CLOSING IN ON IN THIS ISSUE. Immunogenicity Data Patterns Emerging from Cross Trial Comparisons

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1 a Publication of the HIV Vaccine Trials Network Volume 4, issue 2 FEBRUARY, 2013 CLOSING IN ON Immunogenicity Data Patterns Emerging from Cross Trial Comparisons Tracey Day, Barbara Metch, Nicole Frahm, and Cecilia Morgan A major advantage for vaccine studies conducted by the HIV Vaccine Trials Network (HVTN) is the uniformity with which the trials are conducted and evaluated. This includes maintaining similar key study design elements, as well as standardized methods for operational procedures, clinic procedures, data capture, and immunogenicity assessments. This consistency facilitates efficient trial implementation and ensures robust immunogenicity results. 1 It also provides the ability to compare immunogenicity data across multiple trials. Enabling such comparability for trials conducted worldwide was a pioneering goal for the Network when it was created in Since then over 60 trials have been initiated, evaluating diverse vaccine platforms from a variety of product developers. Here, we describe several informative patterns in immunogenicity that have emerged from cross trial data comparisons. Heterologous vector prime/boost regimens elicit higher T-cell response rates, while homologous regimens elicit higher antibody response rates Prime/boost regimens administer multiple vaccinations as a means to increase immune responses to a IN THIS ISSUE Immunogenicity Data Patterns Emerging from Cross Trial Comparisons Can We Be More Effective in Designing Pre-efficacy Vaccine Trials? 2200 Strong: Celebrating our Volunteers Broadly Neutralizing Monoclonal Antibodies: Powerful Tools for HIV Vaccine Development Seattle HVTU Helps Pilot HANC Native American Initiative SPECIAL ANNOUNCEMENTS [back cover] CALENDAR [back cover]

2 Immunogenicity Data Patterns Emerging from Cross Trial Comparisons Standardized procedures used by the HVTN clinical sites include isolation and cryopreservation of plasma, serum, and peripheral blood mononuclear cells (PBMCs). Study specimens are shipped to HVTN endpoint laboratories where assays analyzing T-cell and antibody responses are performed. For T-cell responses, validated assays include antigen-specific cytokine expression via intracellular cytokine staining and IFN-γ ELISpot For antibody responses, standardized assays include binding antibody multiplex assay or ELISA for binding antibodies, and the TZM-bl and A3R5 assays for neutralizing antibodies. 19,20 The HVTN Statistical and Data Management Center (SDMC) and the HVTN Laboratory Program have developed statistical methods to determine whether a participant has a positive response in an assay. The percentage of participants with a positive response (ie, positive response rate) is the value often used to compare immunogenicity across trials, as well as between individual arms within a trial. vaccine. Homologous vector prime/boost regimens comprise administrations of the same vaccine vector, whereas heterologous vector prime/boost regimens contain different vaccine products. Several HVTN clinical trials (HVTN 055, 065, and 068) have evaluated whether homologous or heterologous vector prime/boost regimens are better for different types of HIV vaccine platforms. By compiling the positive response rate data from these trials, a similar pattern was observed (see Table 1 for trial descriptions). 2-4 In each study, whether comparing pox virus vector combinations, or DNA plus modified vaccinia Ankara (MVA) or recombinant adenovirus serotype 5 (rad5) vectors, the heterologous boost regimen arms elicited higher HIVspecific T-cell response rates, and the homologous regimen arms elicited higher HIV-specific antibody response rates. An example of this from HVTN 055, which compared MVA and fowlpox vectored vaccines, is shown in Figure 1. This trend was apparent despite the use of different vaccine vectors and inserts, suggesting that this pattern could hold true for other vaccine types. Both antibody and T-cell responses may be important for a successful HIV vaccine. How might we get the best of both worlds with prime/boost regimens? This is not yet known, but a clue may come from results from HVTN 078. This study compared different heterologous Figure 1. Heterologous vector prime/boost regimens elicit higher T-cell response rates, while higher binding antibody response rates are induced for homologous regimens. Relative peak immunogenicity response rates for T-cell (blue) and antibody responses (red) for HVTN 055 (mean response rate with 95% confidence intervals). M refers to MVA vector and F to fowlpox vector. The response rate data from the homologous arm with FPV-HIV were omitted due to low immunogenicity of this regimen. 2 FEBRUARY 2013 VOLUME 4:2 HVTNEWS

