REIGNITING RECRUITMENT

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1 a Publication of the HIV Vaccine Trials Network Volume 4, issue 1 july, 2012 REIGNITING RECRUITMENT IN THIS ISSUE ARTICLES Probing the Diversity of Vaccine Elicited HIV-1 Antibodies: Informed by the RV144 Correlates Analysis Assays to Probe the Humoral Response Turning up the Heat in Miami Orlando HVTN 505 Study Site: From Challenges to Accomplishments Assessing Mucosal Immune Responses in HIV Vaccine Trials Highlights from Recent HVTN Publications photo by Sid Niazi Social and Behavioral Science in Clinical Trials of Biomedical HIV Prevention Interventions HVTN Annual Network Award Winners HVTN Protocols CALENDAR [back cover]

2 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 Program and the Thai Ministry of Health, was the first HIV-1 vaccine study in which a modest efficacy (31.2%) of HIV 1 vaccination was shown. The study regimen consisted of a canarypox prime expressing Gag, Pro, and gp120 (ALVAC- HIV), and a gp120 boost (AIDSVAX B/E). These results were published in the New England Journal of Medicine in A concerted effort by an international consortium of investigators, led by Dr. Barton F. Haynes of Duke University, set out to determine if any immune variables correlated with infection risk in the RV144 trial. In the initial phase of the studies, there was an open call to measure vaccine-elicited immune responses on RV144 samples. In the next phase, a selected set of immune variables, chosen from the pilot study results, were formally tested as primary immune variables for the correlates analysis of RV144. The RV144 correlates analysis yielded 2 immune correlates of risk: V1V2 IgG antibodies correlated with decreased risk of infection, while HIV-1 Env IgA antibodies correlated with increased risk of infection in the vaccine arm (higher Env IgA levels seemed to have abrogated the vaccine effect, but the risk of infection in this group was not higher than in the placebo group). 2 The combined results of the original and correlate studies galvanized the HIV-1 vaccine field, and have influenced the direction of subsequent protocols testing HIV-1 vaccine candidates. Notably, these correlates of HIV-1 infection risk from RV144 are not necessarily surrogates of protection nor mechanistic correlates. However, toward the goal of identifying the types of immune responses an efficacious vaccine should elicit, the RV144 study has provided key hypotheses to test in further HIV-1 vaccine trials. Key Hypotheses Generated by RV144 The RV144 correlates analysis evaluated 6 primary variables: the binding of plasma IgA antibodies to Env, the avidity of IgG antibodies for Env, antibody-dependent cellular cytotoxicity (ADCC), HIV-1 neutralizing antibodies (nabs), the binding of IgG antibodies to variable regions 1 and 2 (V1V2) of the gp120 Env, and the level of Env-specific CD4 + T cells. 2 In addition to the 2 identified immune correlates of risk (V1V2 IgG and HIV-1 Env IgA), analysis of the 6 primary variables using interaction models suggested that functional IgG antibody responses (ie, ADCC and nabs) might correlate with decreased risk of infection if present with low levels of Env IgA (higher levels of Env IgA seem to counteract the beneficial effects of IgG). Thus, the balance of antibody types, and the interactions among them -- particularly in those with different Fc receptor binding properties -- merits further study to determine if specific Env antibody isotypes influence the level of vaccine efficacy. The results from the RV144 correlates analysis have significantly informed our understanding of how to analytically probe the HIV-1 vaccine elicited antibody responses, and generated several leading hypotheses that are critical to test in future HIV-1 vaccine candidates. Measurements of the response rates and magnitudes of V1V2 IgG antibodies and Env IgA binding antibodies elicited by different vaccine regimens (different vector, adjuvant, and Env immunogen formulations) are important for understanding the broad applicability of these findings -- ie, whether these correlates can be confirmed with other regimens. Durability of HIV-1 Env Immune Responses One of the most overarching lessons from RV144, independent of the immune correlates results, comes from the observation that there was decreased vaccine efficacy over time. This waning of vaccine efficacy corresponds to decreasing vaccinespecific immune responses (anti-env antibody titers). Thus, the measurement of the durability of immune responses (antibody half-life) is a critical component for evaluating improvements in HIV-1 vaccine strategies going forward. Antibody Responses to Circulating vs. Vaccine Strain HIV-1 Env The 2 correlates of risk identified in RV144, along with corresponding secondary analyses, highlight an important feature for assessing vaccine efficacy. The measurements that correlated most strongly with infection risk were not those against the vaccine strain Env in the boost, but rather antibodies against cross-clade responses: The strongest statistical correlate was the association of risk with IgA responses to the C1 region in gp120 of circulating strain in Thailand (CRF01AE). Moreover, IgG responses to the V2 region of CRF01AE Env significantly correlated with decreased risk of infection among the different clades of V2 responses tested in the peptide microarray assay (Gottardo, Montefiori et al., unpublished). 2 This Continued on page JUNE 2012 VOLUME 4:1 HVTNEWS

3 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 immunogenicity. 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 complexity and potential functionality of vaccine elicited immune responses. Validated Assays for HIV-1 Vaccine Endpoints Neutralizing Binding Antibodies Currently validated assays include the measurement of neutralizing antibodies categorized as Tier 1 or Tier 2 neutralization using the TZM-bl assay. Validated assays to assess binding antibody responses (IgG and IgA) by ELISA and multiplex bead technology are in place and can measure magnitude and breadth of vaccine elicited responses. The goal of ongoing validation efforts include improving upon existing assays (eg, validation of A3R5 neutralization assays) and to select additional binding and functional assays most relevant for vaccine evaluation. Qualified and Standardized Assays IgG Subclasses, Isotypes, and Mucosal Antibodies Antibody isotypes and IgG subclasses (IgG1-IgG4) are currently being measured in response to vaccination. The rationale for measuring antibody isotypes and subclasses is that they provide insights into potential functional responses. IgG1 and IgG3 are considered to be the most functional of the subclasses in that they have been associated with HIV-1 neutralization, complement fixation, FcR binding and antibody dependent cellular cytotoxicity / cell mediated virus inhibition (ADCC/ADCVI). lga antibodies at mucosal sites have been correlated with protection in exposed, uninfected subjects and 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 V= Variable Region C=Constant Region H=Heavy Chain L=Light Chain Structure of the antibody: The variable regions (VH, VL) contain the antigen binding sites; the Fc region mediates the effector activity (eg, neutralization); the constant regions present in the heavy chain define the isotype of antibody (IgA, IgD, IgE, IgG, and IgM) and subclass (eg, IgG1-4). clinical trials include the measurement of both monomeric IgA (miga) and dimeric IgA (diga) that will be extended to the analysis of human mucosal samples. Moreover, the analysis of IgA responses and IgG subclasses, and the respective ratios between different types of antibodies, may provide insights into the role of different adjuvants/priming on the quality of the humoral responses. Antibody Epitope Mapping (Linear and Conformational) A potentially important characteristic of antibodies is their breadth of binding to multiple HIV-1 clades (Group M, Clade A, B, C, AE, etc.). Recent assay developments include the measurement of diverse HIV-1 Envs resulting in a single score for magnitude and breadth. The linear epitope specificities are measured using either binding antibody assays with specific epitopes (eg, V2, C1, alanine scanning mutants) or through an HIV-1 multiclade peptide microarray that can fully measure multiclade gp160 epitope specificity. Conformational epitopes (eg, CD4BS, CD4i, C1, and N-glycan dependent epitopes) can be determined through differential binding to wild type proteins/scaffolds with and without mutation(s) that alter conformational binding sites, or through competitive binding assays with monoclonal antibodies of known specificities (eg, A32 monoclonal antibodies [mab] blocking). Continued on page HVTNEWS VOLUME 4:1 JUNE

