Bonferroni correction, but it was not used in this case because the goal was to generate hypotheses about possible immune correlates for additional studies, as opposed to confirming associations definitively. Instead of the Bonferroni method, the scientists used an approach in which each result is assigned a “q-value,” which represents the estimated chance of a false positive.
Haynes revealed in Bangkok that two of the six measures showed statistically significant associations with HIV acquisition: binding of IgA antibodies in plasma to HIV Env, which was linked to an increased relative risk of HIV infection of 1.54 (p=0.027); in other words, vaccine recipients displaying this response at week 26 of the trial were 54% more likely to subsequently become infected than those without it. Conversely, the presence of binding IgG antibodies to the V1/V2 loops of HIV Env scaffolded onto the MLV gp70 protein was associated with a relative risk of 0.57 (p=0.015), indicating that vaccine recipients who showed this response at week 26 were 43% less likely to acquire HIV than those who did not. For both results, the q value was 0.08, meaning that there is an approximately 8% chance they were false positives. The analyses have been repeated and confirmed by a second, independent group of statisticians. Haynes noted that the presence of IgA antibodies to Env did not enhance HIV infection risk compared to placebo recipients; rather, vaccinated individuals with high levels of these responses experienced the same risk of infection as the unvaccinated group. The opposite pattern was true for binding IgG antibodies to the V1/V2 loops: compared to placebo recipients, vaccinated individuals with the highest levels of these antibodies were around 50% less likely to acquire HIV infection.
In discussing the findings, Haynes took pains to stress that the results are exploratory in nature because there was no pre-specified plan for studying immune correlates in the original RV144 protocol. He also emphasized that further work is needed in order to understand whether there is a causal relationship between these immune responses and the trial outcome. The reason why IgA antibodies to Env would be associated with reduced vaccine efficacy is not yet clear, but there is some suggestive evidence that these antibodies interfere with antibody-dependent cellular cytotoxicity (ADCC), one of the immunological mechanisms that could have been responsible for the apparent protection observed in the trial. Similarly, it is not yet known whether the binding IgG antibodies to the V1/V2 loops were directly responsible for protecting against HIV infection (whether via ADCC or some other mechanism). However, these responses did wane significantly in vaccine recipients over the six months after the final week 24 immunizations, which appears to track with the protective effect of vaccination being almost entirely concentrated in the first year of the trial.
While much work remains, these new results represent potentially crucial clues for researchers working to improve on the marginal efficacy observed in RV144. Most optimistically, they hint that it might be possible to push the efficacy threshold of similar vaccine regimens over 50% by fine-tuning the types of immune responses that are induced. Although the induction of broadly neutralizing antibodies against HIV is still thought to be necessary to achieve a highly efficacious vaccine, the development of a candidate offering greater than 50% protection would be a huge step forward.
AIDS Res Hum Retroviruses. 2011 Sep 9. [Epub ahead of print]
Rolland M, Gilbert P.
U.S. Military HIV Research Program, 1600 E. Gude Drive, Rockville, Maryland, United States, 20850
Since the RV144 vaccine combination showed efficacy in a Phase III trial, it provides an opportunity to generate hypotheses about the immune responses necessary for protection against HIV-1 infection, and these results could help devise vaccine candidates with higher efficacy. Here we describe how researchers can determine the correlates of immune protection for an HIV/AIDS vaccine, particularly in the context of the RV144 trial, and we discuss the terminology used to describe correlates and surrogates.