3 Immunogenicity data Patterns Emerging from Cross Trial Comparisons Protocol HVTN 044 Products BY active arms 4-plasmid DNA; IL-2/Ig DNA; 4-plasmid DNA + IL-2/Ig DNA t0; 4-plasmid DNA + IL-2/Ig DNA t48 COLLABORATORS HIV GENE Inserts / proteins Study Findings* (completed trials)/study Aims (ONGOING AND PLANNED TRIALS) VRC, DAIDS gag, pol, nef, env Plasmid IL-2/Ig significantly increased immune responses when administered 2 days after the DNA vaccine, compared with simultaneous administration. HVTN plasmid DNA VRC, DAIDS gag, pol, nef, env Appeared safe and well tolerated. Three doses of the 4-plasmid DNA product elicited more T-cell responses (50%) than 2 doses (22%), primarily a CD4+ T-cell response. HVTN 055 rfpv; rmva; rmva + rfpv HVTN 060 HVTN 063 DNA gag; DNA gag + IL- 12 DNA; DNA gag + IL-12 DNA + CTL MEP DNA gag; DNA gag + IL- 15 DNA; DNA gag +IL-15 DNA + IL-12 DNA Therion, DAIDS env, gag, tat, rev, nef, RT The MVA-HIV and FPV-HIV vaccines were well tolerated, and heterologous boosting was superior at inducing CD8+ T-cell responses. Wyeth (Pfizer), DAIDS gag The HIV gag DNA with plasmid cytokine adjuvants was well tolerated. There were minimal responses with HIV gag DNA alone and a non-significant increase in response rates only with the intermediate dose of IL-12. Wyeth (Pfizer), DAIDS gag The HIV gag DNA with plasmid cytokine adjuvants was well tolerated. There were minimal responses with HIV gag DNA alone, and no apparent augmentation with IL-12 or IL-15. HVTN 065 rmva; DNA + rmva GeoVax, DAIDS gag, pr, RT, env, tat, rev, vpu, pol HVTN 068 HVTN 070 rad5; 4-plasmid DNA + rad5 DNA; DNA + IL-15 DNA; DNA + IL-12 DNA MVA/HIV62 elicited different patterns of T-cell and antibody responses when administered alone (higher antibody responses) or in combination with the JS7 DNA vaccine (better T-cell responses). VRC, DAIDS gag, pol, nef, env Homologous rad5 boosting enhanced Env-specific antibody responses, but did not increase HIV-specific T-cell responses. Despite limited post prime immune responses, DNA priming enhanced the magnitude of post boost Env-specific antibody and CD4+ T-cell responses and influenced the cytokine and memory marker profiles of the boosted T-cell responses. U Penn, Wyeth (Pfizer), DAIDS env, gag, pol Together with HVTN 080 results, indicates electroporation with cytokine adjuvant increased responses to DNA vaccination significantly. HVTN 071 rad5 Merck, DAIDS gag, pol, nef Last study conducted with Merk rad5 vaccine, specimens from large blood draws and leukaphoresis have been used in well over a dozen ancillary studies to help understand the Step study results. HVTN 076 DNA + rad5 VRC, DAIDS gag, pol, nef, env An ongoing phase 1b clinical trial to evaluate mucosal immune responses following intramuscular injections of an HIV DNA plasmid vaccine prime, followed by an HIV adenoviral vector boost in healthy, Ad5 seronegative HIV-1 uninfected adults. HVTN 077 rad35 + rad5; DNA + rad5; DNA + rad35 HVTN 078 NYVAC + rad5; rad5 + NYVAC HVTN 080 DNA; DNA + IL-12 DNA VGX (Inovio), Profectus, DAIDS HVTN 087 DNA + rvsv; DNA + IL-12 DNA + rvsv VRC, DAIDS env An ongoing phase 1b trial to evaluate the safety and immunogenicity of recombinant adenoviral serotype 35 (rad35) and serotype 5 (rad5) HIV-1 vaccines when given as a heterologous prime-boost regimen, or as boosts to a recombinant DNA vaccine in healthy, Ad5-naïve and Ad5-exposed, low risk, HIV-1 uninfected adult participants. VRC, EuroVacc, DAIDS env, gag, pol, nef An ongoing phase 1b clinical trial to evaluate the safety and immunogenicity of heterologous prime/boost vaccine regimens (NYVAC-B/rAd5 versus rad5/nyvac-b) in healthy, HIV-1 uninfected, Ad5 neutralizing antibody seronegative adult participants. env, gag, pol Together with HVTN 070 results, indicates electroporation with cytokine adjuvant increased responses to DNA vaccination significantly. Profectus, DAIDS gag, pol, nef, tat, vif, env An ongoing trial to evaluate the safety and tolerability of a prime-boost regimen of HIV-MAG vaccine given with and without plasmid human IL-12, delivered with electroporation, followed by VSV HIV gag boost in healthy HIV-1 uninfected adult volunteers. HVTN 088 rgp140 with MF59 Novartis, DAIDS Env An ongoing phase 1 trial to: 1) Evaluate the safety and tolerability of and 2) Evaluate neutralizing antibody responses to HIV-1 Sub C gp140 vaccine with MF59 in healthy, HIV-1 uninfected adults, primed or unprimed with HIV-1 subtype B envelope subunit vaccines with MF59. HVTN 094 DNA with GM-CSF + rmva GeoVax, DAIDS HVTN 097 ALVAC + rgp120 Sanofi Pasteur, Global Solutions for Infectious Diseases, DAIDS pr, RT, tat, rev, vpu, env, gag, pol env, gag, pr; Env An ongoing trial to evaluate the safety and immunogenicity of a heterologous prime-boost vaccine regimen of GEO-D03 DNA and MVA/HIV62B vaccines in healthy, HIV-1 uninfected vaccinia naïve adult participants. A planned phase 1b clinical trial to evaluate the safety and immunogenicity of the vaccine regimen ALVAC-HIV (vcp1521) followed by AIDSVAX B/E in healthy, HIV-1 uninfected adult participants in South Africa. HVTN 098 DNA; DNA + IL-12 DNA Inovio, DAIDS env A, env C, gag, pol A planned trial to evaluate the safety and tolerability of PENNVAX -GP with plasmid IL-12, given by intradermal or intramuscular injection with electroporation, in healthy HIV-1 uninfected adult volunteers. HVTN 204 DNA + rad5 VRC, DAIDS gag, pol, nef, env The regimen was well-tolerated and immunogenic, inducing HIV-specific IFN-ɣ+ T cells in 70.8%, which were balanced CD4+/CD8+ polyfunctional responses and high frequencies (83.7% 94.6%) of multi-clade anti-env binding antibodies. HVTN 205/908 rmva; DNA + rmva GeoVax, DAIDS env, pol, gag, tat, rev, vpu, pr, RT * Text adapted from published manuscript. An ongoing phase 2 trial to evaluate the safety and immunogenicity of a prime-boost regimen of pga2/js7 DNA and MVA/HIV62, or MVA/HIV62 alone in healthy, HIV-1 uninfected, vaccinia-naïve individuals. Abbreviations: VRC, Vaccine Research Center (NIAID, NIH); DAIDS, Division of Aquired Immune Deficiency Syndrome (NIAID, NIH); rmva, recombinant modified vaccinia Ankara; rfpv, recombinant fowlpox virus; rvsv, recombinant vesicular stomatitis virus; rad5, recombinant adenovirus serotype 5; NYVAC, a highly attenuated vaccinia virus strain; GM-CSF, granulocyte-macrophage colony-stimulating factor; ALVAC, live attenuated recombinant canarypox derived virus; rgp120 / rgp140, recombinant glycoprotein 120/140 subunit; Ig, immunoglobulin; IL, interleukin Table 1. Descriptions of HVTN trials mentioned in this article. HVTNEWS VOLUME 4:2 FEBRUARY