4 Antibody Affinity/Avidity Assays Measurements of the strength of specific antibody-antigen interactions may correspond with antibody affinity maturation and specific functional antibody responses that ultimately are linked to preventing HIV-1 acquisition or decreasing viral load. The HVTN labs are utilizing two types of assays for measuring antibody avidity: surface plasmon resonance (SPR) and antibody avidity index assay. Measurement of avidity by SPR allows characterization of antibody on-rate and off-rate to specific epitope and protein immunogens. Avidity index assays allow for bridging and comparison between NHP and human clinical trials. Moreover, there is precedence for utilizing avidity measurements for evaluating vaccines (eg, pneumococcal and hepatitis B vaccines). B Cell ELISpot Long-lived plasma B cells are responsible for continued production of antibodies; whereas memory B cells are responsible for producing an anamnestic response after antigen re-exposure. B cell ELISpots are in place for evaluation of human clinical trials and can be used together with antibody assays (using matched envelope reagents) to characterize the vaccine elicited humoral repertoire. Inhibitory Antibody (Non-neutralizing) Assays Standardized assays used in human clinical trials to assess antibody function include ADCC and ADCVI. Alternative neutralization assays with primary cells, such as peripheral blood mononuclear cells (PBMCs) and monocytes/macrophages may also provide information on vaccine elicited antibody function. Further examination of Fc receptor function of vaccine-elicited antibodies is needed, including the roles of different antibody isotypes and subclasses, antibody glycosylation, and antibody interaction with a range of effector cells. Additionally, defining the epitope specificity and antibody isotypes and subclasses that mediate Fc receptor mediated virus inhibition, neutralization, and virion capture will enable a better understanding of antibody function. Assays for Research and Development B Cell Technology Phenotypic analysis of vaccine elicited antigen specific B cells can provide insights for vaccine immunogenicity. Significant advances have been achieved by probing the B-cell response through the generation of mabs using recombinant DNA technology with memory B-cell cultures and antigen specific cell sorts. These mabs provide a stable source of vaccine elicited antibodies to test for antiviral function in complex assay systems (eg, aggregation, mucus inhibition, inhibition of transcytosis). Furthermore, the analysis of antibody gene sequences can determine whether the vaccine elicited antibody repertoire includes characteristics previously associated with broadly neutralizing antibodies (eg, specific VHVL chain gene usage, high mutation frequency, and CDR3 length within the variable domains). These strategies along with detailed epitope mapping (eg, CD4BS, sensitivity to N-linked glycosylation) can inform whether current vaccines are stimulating epitope specific antibodies that potentially may be driven toward induction of broadly neutralizing or virus inhibitory antibodies. With this knowledge in hand, vaccine strategies may build upon current vaccine approaches by further boosting with novel immunogen designs to achieve higher levels of affinity maturation or specific immune focusing. Functional Mucosal Antibody Assays Vaccine elicited antibodies that can block HIV-1 acquisition at mucosal surfaces might be among the most efficacious types of antibodies. In addition to traditional HIV-1 neutralization and Fc receptor mediated virus inhibition; antibodies may also aggregate virions, inhibit movement through cervical mucus, inhibit transcytosis, mediate intra-epithelial neutralization, block HIV-1 Env gp120 interaction with α4β7 integrin on CD4 + target cells, and inhibit macrophage infection. A variety of assays are in development with the aim to mimic key steps during mucosal transmission. Proper assessment in these assays currently may depend on mabs or purified antibodies from genital fluids and plasma. Further studies using these assays to assess vaccine elicited responses at mucosal sites are needed to determine their role in protection from HIV-1 acquisition. A question remaining from the RV144 correlates analysis, particularly in light of the finding related to monomeric plasma IgA, is whether vaccine elicited immune responses were present at mucosal surfaces, and whether these responses may have contributed to the observed efficacy. Future studies are planned for assessing vaccine elicited mucosal immunity with RV144 vaccine products, as well as other HIV-1 vaccine candidates. NOTE: A fully referenced version can be found at 4 JUNE 2012 VOLUME 4:1 HVTNEWS

5 Probing the diversity of Vaccine Elicited HIV-1 Antibodies: Informed by the RV144 Correlates Analysis assays protocol # DNA Prime / Viral Vector Boost Viral Vector Prime / Boost / IAVI B003 / IPCAVD 004 Various Primes / Protein Boost 073/073E / SAAVI / SAAVI protein only 088 Table1. Vetted Antibody Analyses in HVTN Open / Planned Protocols BAMA ELISA (binding Ab) nab ADCC Ab avidity Epitope analyses Ab repertoire analyses B-cell ELISpot B cell phenotyping Mucosal specimens Periphery (blood) Abbreviations and Acronyms: Ab = Antibody; ADCC = antibody dependent cellular cytotoxicity; BAMA = Binding Abs multiplex array; ELISA = Enzyme-linked immunosorbent assay; nab = Neutralizing antibody secondary analysis supports the finding of the primary V2 IgG variable and goes a step further to show increased significance when the clade is matched to the circulating strain in the vaccine population. The outcomes of these 2 CRF01AE measurements provide substantial rationale to include antibody responses to the circulating strains in the vaccine population as key variables to assess going forward in HIV-1 vaccine evaluation. Ultimately, these specific hypotheses should be examined in future HIV-1 vaccine trials in the broader context of examining multiple immune parameters. As noted by Plotkin,there is a natural redundancy of the immune system. 3 Thus, vaccination may protect through multiple mechanisms that are potentially reliant on host and/or pathogen specific factors. Historically, numerous efficacious vaccines correlate with specific antibody responses, but are not necessarily a mechanistic correlate -- that is, the antibody response is not the protective mechanism, but correlates well with vaccine efficacy (a nonmechanistic correlate). 4 This is an important distinction as it is not yet known if the correlates identified for RV144 are mechanistic or non-mechanistic correlates. Diversity of HIV-1 Vaccine Elicited Immune Responses A striking feature of HIV-1 vaccines, more generally, is that there is significant heterogeneity in vaccine responses among individuals. It is precisely this heterogeneity that allows for an immune correlates analysis to be sufficiently powered, since a dynamic range of immune responses is needed to clearly define a correlate. Measurement of the full repertoire of the Continued on page HVTNEWS VOLUME 4:1 JUNE

6 Jim Maynard Vic Sorrell sings like an angel and he s found a way to make it work for him in Nashville. Vic had a great idea to create excitement and draw attention to HVTN 505. He did it with song! Vic launched a city-wide Karaoke competition that used other people s love of singing to raise money for a local AIDS service agency, while educating his community about the trial and collecting contacts from dozens of interested volunteers. He took Orlando HVTN 505 Study Site: From Challenges to Accomplishments a fresh idea and turned up the heat on recruitment in Nashville and now it was time to spread that fire in Miami and beyond! Recruitment staff from across the United States joined HVTN s Edwin Core DeJesus Community and Engagement Keith Barsky (CE) Project Managers in Miami in January to share their very best ideas and brainstorm new ones, all for the sake of increasing enrollment in HVTN 505. This trial, the only HIV vaccine efficacy trial in the world, has been very difficult to enroll. Vic Sorrell s karaoke recruitment event inspired other recruiters at the Miami Recruiters Retreat. Recruitment staff at 21 sites in 18 U.S. cities has been working to find men and transgender women who have sex with men to take part in this trial, and it hasn t been easy. The original goal of 1350 volunteers was expanded to 2200 after a protocol amendment, making an already challenging job that much more difficult. With a new enrollment target, recruiters felt they were struggling for new ways to engage possible volunteers. Experienced recruiters from New York, Boston, Atlanta, Nashville, San Francisco, Rochester, and Seattle worked with the Core CE team to put together a program that reminded workshop participants how important this study is in the fight against HIV, reenergized them to do the work, and sent them home with fresh practical tools to get the job done. One such fresh tool was singing! In addition to recruitment tools workshops, the study s coprincipal investigators, Drs. Scott Hammer and Magda Sobieszczyk, shared their scientific expertise to help recruiters feel more knowledgeable in talking to potential volunteers. HVTN social scientist, Dr. Michele Andrasik, spoke about her research on barriers to recruitment those things that make people less likely to take part in a trial and led a workshop to help recruiters do their jobs in a more culturally competent way. It was an action packed weekend, but would it pay off in increased enrollments? We got back from the recruiters retreat and whoosh, new enrollments just took off! said Jim Carey, Community Engagement Coordinator at the University of Illinois Chicago (UIC) trials site. We needed some fresh ideas and there is no reason to reinvent the wheel. The other sites are a great resource. But it was more than that, the recruiters came back reenergized. This week we enrolled 3 new participants! The Chicago site s experience is not unique. Sites across the Network are launching new programs and events and increasing their rate of enrollment. HVTN 505 achieved a high water mark the week of April 16, when for the first time, 26 volunteers were enrolled in one week. The goal of 2200 participants is still a challenging one and we need to keep the volume turned up on recruitment, but we are well on our way to reaching it. How are things working out for Vic? He is still singing in Nashville and just recently completed the second annual Karaoke competition. And the fire did spread! Recruiters James Mapson and Jim Higginbotham at the University of Alabama- Birmingham site are in the final rounds of their first annual Karaoke competition, following Vic s model with great success. We hope that soon it will be the Network s turn to sing when we reach our goal of 2200 participants in this important trial. Jim Maynard is Associate Director, Community Engagement Unit, HIV Vaccine Trials Network 6 JUNE 2012 VOLUME 4:1 HVTNEWS

