a Publication of the HIV Vaccine Trials Network VOLUME 4, ISSUE 1 | JULY, 2012 REIGNITING RECRUITMENT IN THIS ISSUE photo by Sid Niazi ARTICLES 02 Probing the Diversity of Vaccine Elicited HIV-1 Antibodies: Informed by the 18 Social and Behavioral Science in Clinical Trials of Biomedical HIV Prevention RV144 Correlates Analysis Interventions 03 Assays to Probe the Humoral Response 22 HVTN Annual Network Award Winners 06 Turning up the Heat in Miami 24 HVTN Protocols 07 Orlando HVTN 505 Study Site: From Challenges to Accomplishments CALENDAR [back cover] 10 Assessing Mucosal Immune Responses in HIV Vaccine Trials 13 Highlights from Recent HVTN Publications Probing the Diversity of Vaccine Elicited HIV-1 Antibodies: Informed by the RV144 Correlates Analysis Georgia D. Tomaras RV144, a trial conducted by the U.S. Military HIV Research IgG antibody responses (ie, ADCC and nAbs) might corre- Program and the Thai Ministry of Health, was the first HIV-1 late with decreased risk of infection if present with low levels vaccine study in which a modest efficacy (31.2%) of HIV-1 of Env IgA (higher levels of Env IgA seem to counteract the vaccination was shown. The study regimen consisted of a beneficial effects of IgG). Thus, the balance of antibody types, canarypox prime expressing Gag, Pro, and gp120 (ALVAC- and the interactions among them -- particularly in those with HIV), and a gp120 boost (AIDSVAX® B/E). These results different Fc receptor binding properties -- merits further study were published in the New England Journal of Medicine in to determine if specific Env antibody isotypes influence the 2009.1 level of vaccine efficacy. A concerted effort by an international consortium of investiga- The results from the RV144 correlates analysis have signifi- tors, led by Dr. Barton F. Haynes of Duke University, set out cantly informed our understanding of how to analytically to determine if any immune variables correlated with infection probe the HIV-1 vaccine elicited antibody responses, and risk in the RV144 trial. In the initial phase of the studies, there generated several leading hypotheses that are critical to test was an open call to measure vaccine-elicited immune responses in future HIV-1 vaccine candidates. Measurements of the re- on RV144 samples. In the next phase, a selected set of immune sponse rates and magnitudes of V1V2 IgG antibodies and Env variables, chosen from the pilot study results, were formally IgA binding antibodies elicited by different vaccine regimens tested as primary immune variables for the correlates analysis (different vector, adjuvant, and Env immunogen formulations) of RV144. are important for understanding the broad applicability of these findings -- ie, whether these correlates can be confirmed The RV144 correlates analysis yielded 2 immune correlates with other regimens. of risk: V1V2 IgG antibodies correlated with decreased risk Durability of HIV-1 Env Immune Responses of infection, while HIV-1 Env IgA antibodies correlated with increased risk of infection in the vaccine arm (higher One of the most overarching lessons from RV144, indepen- Env IgA levels seemed to have abrogated the vaccine effect, dent of the immune correlates results, comes from the observa- but the risk of infection in this group was not higher than 2 tion that there was decreased vaccine efficacy over time. This in the placebo group). The combined results of the original waning of vaccine efficacy corresponds to decreasing vaccine- and correlate studies galvanized the HIV-1 vaccine field, and specific immune responses (anti-Env antibody titers). Thus, the have influenced the direction of subsequent protocols testing measurement of the durability of immune responses (antibody HIV-1 vaccine candidates. Notably, these correlates of HIV-1 half-life) is a critical component for evaluating improvements infection risk from RV144 are not necessarily surrogates of in HIV-1 vaccine strategies going forward. protection nor mechanistic correlates. However, toward the goal of identifying the types of immune responses an effica- Antibody Responses to Circulating vs. Vaccine Strain cious vaccine should elicit, the RV144 study has provided key HIV-1 Env hypotheses to test in further HIV-1 vaccine trials. The 2 correlates of risk identified in RV144, along with cor- Key Hypotheses Generated by RV144 responding secondary analyses, highlight an important feature for assessing vaccine efficacy. The measurements that correlated The RV144 correlates analysis evaluated 6 primary variables: most strongly with infection risk were not those against the the binding of plasma IgA antibodies to Env, the avidity of vaccine strain Env in the boost, but rather antibodies against IgG antibodies for Env, antibody-dependent cellular cyto- cross-clade