4 Immunogenicity Data Patterns Emerging from Cross Trial Comparisons regimens with rad5 and NYVAC vectors. The results of this phase 1b study found that priming with rad5 and boosting with NYVAC was better than the reverse order. 5,6 Importantly, this was true for both T-cell and B-cell responses, with both yielding promising response rates. Further research is needed, but these results suggest the hypothesis that employing a strongly immunogenic prime, such as rad5, in combination with an immunogenic boost may enable the best of both worlds by eliciting balanced T-cell and antibody responses. In addition to heterologous and homologous vector regimens, the HVTN is investigating different insert regimens. HVTN 083, for example is an ongoing study that will examine the effects of heterologous versus homologous inserts on T-cell response breadth. CD3+ T-cell response rates measured by ICS 2 weeks after the final vaccination were 60% for DNA vaccine with plasmid IL-2/Ig given 48 hours later as compared to 44% and 30% for DNA vaccine alone and with simultaneous IL-2/Ig administration, respectively (see Figure 2). HVTN 060 and 063 evaluated the effect of plasmid cytokine adjuvants (IL-12 and IL-15) on the immunogenicity of a truncated HIV-1 gag (p37) DNA vaccine. Though T-cell responses in general were low and there was not a statistically significant difference in response rates, there was an increase in HIV-specific T-cell responses after 3 vaccinations when IL-12 was co-administered at an intermediate dose, compared to DNA alone. T-cell responses were not observed at low and high doses of IL-12 and IL Enhancing responses with adjuvants and electroporation DNA plasmids can be weak immunogens on their own, but several HVTN clinical trials point toward potentially effective ways to enhance DNA vaccine potency. One strategy yielding incremental immunogenicity increases has been the administration of DNA vaccines with plasmid-encoded cytokine adjuvants. For example, HVTN 044 evaluated the effects of the cytokine IL-2 on the immunogenicity of an HIV plasmid DNA vaccine. In the study, IL-2 was delivered as a plasmid-encoded fusion protein (IL-2/Ig) and given either simultaneously or 48 hours after the HIV DNA vaccine. Inclusion of IL-2/Ig resulted in a trend toward increased response rates by intracellular cytokine staining (ICS) when given 48 hours after the DNA vaccine. 7 HIV-specific More recently, potential benefits of using adjuvants in conjunction with electroporation (EP) as a delivery method for DNA vaccines have been observed. EP involves delivery of short electrical pulses after DNA vaccine injection. These pulses serve to increase DNA uptake by cells. In HVTN 080, 3 vaccinations of PEN- Figure 2. Cytokine adjuvants and electroporation increase DNA vaccine response rates. The differences in CD3+ T-cell response rates between DNA vaccine alone and DNA given with a cytokine adjuvant (HVTN 044, 060) or electroporation plus a cytokine adjuvant (HVTN 070, 080) are shown. Note: not all study arms are shown (see Table 1 for study descriptions). 4 FEBRUARY 2013 VOLUME 4:2 HVTNEWS

5 Immunogenicity data Patterns Emerging from Cross Trial Comparisons NVAX TM -B (3 mg dose) delivered via EP elicited a 39% higher positive ICS response rate compared to 3 vaccinations of PENNVAX TM -B (6 mg dose) administered by standard intramuscular injection in HVTN 070. In addition, the use of adjuvants in conjunction with EP appears to be associated with further increase in vaccine potency. For example, in HVTN 080, 3 vaccinations of DNA with GENEVAX TM IL-12 delivered EP elicited a 61% higher positive ICS assay response rate compared to 3 standard intramuscular injections of the DNA vaccine alone (see Figure 2). 9 Ongoing and planned HVTN studies will continue to investigate strategies to enhance vaccine potency via adjuvants or modes of administration. HVTN 087 is an ongoing study evaluating a DNA and vesicular stomatitis virus (VSV) vector prime/boost regimen in which the DNA will be co-administered with IL-12 and an EP device from another developer (see Table 1). A study currently in development, HVTN 098, will compare intramuscular administration of DNA via EP, with or without IL-12, with intradermal delivery via EP. New cytokine adjuvants will also be investigated. For example, HVTN 094 is evaluating the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) co-expressed with HIV genes on a DNA plasmid. Future studies may also evaluate various adjuvants with proteins for their ability to enhance immunogenicity and increase response durability of protein immunogens. Vector and insert enhancements to optimize platforms Many vector and insert design improvements occur through an iterative process, with early phase HVTN trials and cross trial comparisons influencing preclinical and further clinical development. One example is the VRC DNA platform. The early generation product tested in HVTN trials consisted of a mixture of 4 plasmids: 1 with an env clade A gene insert, 1 with an env clade B, 1 with an env clade C, and 1 with a gag-pol-nef fusion gene. Results from HVTN 052 indicated Env-specific T-cell responses of approximately 30%, but responses to the other inserts was limited with 0% to Gag, 0% to Nef, and 3.4% to Pol. 10 The current VRC DNA construct is a mixture of 6 plasmids with each HIV gene insert on a separate plasmid. HVTN 204 demonstrated that separating the inserts onto individual plasmids maintained a T-cell response to Env (48.3%), while increasing the responses to Gag (48.3%) and Nef (27.6%). 11 Another example of clinical trial results informing and improving next generation products comes from a comparison of immune responses to different DNA vaccines in HVTN 080 and HVTN 204. Although overall HIVspecific T-cell response rates to the DNA vaccines were a little higher in HVTN 080, most of these responses were to Gag, and less to Env. In contrast, responses elicited by the DNA prime in HVTN 204 were more balanced against Env and Gag, as described above. These distinct response profiles identified opportunities to further improve vector and insert designs. The HVTN 080 developers indicate that preclinical evaluations of their next generation DNA vaccine, currently in development, demonstrate increased Env responses. Cross protocol analysis of HVTN DNA vaccine trials To date, the HVTN has conducted numerous trials evaluating DNA vaccine products. Jin, Morgan, and Yu et al. compiled data from 10 HVTN DNA vaccine trials that used a validated ICS assay to investigate factors influencing DNA vaccine immunogenicity. 12 Postvaccination responses from DNA only vaccine treatment arms (no adjuvants or EP) were compared. Overall, the response rates varied considerably from study to study, but in all cases CD4+ T-cell responses occurred more frequently than CD8+ T-cell responses. The analysis showed that 3 administrations of a DNA vaccine elicited more frequent HIV-specific CD8+ T-cell responses than 2 administrations, and that a 4 th administration did not have a statistically significant effect on response rates in the regimens examined. In addition, there appeared to be a dose response up to 3 mg per vaccine, but higher doses did not further enhance responses. These findings reveal trends across many DNA studies and suggest potential ways to optimize this vaccine HVTNEWS VOLUME 4:2 FEBRUARY