7 Orlando HVTN 505 Study Site: From Challenges to Accomplishments Edwin DeJesus and Keith Barsky In October 2010, the Orlando Immunology Center (OIC) became an expansion site for the HIV Vaccine Trials Network (HVTN). Although new to the HVTN and the NIH s Division of AIDS (DAIDS), we had over 20 years of experience in recruiting patients into privately-sponsored clinical trials for various pharmaceutical companies and NIHsponsored programs. However, we were faced with the reality that many of the established HVTN programs, services, and strategies had not yet been tested or implemented at an independent private site. Some of those elements required modifications to meet the needs of our clinic and our community. Our hope was that the skills that we had acquired over the years would have prepared us for being an HVTN study site. We quickly discovered that there are tremendous difficulties associated with recruiting HIV-negative volunteers into a government-conducted clinical trial, and asking them to volunteer to take an experimental HIV vaccine. At the starting point we had no recruitment team, no Community Advisory Board, and only limited associations with the local gay community organizations. The hill ahead of us was steep, but our drive, enthusiasm, and desire to be part of something big in our community gave us the impetus we needed to eventually conquer those hurdles. Today, we are happy to see that our original hopes have become a reality, and that it is possible to use similar skill sets learned from enrolling other (HIVinfected) clinical trials and apply them to a study within the HIV-negative population. It is also possible for an institution, having no affiliation with a university hospital or a governmental health program, to successfully participate in these types of clinical trials. Orlando site recruiting event. From right to left: Keith Barsky, Dino Martino, Tomas Devia, Anthony Egreja, Juan Rodriguez, Saul Leon To prepare for the challenges of recruiting for HVTN 505, we prioritized several issues: The need to know and understand our local gay community The need for a thorough under standing of the science and rationale of the study The need to form strong alliances with key community organizations The need to relate to potential volunteers and learn to speak their language In every major city, the men who have sex with men (MSM) community has found different patterns of communication, evolution/growth, and networking. Orlando is a medium-sized metropolitan area, but widely spread out, and with no centralized gay neighborhood. There are only a few gay bars, one bathhouse, and no after-hours clubs. All of these establishments are far from each other, and are not easily accessible via public transportation. Despite the lack of gay-nightlife entertainment, Orlando has been recognized by several organizations as one of the gayest cities in the U.S.. It has the largest gay employee association in the U.S. (Disney and Universal Studios Gay and Lesbian Association), the largest U.S. LGBT softball league, as well as one of the largest State Universities, UCF, which has a very strong and recently expanded LGBT service initiative (ucf.edu/ lgbtq). We knew that the at-risk population was present in Orlando, but that we needed to reach out to them by methods other than just visiting clubs or posting flyers in the bars. It was clear to us from the beginning that the most effective approach was going to require a repetition of the message by many different types of vehicles. We wanted to create a sense of pride in our community by the work being done locally as part of a bigger picture, a National Vaccine Study! We believe that a well-trained staff is the key to patient recruitment and retention. Members of the study team were all required to learn the science behind this vaccine study s development (which included a range of general information about viruses and vaccines to very specific information about HIV), as well Continued on page HVTNEWS VOLUME 4:1 JUNE

8 Orlando HVTN 505 Study Site: From Challenges to Accomplishments as the rationale for the study procedures, so they are able to effectively answer questions from potential volunteers. We also organized several meetings to educate our ancillary staff (including receptionists and any other office staff who may peripherally come in contact with a study volunteer) about the proper way to speak and interact with our volunteers, making them feel important from their very first interaction with OIC. One can never underestimate the importance of a first impression! Prior to HVTN, there was very little collaboration between the city organizations that serve people with HIV infection and the at-risk population in Orlando. We initiated programs that helped us to develop strong affiliations with key community gay organizations. We learned that in dealing with many of these groups, in order to expand the relationships, we needed to give something in order to gain something back. One of these organizations is The Center, which provides programs and services for the GLBT community, including free HIV testing. OIC has made an agreement with The Center to see, free of charge and on the same day of testing, any person who tests positive at their site. Our initial intent was to make sure that individuals newly diagnosed with HIV infections had the opportunity to discuss prognosis, HIV pathophysiology, and/or treatment options with a health care provider, immediately after diagnosis. Engagement in HIV care begins at testing, and recent studies highlight the importance of the HIV counseling, testing, and referral experience as it relates to subsequent linkage to medical care. 1-3 The value of the services we provide to The Center extended far beyond our initial expectations. The Center started to also incorporate (as part of their routine HIV screening program) the option of referring seronegative at-risk potential participants to the HVTN 505 study. For those individuals who test Reaching out to the community. Partnerships formed between the Orlando Immunology Center and community resources and services. *Infectious Disease antibody-positive for HIV, we are often able to intervene early and refer them for continuity of care. This also links us to the many seronegative partners (of those seropositive patients), for whom we are also able to provide further testing, counseling, or even possible study participation. We fostered similar relationships with other key gay organizations in Central Florida, helping us to disperse the HIV preventive message and to increase the interest of participating in the HVTN 505 study. These collaborations, which initially started as HVTN 505 promotions, have now transformed into even more important relationships aimed at implementing other programs in our community. The ties that we have formed with these organizations will last long after the HVTN 505 study is completed, and could potentially benefit future DAIDS programs. Social media, the main vehicle in which most young people now communicate with each other, has played a significant role in the dissemination of HVTN 505 news. We created the V-Force our Facebook identity for daily postings and notifications. To communicate more effectively with potential study participants we needed to learn their language, which for many is spoken through texting and ing, as opposed to traditional face-to-face interactions. Their schedules of communication can be also very unpredictable. Thus, we made sure to extend our phone/text/ availability beyond the regular business hours (including weekends), to ensure quick responses for interested candidates. The success with enrollment and retention at our site is the result of the collaborative effort that begins with our recruiter, is held together by our study coordinators, and is sealed by the direct interactions of the principal investigator and sub-investigators, who frequently get involved at different times during the process. This team effort is equally seen in the community. Potential participants are more likely to identify this 8 JUNE 2012 VOLUME 4:1 HVTNEWS

9 Orlando HVTN 505 Study Site: From Challenges to Accomplishments study as part of an exceptional organization when they are able to see many people, of varying backgrounds and levels, delivering the same message. The path that OIC and HVTN have paved for the gay community in Orlando will continue to grow and the strong relationships formed will ultimately lead to greater awareness about HIV and STDs, in general. Edwin DeJesus is Principal Investigator and Keith Barsky is Study Recruiter/Clinical Research Coordinator at the Orlando HVTN 505 study site. Reference List 1. Garland PM, Valverde EE, Fagan J, et al. HIV counseling, testing and referral experiences of persons diagnosed with HIV who have never entered HIV medical care. AIDS Educ Prev 2011;23(3 Suppl): Gardner EM, McLees MP, Steiner JF, Del RC, Burman WJ. The spectrum of engagement in HIV care and its relevance to test-and-treat strategies for prevention of HIV infection. Clin Infect Dis 2011;52(6): Marks G, Gardner LI, Craw J, Crepaz N. Entry and retention in medical care among HIV-diagnosed persons: a metaanalysis. AIDS 2010;24(17): "Probing the Diversity of Vaccine Elicited HIV-1 Antibodies", continued from page 5 epitope specificities (linear and conformational) of different antibody isotypes/subclasses both systemically and at mucosal sites are needed for vaccine trials to understand the breadth and diversity of responses. In RV144, numerous immune parameters were untested in the correlates analysis due both to inherent assay limitations, sample limitations (lack of mucosal samples) and the need to keep the correlates analysis focused on a limited number of endpoints to achieve statistical power. On the other hand, ultimately the heterogeneity of the HIV-1 vaccine response could become a roadblock to overcome for improving upon the 31.2% efficacy obtained in the RV144 study. Thus, understanding the genetic, environmental, and immunological determinants of heterogeneous HIV-1 vaccine elicited immune responses could potentially become critical for refining immunogen designs that can achieve more uniformity of specific immune responses among vaccinees. The HVTN has a wide breadth of humoral, cellular, and innate immunity assays. Furthermore, we are testing and analyzing a wide variety of regimens. Our ability to look across platforms (regimens, products, delivery mechanisms) allows us to spot patterns that single trials may not expose. The analysis of the RV144 vaccine and planned follow-up studies will continue to inform us, both technically and conceptually, how best to probe the full repertoire of vaccine elicited HIV-1 antibodies. Going forward, the 2 RV144 correlates of risk (V1V2 IgG and Env IgA), in terms of both response rate and magnitude, should be evaluated in HIV-1 vaccine strategies (different vectors, adjuvants, and boosts), in the context of profiling epitope specificities and functional attributes of vaccine elicited antibody responses. Such analyses will enable a better understanding of how to elicit durable virus-inhibitory antibodies that can recognize circulating virus strains and prevent HIV-1 acquisition. In response to the correlates analysis results, the HVTN incorporated additional antibody assays into currently open protocols, and into future protocols currently in development. We are analyzing not only blood samples but also mucosal specimens (see Table 1). The addition of mucosal antibody analyses may expand the knowledge gained from the RV144 correlates analysis, and highlight what differences exist between immune responses in the mucosa and those in circulation. Georgia Tomaras is Director, HVTN Binding Antibody Laboratory Program, and Associate Director of Research, Duke Human Vaccine Institute. Reference List 1. Rerks-Ngarm S, Pitisuttithum P, Nitayaphan S, et al. Vaccination with ALVAC and AIDSVAX to Prevent HIV-1 Infection in Thailand. N Engl J Med Haynes BF, Gilbert PB, McElrath MJ, et al. Immune-correlates analysis of an HIV-1 vaccine efficacy trial. N Engl J Med. 2012;366(14): Plotkin SA. Correlates of protection induced by vaccination. Clin Vaccine Immunol. 2010;17(7): Plotkin SA, Gilbert PB. Nomenclature for immune correlates of protection after vaccination. Clin Infect Dis. 2012;54(11): HVTNEWS VOLUME 4:1 JUNE