responses: The strongest statistical correlate was toxicity (ADCC), HIV-1 neutralizing antibodies (nAbs), the the association of risk with IgA responses to the C1 region in binding of IgG antibodies to variable regions 1 and 2 (V1V2) gp120 of circulating strain in Thailand (CRF01AE). Moreover, of the gp120 Env, and the level of Env-specific CD4+ T cells.2 IgG responses to the V2 region of CRF01AE Env significant- In addition to the 2 identified immune correlates of risk ly correlated with decreased risk of infection among the differ- (V1V2 IgG and HIV-1 Env IgA), analysis of the 6 primary ent clades of V2 responses tested in the peptide microarray variables using interaction models suggested that functional assay (Gottardo, Montefiori et al., unpublished).2 This Continued on page 5... 2 JUNE 2012 VOLUME 4:1 | HVTNEWS Assays to Probe the Humoral Response Georgia D. Tomaras There are several categories of assays used to examine the diverse humoral response to vaccination: 1) validated assays for HIV-1 vaccine endpoints, 2) qualified and/or standardized assays and 3) assays for research and development. Validated assays are currently used in the first line assessment of HIV-1 vaccine trials. Qualified and/or standardized assays are utilized to generate hypotheses and further characterize vaccine im- munogenicity. Assays for research and development are more exploratory in nature and are not suitable as a vaccine study endpoint, but can provide substantial insight into the com- plexity and potential functionality of vaccine elicited immune responses. VALidated ASSAYS FOR HIV-1 Vaccine V= Variable Region C=Constant Region H=Heavy Chain L=Light Chain ENDPOINTS Structure of the antibody: The variable regions (VH, VL) contain the an- Neutralizing Binding Antibodies tigen binding sites; the Fc region mediates the effector activity (eg, neutral- ization); the constant regions present in the heavy chain define the isotype Currently validated assays include the measurement of of antibody (IgA, IgD, IgE, IgG, and IgM) and subclass (eg, IgG1-4). neutralizing antibodies categorized as Tier 1 or Tier 2 neutralization using the TZM-bl assay. Validated assays to clinical trials include the measurement of both monomeric assess binding antibody responses (IgG and IgA) by ELISA IgA (mIgA) and dimeric IgA (dIgA) that will be extended to the and multiplex bead technology are in place and can measure analysis of human mucosal samples. Moreover, the analysis magnitude and breadth of vaccine elicited responses. The goal of IgA responses and IgG subclasses, and the respective ratios of ongoing validation efforts include improving upon existing between different types of antibodies, may provide insights assays (eg, validation of A3R5 neutralization assays) and to into the role of different adjuvants/priming on the quality of the select additional binding and functional assays most relevant humoral responses. for vaccine evaluation. Antibody Epitope Mapping (Linear and QUALIFIED AND StandardiZED Conformational) A potentially important characteristic of antibodies is their ASSAYS breadth of binding to multiple HIV-1 clades (Group M, Clade IgG Subclasses, Isotypes, and Mucosal A, B, C, AE, etc.). Recent assay developments include the measurement of diverse HIV-1 Envs resulting in a single score Antibodies for magnitude and breadth. The linear epitope specificities are Antibody isotypes and IgG subclasses (IgG1-IgG4) are measured using either binding antibody assays with specific currently being measured in response to vaccination. The epitopes (eg, V2, C1, alanine scanning mutants) or through an rationale for measuring antibody isotypes and subclasses is HIV-1 multiclade peptide microarray that can fully measure that they provide insights into potential functional responses. multiclade gp160 epitope specificity. Conformational epitopes IgG1 and IgG3 are considered to be the most functional of (eg, CD4BS, CD4i, C1, and N-glycan dependent epitopes) can the subclasses in that they have been associated with HIV-1 be determined through differential binding to wild type pro- neutralization, complement fixation, FcR binding and antibody teins/scaffolds with and without mutation(s) that alter confor- dependent cellular cytotoxicity / cell mediated virus inhibition mational binding sites, or through competitive binding assays (ADCC/ADCVI). lgA antibodies at mucosal sites have been with monoclonal antibodies of known specificities (eg, A32 correlated with protection in exposed, uninfected subjects and monoclonal antibodies [mAb] blocking). studies in NHPs have indicated a potentially protective role for mucosal antibody responses as well as antibody effector functions and IgA memory B cells. Current assays in human Continued on page 4... www.hvtn.org