6 Immunogenicity data Patterns Emerging from Cross Trial Comparisons Reference group CD4+ (N=288 vaccinees a ) CD8+ (N=309 vaccinees b ) Est. odds ratio c (95% CI) Wald p-value Est. odds ratio c (95% CI) Wald p-value Female Male 2.22 (1.30, 3.80) (0.48, 2.13) 0.96 Body mass index 1 unit change 0.90 (0.85, 0.96) (0.89, 1.04) 0.34 Age 1 year change 0.99 (0.96, 1.02) (0.97, 1.05) 0.55 a) 1 participant was excluded from the CD4+ model due to missing data for BMI and 23 due to lack of evaluable CD4+ T-cell assay data. b) 2 participants were excluded from the CD8+ model due to missing data for BMI and 1 due to lack of evaluable CD8+ T-cell assay data. c) The estimated odds ratios were adjusted for other variables in the model. The models also adjusted for race and number of plasmids in the vaccine, which were nonsignificant. Table 2. Increased Env-specific CD4+ T-cell responses were associated with female gender and lower BMI in multivariable logistic regression models of CD4+ and CD8+ envelope specific T-cell response rates in 5 studies of the NIAID VRC DNA vaccine (N=312 vaccinees, 295 US and 17 Peru). platform. The large number of participants included in the pooled DNA vaccine cross protocol analysis provided an opportunity to examine potential associations between participant demographics and vaccine-elicited immune responses. Identifying host factors that influence vaccine immunogenicity can be useful for vaccine development in a variety of ways. For example, the information could be used to increase statistical power for assessing vaccine efficacy, and to improve the ability to assess immune correlates of protection. Using multivariable logistic regression modeling, the cross protocol DNA vaccine study investigators evaluated whether participants age, gender, and body mass index (BMI) were associated with higher CD4+ and CD8+ T-cell response rates in the DNA vaccine trials. In this study, female gender and lower BMI were associated (independently) with statistically higher HIV-specific CD4+ T-cell response rates than male gender and higher BMI. A complimentary analysis was performed focusing on trials evaluating VRC DNA vaccines. Results from this analysis were similar to the broader analysis, as shown in Table 2. Similar trends have been noted in other studies. 13 These results point to baseline characteristics that should be considered when evaluating vaccines in human populations. Innate immune responses predict T-cell response magnitude Innate immune responses are another study readout that may enable predictions of candidate vaccine immune responses. 14 Innate immune responses to pathogens are involved in triggering and shaping the ensuing pathogen-specific adaptive response. Similarly, it is thought that innate responses to immunogens impact the characteristics of the adaptive responses that develop upon vaccination. The HVTN is working to define innate responses to vaccines that predict beneficial adaptive responses. HVTN 071 was a phase 1b trial in which innate responses were evaluated by measuring gene expression profiles from PBMC over the course of 1 week following rad5 vectored HIV vaccination. 15 Differences in gene expression in PBMC were observed early after vaccination, peaking at 24 hours postvaccination. Increased expression was observed primarily in genes involved in inflammation and interferon pathways, whereas expression of genes involved in lymphocyte trafficking was decreased. Using a systems biology approach, innate gene expression profiles were compared with adaptive response characteristics. This method identified patterns in which levels of certain cytokine gene pairs and inflammatory genes were correlated with HIV-specific CD8+ T-cell response magnitude. These results suggest that certain innate responses to the rad5 vector HIV vaccine were predictive of T-cell response magnitude. 6 FEBRUARY 2013 VOLUME 4:2 HVTNEWS