10 Assessing Mucosal Immune Responses in HIV Vaccine Trials Tracey Day and Florian Hladik HIV infection most frequently occurs via sexual transmission of the virus through the mucosal surfaces of the genitals or rectum. The gut mucosa also serves as a site where the virus inflicts significant damage on the immune system during the first few weeks of infection. A primary focus of HIV preventive vaccine research is therefore the induction of protective immune responses in these crucial early battlefields. Mucosal surfaces serve as a barrier between the body and the environment. From harmless food matter to commensal bacteria to pathogens, the mucosal surfaces of the respiratory, gastrointestinal, and genitourinary tracts are constantly bombarded with foreign particles. The mucosal immune system has the difficult task of differentiating between objects that should be ignored and those that represent a threat. To accomplish this balancing act, distinct anatomical and physiological features, which govern mucosal compartments, evolved as part of the immune system. Like systemic immunity, mucosal immunity employs all major lymphocyte subtypes -- B cells, CD4 +, and CD8 + T cells, γδ T cells and natural killer cells -- as well as their antigen-presenting cellular counterparts, dendritic cells and macrophages. sampling is more invasive than blood draws, and requires more time and training for clinical personnel who take such samples. Sampling procedures also yield a lower amount of fluid or cells compared to blood draws, and processing mucosal samples typically proves more challenging. Despite these challenges, the HIV Vaccine Trials Network (HVTN) has strived to enable mucosal immunity assessments in HIV vaccine trials. The large number of HVTN trials collecting mucosal specimens reflects this priority (see Table 1). The HVTN Laboratory Program has developed procedures to process and analyze mucosal specimens, and actively collaborates with leading mucosal immunologists to further improve these methods. These efforts are increasing the HVTN s capacity to evaluate vaccine-specific mucosal responses to candidate preventive HIV vaccines. Some specialized properties of the mucosal immune system have been identified -- the prevalence of different immunoglobulin subtypes, for example. However, due to technical difficulties in investigating human mucosal immune responses, the mechanisms of mucosal immunity in protection from pathogens are not as clearly understood as those of systemic immunity. While systemic immunity can be measured from peripheral blood, such responses do not necessarily reflect the responses in mucosal compartments. In a recent nonhuman primate vaccination study, Bomsel et al. 1 found that protection from an intravaginal virus challenge was associated with anti-viral antibody responses in the vaginal mucosa that were not detected in blood. These results provide evidence that antiviral mucosal immune responses can be important in preventing HIV acquisition, and that these responses may be missed in peripheral blood analyses. 2 Although HIV vaccine researchers have been interested in assaying vaccine- and HIV-specific mucosal responses for many years, a number of challenges have slowed progress in incorporating such measurements into clinical research. Mucosal Figure 1: Section of a colon biopsy that has been fluorescently stained to visualize the CCR5 + cells (shown in red) and the CD3 + T cells (shown in white). Colors overlapping on the same cell indicate T cells that have the CCR5 HIV-receptor. The nuclei of all cells are also stained (blue) so that we can see the tissue structure and how many total cells there are in the biopsy. 10 JUNE 2012 VOLUME 4:1 HVTNEWS

11 Assessing Mucosal Immune Responses in HIV Vaccine Trials Current assessments aim to evaluate a broad range of mucosal immune responses and answer some key questions for HIV vaccine development. Do vaccines delivered intramuscularly elicit detectable mucosal responses? Which mucosal immune responses may be associated with protection from HIV infection? Which mucosal specimens and assays are most relevant to the detection of responses? The answers will be vital for clarifying what mucosal immune responses are capable of blocking HIV infection, and for identifying vaccines that elicit such responses. Measuring Mucosal Immune Responses To measure mucosal immune responses, a variety of mucosal specimens are collected from some or all trial participants. Tissue biopsies provide samples of mucosal immune system cells and can yield detailed information on cellular responses. Currently, cellular responses are evaluated from foreskin, rectal, colon, and other gastrointestinal tract biopsies (see Table 1). A cervical cytobrush may be used to collect cells from the cervix, a vulnerable site for HIV infection in women. T-cell, B-cell, and innate immune cell phenotypes can be analyzed from cellular samples by evaluating cell surface markers via flow cytometry, and by measuring gene expression using transcriptional profiling. HIV-specific T- and B-cell functionality may be assessed from fresh cellular samples through intracellular cytokine staining and B-cell enzyme-linked immunosorbent spot (ELISpot) assays. The organization of immune cells within MUCOSAL SPECIMENS TRIAL hvtn 076 saliva semen rectal secretions cervical secretions cervical cytobrush rectal biopsy colon biopsy small intestine biopsy* foreskin hvtn 077 hvtn 078 hvtn 086/SAAVI 103 hvtn 088 hvtn 096 hvtn 097 hvtn 205 hvtn 505 hvtn 914 * Terminal ileum Table 1: HVTN Opened / Planned Protocols that Include Vetted Mucosal Analyses. HVTNEWS VOLUME 4:1 JUNE

12 Assessing Mucosal Immune Responses in HIV Vaccine Trials the tissues can also be examined from tissue biopsies using immunohistochemistry. Figure 1, for example, shows a colon biopsy sample in which T cells are identified by staining with CD3 antibodies (in white). By simultaneously staining with a CCR5 antibody (in red), the numbers of T cells expressing the CCR5 HIV co-receptor are visualized, as well as their locations in the tissue. Mucosal surface secretions can provide important information as well. HIV-specific mucosal antibodies and mucosal cytokine response profiles, for example, may be obtained from vaginal and rectal secretions, saliva, and semen. A variety of approaches exist to collect vaginal and rectal secretions including adsorbent wicks and sponges. In the case of vaginal secretions, cups that collect small amounts of fluid from volunteers can also be used during clinic visits. Peripheral blood samples that are collected during HVTN clinical trials are typically cryopreserved and shipped to the central laboratory for analysis. Performing immunogenicity analyses in a centralized lab ensures optimal data quality and allows data from multiple trials to be more readily compared. Adopting this approach for mucosal specimens, however, is challenging because the small number of immune cells present in mucosal samples, in conjunction with tissue-derived proteases, make these samples particularly vulnerable during cryopreservation and subsequent thawing. In addition, the large variety of specimen types requires the development of customized cryopreservation methods to optimize cell viability for each specimen type. Maximizing cell viability during the freezing and thawing process is critical for some immunogenicity assays, such as intracellular cytokine staining to detect HIV-specific T-cell responses. This key assay requires that a certain number of cells remain viable during assaying, which currently limits its application to fresh mucosal samples. To develop improved cryopreservation methods and assays for assessing mucosal immune responses in conjunction with HIV vaccine trials, the HVTN Laboratory Program initiated a collaboration with leading mucosal immunologists, the Mucosal Immunology Group (MIG). 3 With support from the National Institute of Allergy and Infectious Diseases (NIAID), MIG is tasked with developing, optimizing, and standardizing mucosal specimen preserving and processing techniques. MIG also aims to develop standardized assays to measure and characterize mucosal immune responses. A major benefit that standardized methods provide is that they permit comparison of results across many studies, providing an opportunity for novel insights. Cutting Edge Technologies Optimize Output from Mucosal Analyses Several U.S. and African MIG laboratories are using new technologies to maximize the information obtained from mucosal samples collected in HVTN trials. For example, newer multilaser flow cytometers and, recently, single-cell mass spectrometry cytometers that simultaneously detect more than 30 labeled antigens enable researchers to gain much more information from the same number of cells. 4 Other new methods include bead-based immunosorbent approaches to measure small quantities of proteins in mucosal samples, and the use of computerized image analysis of antibody-stained biopsy sections (immunohistochemistry). Computers can measure 10- to 100-fold more cells, offering far more sensitivity than would manual analysis. Figure 1 shows a sample from one such analysis, in which CCR5 expression on T cells was digitally quantitated within colon biopsy tissue sections using this method. Single-cell, subnanoliter well arrays, under development by Chris Love at the Massachusetts Institute of Technology and his collaborators, incorporates 80,000 to 250,000 wells onto microscopy slides, with each well holding up to 125 pl of reagents along with one cell. 5 Those cells are labeled with specific antibodies for phenotyping via multicolor cytometry. Because the cells remain alive, they can be tested further for other functions, such as whether they secrete particular cytokines. This combined phenotypic and immunofunctional analysis offers great advantages over both flow cytometry and in situ analysis. Flow cytometers query each cell only once before it is discarded, and imaging of processed and sectioned tissue is limited to nonfunctional assays. The single-cell platform permits quantitative analysis of antigen-specific T-cell responses among precisely phenotyped T-cell subtypes, making it ideal for assessing mucosal cell immune responses to vaccines. In acknowledgement that mucosal analyses are indispensable for defining the immunological impact of HIV vaccines and microbicides, innovative leaps are being made to optimize their use in clinical trials. Recent findings of highly cross-reactive antibody responses in HIV-infected individuals, and the uncovering of systemic correlates of risk among those who received the RV144 vaccine, put us closer to knowing what a vaccine should do to protect against HIV. From here, we still require an iterative process to identify a vaccine that will induce the necessary immune responses. It is truly an exciting time for those working to prevent HIV infections. Continued on page JUNE 2012 VOLUME 4:1 HVTNEWS

13 Highlights from Recent HVTN Publications Tracey Day From primary protocol results to auxiliary research findings to commentaries and review articles, HVTN researchers and collaborators have published a large number of articles over the past year. Here we present a tabular summary of our publications from February 2011 February 2012 and highlight several in more detail below. Heterologous vs. Homologous Boosting (HVTN 068) Vaccine efficacy can be increased by administering multiple vaccinations. This strategy, known as a prime/boost regimen, is used for many licensed vaccines and by several candidate HIV-1 vaccines. De Rosa et al. hypothesized that heterologous vaccination could improve the quality of T-cell responses for recombinant adenoviral serotype 5 (rad5) vaccines. To test this hypothesis, immune responses in Ad5-seronegative adults were compared for a homologous prime boost regimen (rad5 prime/ rad5 boost) and a heterologous prime boost regimen (DNA prime/rad5 boost). The heterologous DNA prime/rad5 boost regimen induced higher levels of CD4 + T cells and altered the cytokine and memory marker profiles of the T-cell responses. In contrast, homologous boosting did not increase T-cell responses, but enhanced Env-specific antibody responses. This suggests that to optimize both T-cell and antibody responses, vaccine regimens may need to include both heterologous and homologous vaccine modalities. This study also evaluated the detailed kinetics of prime/boost responses. By assessing responses at many time points, researchers observed that although limited responses were detected immediately following DNA priming, the presence of a DNA prime influenced postboost responses. The authors therefore propose that evaluations of DNA vaccine immunogenicity should include assessment after boosting. 1 Neutralizing Exposure (HVTN 049) Vaccines that elicit broadly cross-neutralizing antibodies (bnabs) against HIV-1 may be effective in preventing infection. However, immunogens capable of inducing bnabs have not yet been identified. Alternative froms of HIV envelope glycoproteins (Env) that expose conserved regions are being investigated as a means to generate bnabs. Spearman et al. examined whether a modified trimeric Env vaccine could induce bnabs when used together with a DNA prime. To enhance the exposure of the coreceptor binding site, a deletion was introduced in the V2 loop of Env. Although some bnabs target regions of the V2 loop, its deletion was found to render the virus more susceptible to neutralization in previous studies and thus warranted testing in human trials. In this trial, the trimeric recombinant protein was administered in MF59 adjuvant, following priming with gag and env DNA. The DNA vaccine component consisted of plasmid DNA adsorbed onto polylactide coglycolide microspheres, which have been shown to improve immunogenicity of DNA vaccines in animal models. Remarkably, 100% of vaccinees developed nab responses against the homologous virus isolate, SF162. Neutralization against heterlogous tier 2 virus isolates, which are typically more difficult to neutralize, however, was weak. Env-specific CD4 + T-cell responses were also detected in the majority of vacinees after boosting. The magnitude and potency of neutralizing antibody responses in this trial were encouraging, but the challenge of eliciting bnabs remains. 2 Multiclade Prime/Boost Regimen (HVTN 204) The large genetic diversity of circulating HIV-1 strains makes development of a universal HIV vaccine challenging. To induce responses against a variety of virus subtypes, the Vaccine Research Center (VRC) of the National Institute for Allergy and Infectious Diseases developed a DNA prime rad5 boost vaccine regimen containing inserts from HIV-1 clades A, B, and C. Churchyard et al. report on the results of evaluating this regimen in a phase 2a clinical trial conducted in healthy adults from South Africa, the United States, Latin America, and the Caribbean. Similar to other studies involving this regimen, the vaccines were well tolerated and appeared safe. The results from intracellular cytokine staining revealed that this regimen induced concurrent, polyfunctional CD4 + and CD8 + T-cell responses, as well as central and effector memory CD8 + T-cell responses with antiviral activity. High frequencies of durable Env-specific binding antibody responses were also generated; however, little virus neutralization activity was detected from these antibody responses. The promising results of this and other clinical and preclinical studies contributed to the advancement of this candidate to clinical efficacy testing in the phase 2b trial, HVTN Tables on following page, text continued on page HVTNEWS VOLUME 4:1 JUNE

14 Tabular Summary of Recent HVTN Publications Primary Trial Publications: Protocol First Author Aim* HVTN 060, HVTN 063 Kalams, S. This first in humans study evaluated a DNA plasmid vaccine expressing HIV-1 Gag-p37 with or without a plasmid cytokine adjuvant (IL-12- or IL-15). The adjuvant addition was an attempt to augment the immunogenicity of the DNA vaccine. HVTN 044 Baden, L. HVTN 068 DeRosa, S. HVTN 069 Koblin, B. HVTN 204 Churchyard, G. This study investigated IL-2 as an adjuvant by comparing HIV-1 DNA vaccine (VRC-HIVDNA VP) alone and with a plasmid coding for a fusion protein of IL-2 and the Fc protein of immunoglobulin G (IL-2/Ig; VRC-ADJDNA004-IL2-VP). The study compared immune responses and kinetics of the responses of "homologous priming and boosting with a rad5 vaccine (VRC-HIVADV VP) to heterologous DNA prime (VRC-HIVDNA VP) and rad5 boost vaccination." A comparison of a DNA vaccine prime (VRC-HIVDNA VP) given intramuscularly and a rad5 vaccine boost (VRC- HIVADV VP) given one of 3 routes: intramuscularly, intradermally, or subcutaneously. A phase II trial that evaluated the safety and immunogenicity of a multiclade DNA prime (VRC-HIVDNA VP) and a multiclade rad5 boost (VRC-HIVADV VP) vaccine in healthy adults from various geographical locations. HVTN 503/ Phambili Gray, G. Assessed safety and efficacy of the MRKAd5 HIV-1 gag/pol/nef subtype B vaccine in South Africa, a subtype C region. HVTN 049 Spearman, P. HVTN 065 Goepfert, P. HVTN 055 Keefer, M. The first human trial of a vaccine regimen consisting of a DNA prime (pcmvgagb, psinenvb; PLG) and a trimeric, V2- deleted Env protein boost (gp140sf162dv2; MF59) aimed to elicit T-cell responses and bnabs. A phase I trial that Compared the safety and immunogenicity of a recombinant MVA vaccine (GeoVax MVA/HIV62) administered alone or with a DNA vaccine prime (GeoVax pga2/js7 DNA). This first in humans study examined the safety and immunogenicity of homologous and heterologous prime-boost regimens with different replication-defective poxvirus vectors (MVA-HIV and FPV-HIV) containing identical HIV inserts. Review, Commentary, and Methods Publications: Protocol First Author Aim* Review Kublin, J. HIV Vaccine Trials Network: activities and achievements of the first decade and beyond. Commentary Gilbert, P. Commentary on "Principal stratification - a goal or a tool?" by Judea Pearl. Review Rolland, M. Evaluating immune correlates in HIV type 1 vaccine efficacy trials: what RV144 may provide. Review Sobieszczyk, M. Host genetic polymorphisms associated with innate immune factors and HIV-1. Methods Pollara, J. High-throughput quantitative analysis of HIV-1 and SIV-specific ADCC-mediating antibody responses. Methods Piehler, B LabKey Server nab: a tool for analyzing, visualizing and sharing results from neutralizing antibody assays. Methods Huang, Y Comparing biomarkers as principal surrogate endpoints. Review Corey, L. HIV-1 vaccines and adaptive trial designs. Other Gilbert, P. Statistical interpretation of the RV144 HIV vaccine efficacy trial in Thailand: a case study for statistical issues in efficacy trials. Other Landers, S Community perspectives on developing a sexual health agenda for gay and bisexual men. 14 JUNE 2012 VOLUME 4:1 HVTNEWS

15 Article highlighted in HVTNews article on page 13. * Text adapted or quoted from original articles. Result* The vaccine and adjuvants were well tolerated. The vaccine's immunogenicity was not improved by the addition of the adjuvants. Citation PLoS One. 2012;7(1):e29231 Plasmid IL-2/Ig increased immune responses when administered 2 days after the DNA vaccine, compared with simultaneous administration." This study was the first to demonstrate that cytokine adjuvants can improve the immunogenicity of a DNA vaccine in humans. J Infect Dis. 2011;204(10): "Homologous boosting greatly enhanced Env-specific Ab responses, but did not increase T-cell responses. Despite limited postprime immune responses, DNA priming significantly enhanced the magnitude of postboost Env-specific Ab and CD4 + T-cell responses and influenced the cytokine and memory marker profiles of the boosted T-cell responses." The vaccine's immunogenicty was not affected by giving the rad5 boost via different administration routes. In addition, there was a higher frequency of local reactions after intradermal or subcutaneous administration. The regimen was well-tolerated and induced IFN-γ + T cells in 70.8% of participants. T-cell responses were predominantly balanced CD4 + / CD8 + polyfunctional responses. High frequencies (83.7% 94.6%) of multi-clade anti-env binding antibodies were also observed. This trial was stopped early because the vaccine was not efficacious in another study (Step study). The available data suggested no protection from acqusition or reduction of viral load setpoint for vaccine recipients. The vaccine induced IFN-γ-secreting T-cell responses to clade B (89%) and C peptides (77%). A trend for a lower viral-load setpoint and a slower decline in CD4 count was observed in vaccinated women. This regimen generated Env-specific CD4 + T cell but not CD8 + T-cell responses and high titers of NAbs against the homologous SF162 virus. The Ab responses however, had limited breadth and did not neutralize heterologous, tier 2 clade B virus strains. The MVA62 vaccine was well tolerated and appeared safe. Different patterns of immune responses were induced when MVA62 was administered alone (higher Ab responses) or together with the JS7 DNA vaccine (better T-cell responses). Both the homologous and heterologous prime-boost regimens were well tolerated. Balanced HIV-specific CD4 + and CD8 + T-cell responses were more reliably elicited by the heterologous prime boost strategies. J Immunol. 2011;187(6): PLoS One. 2011;6(9):e24517 PLoS One. 2011;6(8):e21225 Lancet Infect Dis. 2011;11(7): J Infect Dis. 2011;203(8): J Infect Dis. 2011;203(5): Vaccine. 2011;29(10): summary* Describes strategic accomplishments made by the HIV Vaccine Trials Network. These acomplishments improve the process of designing and implementing HIV vaccine clinical trials, as well as analyzing their results. Articulates the scientific value of principal stratification estimands for randomized, placebo-controlled, preventive HIV vaccine efficacy trials. Describes how immune correlates may be determined from vaccine efficacy trials and from the RV144 trial in particular. Discusses how clinical trial designs may be augmented to improve the assessment of correlates. Genetic studies have identified polymorphisms in genes associated with the innate immune response to HIV infection, including TLR4, TLR9, IRF-3, TRIM5α and the ABOBEC3 gene family, that contribute to protection from disease progression. The assay, based on the hydrolysis of a fluorogenic peptide substrate by granzyme B, provides "rapid quantification of HIV-1 or SIVspecific ADCC-mediating antibodies that develop in response to vaccination, or in the natural course of infection." The use of standardized software, such as LabKey Server's neutralizing antibody (nab) tool, for analyzing, sharing, and archiving assay results improves "the reproducibility, reliability, and comparability of data obtained across many labs." Proposes to characterize a marker s "principal surrogate value based on the distribution of risk difference between interventions" and develops "a semiparametric estimated-likelihood method to estimate the joint surrogate value of multiple biomarkers." Discusses the rationale behind adaptive trial designs, in which multiple candidates are initially tested and only those showing efficacy signals are carried through the extended study. This allows a more rational faster paced strategy for vaccine development and improves the ability to identify immune correlates. Discusses the interpretation of statistical results from RV144 generated using frequentist statistics and Bayesian framework analyses. Discusses the history and next steps for advocacy for the health of gay and bisexual men of color. Citation Clinical Investigation. 2012; 2(3): Int J Biostat. 2011;7(1):Article 36 AIDS Res Hum Retroviruses. 2012;28(4): Curr Opin HIV AIDS. 2011;6(5): Cytometry A. 2011;79(8): BMC Immunol May 27;12:33 Biometrics. 2011;67(4): Sci Transl Med. 2011;3(79):79ps13 J Infect Dis. 2011;203(7): AIDS Behav. 2011;15 Suppl 1:S HVTNEWS VOLUME 4:1 JUNE