7 Immunogenicity data Patterns Emerging from Cross Trial Comparisons The HVTN Laboratory Program has developed a suite of innate response assays that may be used to assess innate immune responses in HVTN trials. Sampling time points allowing for these assessments have been incorporated in multiple ongoing and planned trials evaluating different products and regimens including HVTN 071, 076, 087, 088, 094, 097, and 205/908 (see Table 1 for trial descriptions). These assays will likely serve as a future platform for comparing cross trial data, and may help further identify innate responses that shape adaptive responses and potentially indicate induction of mucosal responses. Conclusion Immunogenicity patterns emerging from cross protocol analyses of HVTN trials are providing clues for optimizing candidate vaccine platforms and regimens. These insights are possible because the studies are conducted within a single global network in which standardized procedures and immune response assays are utilized. Information gleaned from these insights could accelerate the development of vaccines for HIV, and may be applied to vaccine research for other disease areas as well. The authors would like to thank Marnie Elizaga, Yunda Huang, Georgia Tomaras, HVTN 044, 052, 055, 060, 063, 065, 068, 070, 071, 076, 077, 078, 080, 087, 088, 094, 097, 098, 204, 205/908 Protocol Teams and participants. Tracey Day is HVTN Senior Science Writer, Barbara Metch is Senior Statistical Analyst working with the HVTN, Nicole Frahm is Associate Director, Laboratory Sciences at the HVTN Laboratory Program, Cecilia Morgan is HVTN Associate Director, Scientific Development. Reference List 1. Day T, Morgan C, Ferrara A, Kublin J. The HVTN: Strategic accomplishments of the first decade and beyond. HVTNews. 2012;3(2): Keefer MC, Frey SE, Elizaga M, et al. A phase I trial of preventive HIV vaccination with heterologous poxviral-vectors containing matching HIV-1 inserts in healthy HIV-uninfected subjects. Vaccine. 2011;29(10): Goepfert PA, Elizaga ML, Sato A, et al. Phase 1 safety and immunogenicity testing of DNA and recombinant modified vaccinia Ankara vaccines expressing HIV-1 virus-like particles. J Infect Dis. 2011;203(5): De Rosa SC, Thomas EP, Bui J, et al. HIV-DNA priming alters T cell responses to HIV-adenovirus vaccine even when responses to DNA are undetectable. J Immunol. 2011;187(6): Montefiori D, Huang Y, Karuna M, et al. rad5/nyvac-b is superior to NYVAC-B/rAd5 and is dependent on rad5 dose for neutralizing antibody responses against HIV-1. Poster presented at: AIDS Vaccine 2012; September 9-12, 2012; Boston, MA. 6. Bart P, Huang Y, Frahm N, et al. rad5 prime/nyvac-b Boost Regimen is Superior to NYVAC-B prime/rad5 Boost Regimen for Both Response Rates and Magnitude of CD4 and CD8 T-Cell Responses. Poster presented at: AIDS Vaccine 2012; September 9-12, 2012; Boston, MA. 7. Baden LR, Blattner WA, Morgan C, et al. Timing of plasmid cytokine (IL-2/Ig) administration affects HIV-1 vaccine immunogenicity in HIV-seronegative subjects. J Infect Dis. 2011;204(10): Kalams SA, Parker S, Jin X, et al. Safety and immunogenicity of an HIV-1 gag DNA vaccine with or without IL-12 and/or IL-15 plasmid cytokine adjuvant in healthy, HIV-1 uninfected adults. PLoS ONE. 2012;7(1):e Kaldor J, Edupuganti S, Elizaga M, et al. Robust immunogenicity after intramuscular HIV DNA vaccination with IL-12 plasmid cytokine adjuvant delivered via electroporation in HIV uninfected adults (HVTN 070 & 080). Poster presented at: AIDS Vaccine 2011; September 12-15, 2011; Bangkok, Thailand. 10. Morgan C, Peiperl L, McElrath MJ, et al. DNA prime followed by adenoviral vector boost elicits HIV-1 specific CD8+ T cell responses in healthy HIV-1 uninfected adults (HVTN 052 and 057). In: AIDSVaccine 2007; August 20-23, 2007; Seattle, WA. Abstract P Churchyard GJ, Morgan C, Adams E, et al. A phase IIA randomized clinical trial of a multiclade HIV-1 DNA prime followed by a multiclade rad5 HIV-1 vaccine boost in healthy adults (HVTN204). PLoS ONE. 2011;6(8):e Morgan C, Jin X, Yu X, et al. DNA plasmid HIV vaccine design, HVTNEWS VOLUME 4:2 FEBRUARY

8 Immunogenicity Patterns Emerging from Cross Trial Comparisons number of doses, participant gender, and body mass index affect T- cell responses across HIV vaccine clinical trials [Abstract]. Retrovirology. 2012; 9 (Suppl 2):P Corey L, Langenberg AG, Ashley R, et al. Recombinant glycoprotein vaccine for the prevention of genital HSV-2 infection: two randomized controlled trials. Chiron HSV Vaccine Study Group. JAMA. 1999;282(4): Heit A, Andersen EA. The role of innate immunity in vaccinology. HVTNews. 2012;3(2): Zak D, Andersen-Nissen E, Peterson E, et al. Merck Ad5/HIV induces broad innate immune activation that predicts CD8+ T-cell responses but is attenuated by preexisting Ad5 immunity. [published online November 14, 2012]. Proc Natl Acad Sci. doi: / pnas Moodie Z, Price L, Gouttefangeas C, et al. Response definition criteria for ELISPOT assays revisited. Cancer Immunol Immunother. 2010;59(10): Moodie Z, Huang Y, Gu L, Hural J, Self SG. Statistical positivity criteria for the analysis of ELISpot assay data in HIV-1 vaccine trials. J Immunol Methods. 2006;315(1-2): Horton H, Thomas EP, Stucky JA, et al. Optimization and validation of an 8-color intracellular cytokine staining (ICS) assay to quantify antigen-specific T cells induced by vaccination. J Immunol Methods. 2007;323(1): Li M, Gao F, Mascola JR, et al. Human immunodeficiency virus type 1 env clones from acute and early subtype B infections for standardized assessments of vaccine-elicited neutralizing antibodies. J Virol. 2005;79(16): Montefiori DC, Karnasuta C, Huang Y, et al. Magnitude and breadth of the neutralizing antibody response in the RV144 and Vax003 HIV-1 vaccine efficacy trials. J Infect Dis. 2012;206(3): FEBRUARY 2013 VOLUME 4:2 HVTNEWS