16 Tabular Summary of Recent HVTN Publications (cont.) Auxiliary Study Publications: Protocol First Author Aim* HVTN 071, HVTN 502 Frahm, N. Analyzed adenovirus-specific T cells in recipients of the MRKAd5 HIV-1 gag/pol/nef vaccine and evaluated their impact on vaccine-induced HIV insert-specific T-cell responses. HVTN 502/ Merck 023/ Step Study Barouch, D. The study determined nab titers for alternative adenovirus serotypes (Ad5, Ad26, Ad35, and Ad48) in individuals from the Americas, sub-saharan Africa, and Southeast Asia. HVTN 050, HVTN 054 Friedrich, D. Investigated the epitopes and HLA molecules utilized to mount HIV-specific CD8 + T-cell responses in healthy HIV-seronegative individuals who participated in clinical trials of MRKAd5 HIV-1 gag and VRC-HIVADV VP rad5 vaccines. HVTN 502/ Merck 023/ Step Study HVTN 502/ Merck 023/ Step Study HVTN 502/ Merck 023/ Step Study Voytek, C. Li, F. Zhang, X. Examined the motivators and deterrents to HIV vaccine trial participation among women at 'high risk' for HIV acquisition. Evaluated "whether the T-cell epitope response specificities elicited by vaccination were within conserved or variable regions of the HIV proteins and whether the vaccine elicited the type of T-cell coverage likely to be effective after encountering the diversity of HIV-1 circulating in the regions where the vaccine trial was conducted". Evaluated a novel method for predicting HLA alleles using unphased flanking single-nucleotide polymorphism genotypes in ethnically diverse populations. HVTN 050 Pine, S. Devised a novel assay to measure 30 secreted factors from ex vivo-stimulated PBMC to examine "the relationship between pre-existing Ad5 immunity and T-cell cytokine response profiles in healthy, HIVuninfected recipients of MRKAd5 HIV-1 gag vaccine." HVTN 502/ Merck 023/ Step Study HVTN 502/ Merck 023/ Step Study HVTN 502/ Merck 023/ Step Study HVTN 502/ Merck 023/ Step Study HVTN 071, HVTN 502/ Merck 023/ Step Study, HVTN Barnabas, R. Fellay, J. Fitzgerald, D. Rolland, M. Migueles, S. Investigated the epidemiology of herpes simplex virus type 2 (HSV-2) infection "and its impact on HIV incidence, viral load, and time to antiretroviral therapy initiation among Step study participants." Investigated the mechanisms associated with variability in HIV-specific T-cell responses to the MRKAd5 HIV- 1 gag/pol/nef vaccine using whole-genome genotyping and HLA class I typing. Evaluated the impact of the MRKAd5 HIV-1 gag/pol/nef vaccine on disease progression in participants who became HIV-1 infected during a 2 year follow-up period. Performed a sieve analysis of breakthrough virus sequences from infected Step study participants to determine whether the MRKAd5 HIV-1 gag/pol/nef vaccine put immune pressure on the infecting viruses. Compared HIV-specific CD8 + T-cell cytotoxic capacity (granzyme B target cell activity and HIV-infected CD4 + T-cell elimination) for uninfected MRKAd5 HIV-1 gag/pol/nef vaccine recipients, HIV-negative individuals, and chronically infected patients. 16 JUNE 2012 VOLUME 4:1 HVTNEWS

17 Article highlighted in HVTNews article on page 13. * Text adapted or quoted from original articles. Result* Higher frequencies of adenovirus-specific CD4 + T-cell responses prior to vaccination were associated with decreased levels of HIV-specific CD4 + T-cell responses, and decreased breadth of CD8 + T-cell responses. These effects were observed to be independent of pre-existing adenovirus-specific nab titers. "Ad26, Ad35, and Ad48 nab titers were substantially lower than Ad5 nab titers in multiple large international human populations." "Certain HLA alleles (B27, B57, B35, and B14) are preferentially utilized to mount HIV-specific responses, whereas other alleles (A02 and B07) are rarely utilized (P < 0.001)." The majority of women interviewed were positive about participating in HIV vaccine trials but were ultimately deterred by concerns about research procedures, potential consequences of participation, and/or their ability to attend study appointments. Citation J Clin Invest. 2012;122(1): Vaccine. 2011;29(32): J Acquir Immune Defic Syndr. 2011;58(3): Vaccine. 2011;29(36): The study found that "the Merck gag/pol/nef vaccine tended to elicit responses to fewer highly conserved epitopes and more lessconserved epitopes than would be predicted from the vaccine sequences alone." PLoS One. 2011;6(6):e20479 The data support the application of this approach for predicting HLA alleles with genome-wide association study-derived single-nucleotide polymorphism data. The predictive models and the program used are available online. "The cytokine response profile of Gag-specific T cells mirrored the Ad5-specific response present in all subjects before vaccination, and included a number of Th1- and Th2-associated cytokines not routinely assessed in current vaccine trials, such as IP-10, IL-10, IL-13, and GM-CSF." Pre-existing HSV-2 infection was a significant risk factor for HIV acquisition in MSM participating in the Step study. However, past HSV-2 infection was not associated with increased HIV viral load or early disease progression. No genetic variant was found to reach significance; however polymorphisms in MHC alleles HLA-B*2705, B*5101, and B*5701 were associated with higher Gag responses, whereas HLA-B*0801 and B*4501 were associated with lower responses. No significant differences between vaccine and placebo recipients were found with regard to HIV RNA levels, CD4 + T-cell counts, time to initiation of anti-retroviral therapy, and AIDS-free survival. The data hinted that vaccinees with protective HLA types (B*27, B*57, B*58) had lower HIV RNA levels than placebo recipients with those HLA types. The study found that HIV sequences from infected vaccinees had greater distances to the vaccine sequence than those for infected placebo recipients. The first evidence of selective pressure from vaccine-induced T-cell responses on HIV-1 infection in humans. Vaccine recipients with "HLA class I alleles B*27, B*57, or B*58, which have been associated with immune control over HIV replication in chronic infection," had higher T-cell cytotoxic capacity, suggesting these alleles allow better cytotoxic priming in infection and vaccination. BMC Genet Apr 25;12:39 PLoS One. 2011;6(4):e18526 J Acquir Immune Defic Syndr. 2011;57(3): J Infect Dis. 2011;203(6): J Infect Dis. 2011;203(6): Nat Med. 2011;17(3): PLoS Pathog. 2011;7(2):e Abbreviations: Ab = antibody, Ad5 = adenovirus type 5, ADCC = antibody dependent cell-mediated cytotoxicity, bnab = broadly neutralizing antibody, CTL = cytotoxic T lymphocyte, FPV = fowlpox virus, MSM = men who have sex with men, MVA = modified vaccinia Ankara, nab = neutralizing antibody, PBMC = peripheral blood mononuclear cell, PLG = polylactide coglycolide microspheres, rad5 = recombinant adenovirus type 5, VE = vaccine efficacy, Continued on page HVTNEWS VOLUME 4:1 JUNE