9 Can We Be More Effective in Designing Pre-efficacy Vaccine Trials? Yunda Huang, Zoe Moodie, and Cecilia Morgan The HVTN s mission is to drive, as rapidly and scientifically rigorously as possible, the development of safe and effective vaccines for prevention of HIV infection globally. To that end, the Network must develop tools that enable it to answer the critical questions in this rapidly evolving field. These tools include novel clinical trial designs, from early phases through efficacy studies. In a previous HVTNews article, 1 Gilbert and Grove described innovative sequential designs for HIV vaccine efficacy trials in which the primary objective is the assessment of vaccine efficacy in reducing the rate of HIV infection (see also Gilbert, Grove, et al., 2011). 2 But what about the design of pre-efficacy trials, where the primary objectives involve safety and immunogenicity assessments? As some traditional design strategies, used in other clinical trial settings, may not be feasible in the context of HIV vaccine evaluations, innovative statistical designs are particularly important to HVTN trials. There are 2 major goals for the HVTN s agenda in early phase, pre-efficacy trials. One is first-in-human safety and immunogenicity evaluation of vaccine candidates. The other is selection of vaccine regimens to move onto the next level of testing. For the former, often only a relatively small number of participants is needed to establish initial vaccine safety. In dose escalation studies, for example, 10 participants are often evaluated at each dose level to determine the maximally tolerated dosage (MTD). For further characterization of the vaccine s safety and immunogenicity, additional subjects (n=15-30) are enrolled at the MTD. Such a design provides a reasonably high probability of moving a truly promising vaccine candidate forward, or stopping the study of a weak one, with a low chance of missing a promising vaccine candidate or wrongly selecting a weak one. An immunogenicity checkpoint can be instituted; for example, if 3 positive responses are seen out of 25 at the MTD, continue evaluation, else (ie, 0-2 out of 25) stop. The principle of such stopping rules is to stop only if some maximum immune response rate can be ruled out with high probability, while the choice of maximum immune response rate often depends on the portfolio of candidate vaccine regimens being evaluated. When feasible, multiple vaccine products at similar stages of development from different vaccine developers may be tested in the same study, sharing common placebo samples and reducing trial development overhead. Once initial safety and immunogenicity are established, there may be interest in further regimen optimization among more than 2 regimens in terms of immunogenicity. When it is not critical to establish superiority or non-inferiority of one vaccine candidate compared to the others, and the study vaccine regimens and objectives do not lend themselves to a more structured statistical design, such as factorial or Latin square (discussed below), a rank-and-select design can be an efficient approach to advancing regimens. 3 Such a design often ranks all active comparator arms and selects the arm or arms with the best immune response, such as response rates and/or response magnitudes. Rank-and-select designs in general require a smaller sample size than superiority and noninferiority trials. A rank-and-select design may be useful, for example, in a dose de-escalation study of an adjuvant, to evaluate the optimal adjuvant dose for eliciting a certain immune response. The above mentioned approaches have been implemented in multiple HVTN trials. As the HIV vaccine field continues to mature, there are currently more HIV vaccine products available for testing in various combinations than ever before. For example, there are different DNA constructs, viral vectors, vector inserts of HIV genes, and proteins. Furthermore these products could also be combined with different types of adjuvants, given at different dose levels, and administered through various routes and delivery methods. There are HVTNEWS VOLUME 4:2 FEBRUARY

10 Can We Be More Effective in Designing Pre-efficacy Vaccine Trials? also different factors that may play an important role in vaccine evaluation, including demographics, host genetics, and environmental factors such as prior exposure to the vaccine vector, co-infection with other pathogens, or background use of non-vaccine prevention modalities. In selecting the best regimen(s) for the next stage of testing, one primary challenge comes from this vast number of combinations that must be taken into account in an early-phase trial design. The biggest advantage of using a factorial design relies on the assumption that results about any given factor can be combined over the levels of the other factors for the assessment of main effects. However, care is needed in factorial designs when antagonism may occur with combinations of products. Large sample sizes are needed to assess the effect of interactions, and the power to assess the main effects may be compromised by negative interactions. From a trial design perspective, such challenges may be ameliorated by more systematic, structured evaluations of these different types of factors. One example of a systematic structured design is the factorial design, where more than 1 factor with multiple levels can be studied simultaneously. In a factorial design, study participants FACTOR B: +/- DNA IN REGIMEN Figure 1. 2x2 Factorial design example FACTOR A: ADJUVANTS adjuvant a adjuvant b DNA + Viral Vector + Protein N=25 N=25 Viral Vector + Protein N=25 N=25 are randomized across combinations of factor levels, each representing 1 vaccine regimen of interest for evaluation. As an example, a 2 2 factorial design could be used to randomize participants into 1 of 4 groups defined by 2 novel adjuvants combined with 2 vaccine regimens such as a DNA + viral vector + protein vs a viral vector + protein (see Figure 1). Multiple questions can be addressed in such a factorial trial including: (1) How do the adjuvants differentially impact immunogenicity? (2) How does the DNA component impact immunogenicity? (3) Does the adjuvant effect differ with and without DNA? (4) Does the DNA effect differ between the adjuvants? In contrast, to obtain the same precision for estimating main effects of each factor, single-factor designs would require twice as many subjects, while only being able to answer the first 2 questions. ROW FACTOR: delivery method When certain factors are not of particular interest for direct comparisons, but can potentially influence immunogenicity, randomized block designs, which randomize treatments within blocks of participants, can be used to systematically control for variability arising from specific factors, and improve efficiency when the blocking factor(s) predict immunogenicity. Instead of randomizing across all combinations of factors, participants are not randomized across the blocking factors. Therefore, caution is needed in the interpretation of main effects of (nonrandomized) blocking factors, and interactions between randomization and blocking factors. The optimally efficient randomized block design for detecting main effects of treatment is the Latin squares design, which minimizes nuisance sources of variability in immunogenicity outcomes by systematic blocking in 2 directions. For example, the replicated Latin squares design shown in Figure 2 Figure 2. 3x3 Latin square design example COLUMN FACTOR: prep usage ORAL TOPICAL COMBINATION IM A (n=30) B (n=30) C (n=30) EP-IM C (n=30) A (n=30) B (n=30) ID B (n=30) C (n=30) A (n=30) evaluates 3 vaccine candidates -- A, B, and C -- under different types of pre-exposure prophylaxis (PrEP) usage (oral, topical, and combination) and different delivery methods of the vaccine (intramuscular by needle (IM), intramuscular by electroporation (EP-IM), and intradermal by needle (ID). Instead of being randomized into FEBRUARY 2013 VOLUME 4:2 HVTNEWS

11 Can We Be More Effective in Designing Pre-efficacy Vaccine Trials? plausibility of interactions interest in interactions interest in main effects # of factors, # of treatments recommended design examples High High High >=2, >=4 Factorial designs powered to examine interactions Medium/Low Low High for vaccine >=2, >=4 Factorial designs only powered to examine main effect Medium/Low Low High for treatment; Low for blocking factors =2, >=3 Replicated Latin square designs DNA x viral vector (with or without protein) Vaccine x adjuvant Vaccine x host genetics x dose Low Low Low N/A, >=3 Rank-and-select Vaccine regimen comparison Table 1. Paradigm for selecting an appropriate structured design for phase 1b vaccine trials categories (3x3x3, determined by PrEP usage, delivery method, and vaccine) as a factorial design would, study participants falling into any of the 9 combinations of PrEP usage and delivery method are randomized to receive 1 of the 3 tested vaccines. This design can answer 3 questions: (1) Which vaccine works best overall? (2) For each given vaccine delivery method or PrEP usage type, which vaccine is best? (3) Does vaccine delivery method or PrEP usage type affect immunogenicity? This design, however, cannot be used to assess interactions of vaccine regimen with the row and column factors; thus the niche for this design is in an assessment of vaccine effects when it is not critical to assess interactions. The HVTN has entered into a very exciting, yet challenging, era where many interesting vaccine concepts are available, and more novel options are forthcoming. Innovative designs in early phase trials can improve efficiency in evaluating the large number of vaccine candidates (Table 1). To fully utilize the advantages that these and other innovations afford us, continued close collaboration among a range of experts including researchers, community leaders, and biostatisticians is needed, so that the Network can carry out its important task of moving the field forward, and working towards finding an effective HIV vaccine. Yunda Huang and Zoe Moodie are Senior Staff Scientists and statisticians, Statistical Center for HIV/AIDS Research and Prevention (SCHARP), working with the HVTN; Cecilia Morgan is HVTN Associate Director, Scientific Development. Reference List 1. Gilbert P, Grove D. A sequential two-stage trial design for evaluating efficacy and immune correlates for multiple vaccine regimens. HVTNews. 2011;3(1): Gilbert PB, Grove D, Gabriel E, et al. A sequential phase 2b trial design for evaluating vaccine efficacy and immune correlates for multiple HIV vaccine regimens. Statistical Communications in Infectious Diseases. 2011;3(1), Article Moodie Z, Rossini AJ, Hudgens MG, Gilbert PB, Self SG, Russell ND. Statistical evaluation of HIV vaccines in early clinical trials. Contemp Clin Trials. 2006;27(2): HVTNEWS VOLUME 4:2 FEBRUARY

12 2200 STRONG CELEBRATING OUR VOLUNTEERS HVTN CONFERENCE RECEPTION, OCTOBER 29, 2012 At the 2012 HVTN Fall Full Group Meeting in Seattle, the first night s reception included a special celebration for reaching an enrollment milestone of 2200 participants in HVTN 505. At the time the reception was planned, 2200 was the trial s target enrollment, and given the importance of this trial (currently the only preventive HIV vaccine efficacy trial worldwide), an acknowledgement of this achievement was in order. Special guests at the reception were models from the Seattle HIV Vaccine Trials Unit s successful recruitment campaign, which featured local well-known members of the community in ads seeking volunteers for the Unit s vaccine trials. This strategy has also been used successfully at several other HVTN sites. 12 FEBRUARY 2013 VOLUME 4:2 HVTNEWS

13 (facing page, left) Blue Bear Wharton (center) poses with Davora Lindner and her partner Ro Yoon of Seattle's HIV Vaccine Trials Unit. Blue Bear as Debrianna modeled for the HVTU's promotional campaign. (facing page, right) Entertainer and community activist Aleksa Manila looks beautiful for the camera. (right) Photographer Nate Gowdy poses alongside Glo Euro N'Wei, Yuriko Lomein and Marilyn Moan-roe of the Sisters of Perpetual Indulgence, The Abbey of St. Joan. The Sisters donated their time to help Seattle's HIV Vaccine Trials Unit with outreach and even modeled for the promotional campaign. (below) Seattle HVTU Outreach Specialist Jacob McIntyre likes the HVTU s recruitment poster featuring Tanya, Miss U.T.O.P.I.A (U.T.O.P.I.A seeks to create a safe space for Pacific Islanders LGBTQI communities, advocating for social justice, education, and overall wellness.) (backgound) Chihuly gardens illuminate the night. Reception photos: Nate Gowdi; Recruitment Posters: Nate Gowdy and Carlos Paradinha. HVTNEWS VOLUME 4:2 FEBRUARY

14 Broadly Neutralizing Monoclonal Antibodies: Powerful Tools for HIV Vaccine Development Tracey Day An exciting milestone for HIV vaccine research has been the identification of antibodies capable of neutralizing diverse HIV strains in chronically infected patients. Although somewhat rare, the existence of these broadly neutralizing antibodies (bnabs) hints that the immune system is capable of developing antibodies with sufficient cross-reactivity to combat HIV s extreme variability. In the last several years, new technologies have been applied to bnab discovery, resulting in a surge of new antibodies with even greater neutralization breadth and potency. These bnabs provide valuable information for guiding immunogen design in classic vaccination approaches. 1 Several novel applications are also possible for bnabs that are manufactured as monoclonal antibodies (mabs). These are based on the ability to administer mabs to humans and thereby establish immunity passively. Multiple delivery approaches are under investigation, including direct administration via infusion or injection, or through vector-mediated gene transfer. Clinical studies using bnab transfer provide the opportunity to test in humans, for the first time, whether bnab presence can prevent HIV acquisition. Such proof of concept testing would establish an important immunological benchmark for efficacious neutralizing activity levels. If proven, such studies could lead to the development of bnab transfer as a strategy for prevention of HIV infection or disease progression. In addition, this would enable the investigation of HIV neutralization mechanisms to inform immunogen design. The HIV Vaccine Trials Network (HVTN) has great interest in moving forward with clinical evaluations of bnabs because of the power these studies could have to advance HIV vaccine development. Challenges for induction of bnabs against HIV During natural HIV infection, HIV-specific antibodies develop during the first several weeks of infection. 2 Some of these early antibodies possess neutralizing activity as measured in vitro, but their reactivity is limited to closely related HIV strains. After several years of infection, a proportion (10-30%) of individuals develops antibodies with broader reactivity, as judged by neutralization of a panel of diverse viral isolates in vitro. 3-7 The failure of HIV infected hosts to develop bnabs in a timely manner is due to the numerous evasion strategies employed by the virus. For example, neutralizing antibodies impede viral entry into host cells by targeting the HIV envelope protein (Env), which is hypervariable across virus strains and even among isolates within an infected individual. 8,9 Env has multiple unique properties that serve to effectively avoid host bnab development. The Env spike on the HIV surface is a membrane-bound heterotrimer comprising the glycoprotein subunits gp41 and gp120 (see Figure 1). 10 Env conformational instability leads to non-native forms on virion surfaces that misdirect immune responses toward non-neutralizing responses. 11,12 Several bnab binding epitopes, such as the CD4 binding site, are buried in the native structure impeding antibody binding. 13,14 In addition, glycan residues form a shield on the surface further masking antibody binding epitopes. 15,16 Neutralizing antibodies that develop can target variable regions, and may therefore be strain specific. 17 Properties of bnabs targeting HIV bnabs have not yet been induced via vaccination in humans largely due to the challenges posed by Env and described above. Nonetheless, numerous bnabs have been identified in chronically infected patients and are being used to understand how antibody mediated viral neutralization is accomplished. 1,18 Early efforts identified 5 bnabs targeting 3 different Env epitopes: 2G12 recognizing a glycan-associated epitope, 19,20 b12 recognizing an epitope in the CD4 binding site, 21,22 and 2F5, 4E10, and Z13 recognizing regions 14 FEBRUARY 2013 VOLUME 4:2 HVTNEWS