18 Social and Behavioral Science in Clinical Trials of Biomedical HIV Prevention Interventions Beryl Koblin, Michele Peake Andrasik, and Judy Austin The HIV prevention toolbox has been a mixed bag in recent years, with the success of several randomized clinical trials of biomedical interventions in reducing HIV infections, and the failure of others to show efficacy. A trial of a recombinant canarypox vector vaccine prime with a recombinant glycoprotein 120 subunit vaccine boost found a modest efficacy (31%) among a general population sample in Thailand (RV144), 1 but the Step study found that a replication-defective adenovirus type 5 (Ad5) vector vaccine with subtype B HIV-1 gag/pol/nef inserts did not protect against HIV infection, and appeared to increase the risk of HIV acquisition among MSM who were uncircumcised or had neutralizing antibodies to Ad5 at enrollment. 2 The Partners PrEP study, which looked at daily oral tenofovir or tenofovir with emtricitabine, provided a 67% and 75% reduction in HIV infection rates, respectively, among serodiscordant heterosexual couples. 3 Conversely, a study of daily oral use of tenofovir and emtricitabine among heterosexual women (FEM-PREP) did not find any reduction in HIV infection (HR= 0.94; 95% CI, ). The range of efficacies observed with different biomedical prevention interventions could be explained by a number of factors. The biomedical intervention itself may not be of sufficient strength or appropriately targeted to have an impact on HIV. Host factors, such as genetics or gender, may influence efficacy. 4 Insufficient dosing levels, as a result of low adherence, has been the proposed mechanism for the range of efficacies reported in pre-exposure prophylaxis (PrEP) studies. 5 Finally, if HIV exposure is not equivalent in the different treatment arms in a clinical trial, the ability to detect an efficacious intervention may be undermined. Behavioral science provides the backbone for investigating several of these factors at the individual level. Social science provides the structure to investigate context at the dyadic, situational and community levels in which individuals operate. 6 Both individual and contextual factors may influence participation and retention rates, adherence, and risk of HIV exposure, as well as dissemination and uptake of highly efficacious interventions. Documentation of HIV exposure, through self-report of risk behaviors, is used to determine if exposure to the virus is equivalent in the different arms of the study at baseline and during followup, and if treatment assignment is associated with any changes in exposure. As mentioned above, differential exposure may obscure efficacy. Collection of risk behavior data can be used to determine if the level of HIV exposure could act as an effect modifier to biomedical intervention efficacy. Randomization and blinding are in place in clinical trials to Both individual [behavioral] and contextual [social] factors may influence participation and retention rates, adherence, and risk of HIV exposure, as well as dissemination and uptake of highly efficacious interventions. eliminate imbalances in exposure by treatment arms. 7 However, randomization does not preclude a systematic or differential shift in risk occurring during follow-up. Unblinding of treatment assignment during the study, or perceptions of treatment assignment while blinded, may contribute to changes in risk behaviors, leading to changes in exposure to HIV. 8 Recent studies within the HVTN illustrate the critical role of social and behavioral science work in the conduct and interpretation of recent HIV vaccine trials. The HVTN has utilized behavioral investigations in the Step study, to determine if the increased risk of HIV acquisition among subgroups of vaccinees, compared to placebo recipients, could be explained by differences in HIV exposure. The analysis found few differences in sexual risk behaviors by treatment arm, and vaccine recipients remained at higher risk of HIV in- 18 JUNE 2012 VOLUME 4:1 HVTNEWS

19 Social and Behavioral Science in Clinical Trials of Biomedical HIV Prevention Interventions fection compared to placebo recipients, even when controlling for sexual risk behaviors and other potential confounders (HR =2.8; 95% CI, ). Thus, increased risk of HIV infection among subgroups of MSM who received the vaccine was not explained by an increase in HIV exposure in these groups of MSM, ruling out behavioral differences and lending support to a biological mechanism. 9 Participant recruitment, as any recruiter can attest, is an art and, importantly, a science. Recent social science work in the HVTN provides important insights into recruitment of MSM and transgender women into HVTN 505. Data from surveys (n=1835) and 6 focus groups (n=62) with MSM in 6 cities (Boston, Chicago, Denver, Houston, Los Angeles and New York) indicated that more than 70% of eligible MSM would consider participating in an HIV vaccine study. Lack of knowledge and information about HIV vaccine trials emerged as the primary barrier to participation. Almost 40% of survey respondents indicated having never heard about a vaccine trial, and another 20% indicated hearing about it as a result of taking the survey. Among participants who were unsure of their interest in participating (n=254), 63% indicated that interest in participating would increase if they had more information. Additional barriers to participation included: concerns about side effects including vaccine induced seropositivity (VISP) (77%); concerns about privacy (54%); concerns about others perceiving them as risky (55%); and acquiring HIV from the vaccine (43%, with 31% endorsing not sure). Participation facilitators included: safety (92%); helping to end the epidemic (90%); and potential protection from HIV (60%). The increased dissemination of communitylevel information about the progress that has been made in HIV vaccine research is sorely needed, as 64% of respondents endorsed believing that there was very little chance of finding a vaccine that works. Two focus groups with African American gay male leaders (N= 15), 4 focus groups with male-tofemale transgender women (N=42) and 6 focus groups with HVTN Clinical Research Site (CRS) staff (N= 74) were also conducted. Data from these groups highlighted the need for improved cultural responsiveness throughout the Network and the identification of innovative approaches to strengthen community and research institution partnerships. Based on these results, the HVTN is planning to enhance VISP and other adverse event educational materials available to the community, focus on including participant testimonials showcasing participants experience in the study, and collect survey data on experiences of current trial participants. In addition, the HVTN has developed 2 cultural responsiveness curricula for use at clinical research sites (CRS) and with Core staff. A 5 hour transgender specific cultural responsiveness workshop was provided at the Spring 2012 HVTN Full Group Meeting. A more general full day cultural responsiveness workshop was pilot tested and evaluated with CRS staff at 2 sites. Evaluation results were utilized to improve the curriculum and the workshop was then provided to all HVTN Core staff. The evaluations and feedback from these workshops will lead to additional improvements in the overall curriculum and plans to make the training available to all sites are underway. The HVTN has made a significant commitment to inclusion of social and behavioral science into its work. In support of The HVTN has made a significant commitment to inclusion of social and behavioral science into its work. the mission and goals of the HVTN, the newly formed Social and Behavioral Working Group (SBWG) examines social and behavioral science questions that have an impact on the design, implementation and interpretation of vaccine trials. These questions include behavioral risk assessment, adherence measures for protocols that would involve the use of PrEP (such as HVTN 095-MTN 022), and assessment of understanding of the informed consent. The SBWG is identifying social and behavioral science research priorities within the Network, to facilitate the integration of behavioral and social science work into existing and future HVTN efforts. Another goal is to assist investigators in incorporating social and behavioral science into their research agenda. These efforts will be defined as specific objectives related to the overall protocols, employing well-established and appropriate behavioral and social science methodologies for the questions at hand. The current research priorities of the SBWG include: measuring and describing PrEP use among HVTN 505 participants, HVTNEWS VOLUME 4:1 JUNE

20 Social and Behavioral Science in Clinical Trials of Biomedical HIV Prevention Interventions examining biomarkers of sexual exposure to HIV through collaboration between the SBWG and laboratory scientists, developing methods and measures for optimal community engagement, evaluating recruitment and retention strategies, developing new approaches to informed consent assessment of understanding, improving assessment of understanding, evaluating risk factors for HIV infection in different populations (including substance users), providing data for efficacy trial design, and collaborating closely with the NIH's Division of AIDS' and the Office of HIV/AIDS Network Coordination's efforts to develop core measures of risk behaviors. An overarching principal is to incorporate social and behavioral science early in study design and idea generation, to ensure appropriate integration of the necessary expertise. As PrEP use becomes more common, the issues of acceptability, adherence, and measurement of HIV exposure through different routes (eg, vaginal vs. anal sex) will become even more complicated. The upcoming HVTN 095-MTN 022 trial, testing HIV vaccines in combination with oral and topical antiretrovirals, will begin to explore some of these issues. Thus, biomedical and social-behavioral sciences, often perceived as opposites, are, in essence, complementary and interactive within a greater whole. Beryl Koblin is Principal Investigator, Laboratory of Infectious Disease Prevention, New York Blood Center; Michele Peake Andrasik is the HVTN Social Scientist; Judy Austin is a doctoral student in Epidemiology at the Mailman School of Public Health, Columbia University. Reference List 1. Rerks-Ngarm S, Pitisuttithum P, Nitayaphan S, et al. Vaccination with ALVAC and AIDSVAX to Prevent HIV-1 Infection in Thailand. N Engl J Med Buchbinder SP, Mehrotra DV, Duerr A, et al. Efficacy assessment of a cell-mediated immunity HIV-1 vaccine (the Step Study): a double-blind, randomised, placebo-controlled, test-of-concept trial. Lancet. 2008;372(9653): Baeten JM, Donnell D, Ndase P, et al. ARV PrEP for HIV-1 prevention among heterosexual men and women. 19th Conference on Retroviruses and Opportunistic Infections, Seattle, WA, March 5-8 Section 8, Paper # Stanberry LR, Spruance SL, Cunningham AL, et al. Glycoprotein-D-adjuvant vaccine to prevent genital herpes. N Engl J Med. 2002;347(21): Grant RM, Lama JR, Anderson PL, et al. Preexposure chemoprophylaxis for HIV prevention in men who have sex with men. N Engl J Med. 2010;363(27): Frye V, Latka MH, Koblin B, et al. The urban environment and sexual risk behavior among men who have sex with men. J Urban Health. 2006;83(2): Schulz KF, Grimes DA. Generation of allocation sequences in randomised trials: chance, not choice. Lancet. 2002;359(9305): Bartholow BN, Buchbinder S, Celum C, et al. HIV sexual risk behavior over 36 months of follow-up in the world's first HIV vaccine efficacy trial. J Acquir Immune Defic Syndr. 2005;39(1): Koblin BA, Mayer KH, Noonan E, et al. Sexual risk behaviors, circumcision status and pre-existing immunity to adenovirus type 5 among men who have sex with men participating in a randomized HIV-1 vaccine efficacy trial: Step Study. J Acquir Immune Defic Syndr JUNE 2012 VOLUME 4:1 HVTNEWS

21 Highlights from Recent HVTN Publications "Highlights from Recent HVTN Publications", continued from page 13 Hint on Helpful HLAs (Step Study) T-cell responses play an important role in controlling virus replication during natural HIV-1 infection. Vaccines that elicit strong T-cell responses, therefore, may lower viral loads and slow disease progression. The Merck adenovirus type 5 trivalent HIV-1 vaccine (MRKAd5/HIV-1 gag/pol/nef), which elicits T-cell responses, was evaluated in an efficacy trial, known as the Step study. In 2007, the study was stopped when the first interim analysis indicated that the vaccine would not achieve efficacy for preventing HIV-1 acquisition or reducing plasma HIV-1 RNA levels. Fitzgerald et al. evaluated the effect of the vaccine on disease progression in participants who became HIV-1 infected during a 2 year follow-up period. Among 87 male study participants who became HIV-1 infected no significant differences between vaccine and placebo recipients were found with regard to several disease progression measures (HIV-1 RNA levels, CD4 + T-cell counts, time to initiation of anti-retroviral therapy, and AIDS-free survival). Given the magnitude of CD8 + T-cell responses elicited by the vaccine these results point to a need for additional research to elucidate why the vaccine failed to control viral replication. Interestingly, a subanalysis of the data hint that the vaccine may have reduced HIV-1 RNA levels in participants with certain HLA types (HLA class I alleles B*27, B*57, and B*58) known to be associated with lower viral load setpoints and delayed disease progression. These observations suggest that reduction of viral loads may be possible if the right CD8 + T-cell responses are induced. Further research is needed to identify what these responses are and how to induce them via vaccination. 4 Clade Crisscross (Phambili) Cross-clade immunity has been shown to develop in individuals infected with HIV-1 and in uninfected individuals vaccinated with a HIV-1 clade-specific vaccine. A test of concept study was initiated to assess efficacy of the MRKAd5/ HIV-1 clade B vaccine in South Africa, where there is a high prevalence of HIV-1 and the major circulating strain is clade C. The trial was stopped after the same vaccine was found to be ineffective in the Step study. Gray et al. report that, like the Step study, the MRKAd5/HIV-1 vaccine did not prevent HIV-1 infection or lower viral load setpoint among South African participants. This was despite having induced a high frequency and magnitude of T-cell responses as measured by interferon-γ ELISpot. Although vaccine recipients developed greater responses to the vaccine-matched clade B antigens, most developed clade C responses as well. Although premature cessation of the trial limited what conclusions could be drawn, unlike the Step study, there was no increase in risk of HIV-1 infection observed in Ad5-seropositive or uncircumcised men. Post-hoc analyses indicated a trend toward lower viral load setpoints and slower CD4 + T-cell decline for female vaccinees, as compared to placebo recipients. These data suggest that future efficacy trials could stratify by sex to examine potential differences in vaccine efficacy for females and males. 5 Panning for Vaccine-Induced Pressure (Step Study) The MRKAd5/HIV-1 vaccine evaluated in the Step study induced HIV-specific T-cell responses in more than 75% of vaccinated subjects, yet it did not prevent infection or reduce viral load set point. Rolland et al. investigated whether the vaccine-elicited T-cell responses affected virus strains of the breakthrough infections. A sieve analysis approach was used to test whether immunization had the effect of sifting out certain virus strains. HIV-1 sequences isolated from 68 infected vaccine and placebo recipients were compared with the vaccine insert sequence. Viruses infecting vaccine recipients were more likely to encode epitopes that differed from those present in the vaccine. The most significant site distinguishing vaccine and placebo recipients was Gag amino acid 84. Differences between sequences of viruses infecting vaccine versus placebo recipients were confined to Gag, Pol, and Nef, the portions of the virus that were included in the vaccine. These data support the notion that vaccine-induced T-cell pressure led to a high rate of mismatch among vaccine recipient founder sequences. The data represent the first demonstration of a vaccine exerting immune pressure on HIV-1 and provide impetus for designing vaccines to elicit cytotoxic T lymphocyte responses. The researchers propose that new vaccines should aim to trigger mutations in vulnerable regions of HIV-1, potentially cornering the virus into adopting attenuated forms. 6 Troubling T-cells (Step Study/HVTN 071) HIV-1 insert-containing adenovirus serotype 5 (Ad5) vector vaccines induce strong HIV-specific T-cell responses. However, pre-existing Ad5 immunity can dampen the immune responses induced by these vaccines. Research has focused more on the role of Ad5-specific antibodies in reducing Ad5 vaccine immunogenicity. Using samples from clinical stud- Continued on page HVTNEWS VOLUME 4:1 JUNE

22 HVTN Annual Network Award Winners On May 30th 2012 in Washington DC, the HVTN honored individuals who perform outstanding work at our sites Keith Barsky (Center), HVTN Service Award winner. Award Recipient: Keith Barsky Recruiter, Orlando HVTN service Award The HVTN Service Award recognizes individuals who have creatively responded to a particular challenge arising in their CRS work, and by which value has been added to the CRS / HVTN enterprise. Accolades for Keith Keith is the only recruiter at OIC [Orlando Immunology Center] for the HVTN, which means he has spoken to almost every person that has been screened or enrolled at our site. His rate of converting a study contact into a screen is among the highest of all the HVTN centers; all thanks to his excellent communication skills. The path that Keith has paved between our center and the gay community in Orlando has resulted in strong relationships which I suspect will ultimately help provide more awareness about HIV and STD prevention in Orlando. Prior to the HVTN, there was very little collaboration between city organizations that serve the HIV-infected and at-risk populations in Orlando. Keith was able to form strong ties with the HIV community and gay organizations. HVTN Mentoring Award Award Recipient: Magdalena Sobieszczyk, MD, MPH Co-Principal Investigator, New York The HVTN Mentoring Award recognizes an individual who has demonstrated extraordinary commitment as a mentor by promoting research, training/education, and professional development of those they have mentored. Accolades for Magda What I really appreciate about her is that she is completely non-intimidating and welcoming, which allows me to approach her with any question or concern that I have with my work and research. I think this is a particularly important quality for people mentoring early researchers, who may find the whole process and experience very intimidating. Having someone like Dr. Sobieszczyk as a mentor and guide has really helped me grow as a researcher, as well as know how to be a good mentor myself for those I will work with in the future. Magda makes training and mentorship a priority by providing incredible support for her staff and seeking out educational and training opportunities. She challenges us to become analytical and thoughtful researchers. Regardless of her own workload, she is always available as a compassionate listener and to offer her knowledge and expertise. She sets a marvelous example. Her kindness and generosity equal her intelligence and hard work! 22 JUNE 2012 VOLUME 4:1 HVTNEWS

23 Kyle Rybczyk (Center), HVTN Citizenship Award winner. HVTN Citizenship Award Award Recipient: Kyle Rybczyk, FNP-BC Clinic Coordinator, Nashville The HVTN Citizenship Award is for individuals deserving special recognition for good citizenship with regard to consistent, reliable, and quality performance of CRS duties and activities. Accolades for Kyle In my entire career, I have not had the pleasure of working on a team led by someone more vision-oriented and committed to excellence than Kyle Rybczyk. I am beyond impressed by the diligence he consistently displays in ensuring a quality experience for each and every volunteer who walks through our doors. Furthermore, our site auditor, on several occasions, has remarked of the near unparalleled excellence that Kyle inspires with regards to documentation and accurate clinical process at our site. Kyle is an outstanding citizen within the HVTN and also within our community here in Nashville. He encourages those around him to identify the best within themselves and to strive for consistently making that best of themselves a reality. I am honored to nominate Kyle for this award. Octavio Valente, Jr. Volunteer Service Award Award Recipient: Eduviges Cuello Community Advisory Board (CAB) member, Dominican Republic Octavio Valente, Jr. was a dedicated CAB member from Rio de Janeiro, Brazil who passed away on March 21, Octavio served many roles in the HVTN and in the HIV/AIDS community through his advocacy, dedication and spirited energy. In his honor, this service award is given to a CAB member who has demonstrated exemplary leadership and dedication in the HVTN. Accolades for Eduviges Eduviges was the First HIV Vaccine volunteer in the Dominican Republic. Motivated by that experience, she joined the CAB of the Instituto Dermatológico y Cirugía de Piel (IDCP) in Santo Domingo. She has advocated on the importance of community participation in prevention research at several different international forums, and has been given a national award in the Dominican Republic for her contribution to the advancement of public health. Eduivges has served on the HVTN Global CAB, its Ethics and Scientific Working Groups, HANC Partners, and the HVTN 907/906 protocol team. All of this is a testament to her commitment to the defense and protection of the rights of volunteers participating in vaccine studies, contributing significantly to shine the light on the role of activists in the fight against the HIV/AIDS. HVTNEWS VOLUME 4:1 JUNE