15 Broadly Neutralizing Monoclonal Antibodies: gp120 gp41 Glycan shield V1-V2 loop V3 loop C4 binding site MPER viral envelope spike Figure 1. Broadly neutralizing antibody (bnab) binding sites identify vulnerable regions of the HIV Envelope spike that may be targeted for immunogen design. bnab binding sites have been localized to the V1/V2 loop (PG9, PG16), V3 loop (PG9, PG16), CD4 binding site (b12, VRC01, HJ16), and MPER (2F5, 4E10, Z13e1) regions. near the membrane attachment site of gp41 (membrane proximal external region, MPER) (see Figure 1) These antibodies neutralized virus strains from multiple clades in vitro, and possessed a number of unusual features, such as exceptionally high levels of somatic mutation. During the last several years new technologies have been applied to bnab research and resulted in a large increase in the number of bnabs identified. For example, resurfaced glycoprotein probes have been constructed and used to screen sera for epitope-specific neutralizing activity Other new methods, such as single B cell cloning, deep sequencing, and high-throughput microneutralization assays facilitated the detection and cloning of bnab expressing B cells Several of these new bnabs possess improved characteristics, such as exceptionally broad neutralization activity (80-100% of tested virus isolates), and vastly increased potency. 1 Some of the new bnabs, such as VRC01, bind to epitopes within the CD4 binding site, but have greater neutralization breadth than the early antibodies that target this epitope. 29 Many of the recently identified bnabs, however, bind to new epitopes (see Figure 1). For example, 2 clonally related bnabs, PG9 and PG16, bind to a conformational epitope made up of V1 and V2 loop quaternary structure residues. 36 Other examples include PGT121, a member of the V3 loop carbohydrate epitope-specific group. 37 Together, these bnab epitopes reveal multiple vulnerable sites on Env that may be targeted with increasing precision for vaccine immunogen design. 38 The humanized mouse monoclonal antibody, ibalizumab (TaiMed/Aaron Diamond AIDS Research Center), is an example of alternative concept for HIV neutralization. This antibody potently blocks entry of diverse HIV strains into host cells by binding to CD4, the primary host cell receptor utilized by HIV. 39 Bispecific antibodylike molecules, comprising ibalizumab in combination with other bnabs described above, have been engineered and shown to achieve remarkably high potency and neutralization breadth in vitro HVTNEWS VOLUME 4:2 FEBRUARY

16 Broadly Neutralizing Monoclonal Antibodies Collectively, these bnab studies reveal multiple locations that are vulnerable to antibody neutralization. Researchers are applying this knowledge toward immunogen design in a variety of ways. One strategy is to develop methods to present Env in more native-like or exposed conformations through the use of trimeric proteins or virus-like particles displaying membrane-bound Env on their surface. In addition, studies are being conducted to learn how bnabs develop in chronically infected patients in hopes of shepherding bnab development via stepwise vaccinations with increasingly specific immunogens. 1 That bnabs develop in humans indicates the possibility that immunogens may be designed to elicit bnabs via classic vaccination approaches. Immunization with bnabs Passive immunization involves the transfer of antibodies to achieve temporary or conditional protection from infection. This is in contrast to classic active vaccination approaches, in which pathogen-specific antigen delivery elicits a long lasting protective immune response. Antibody transfer for passive immunization may be accomplished by direct administration of mabs. To maintain sufficient antibody levels for protection against HIV, repeated administrations of broadly neutralizing mabs (bnmabs) would be needed, for example on a monthly or quarterly basis. mab delivery is a well-established platform for immunotherapy against several cancers and in one case to prevent infectious disease (respiratory syncytial virus in infants). Alternatively, vector-mediated gene delivery can be used to administer bnab. In this case, the genes encoding the antibody are inserted into a viral vector, for example adeno-associated virus (AAV), and this is used to transduce host cells in vivo. This approach renders the person immunized so long as the transduced gene is expressed and therefore antibody is produced. Both direct and vector-mediated approaches have been used to transfer bnabs to non-human primates. Using different simian HIV (SHIV) challenge models, bnabs have successfully protected animals from SHIV infection to various degrees. 38,41 These findings support the notion that transfer or induction of bnabs in humans could reduce HIV infection rates; however clinical studies in humans are needed to verify this concept. Clinical trials with bnab to advance vaccine development A major outstanding question for HIV vaccine research is whether bnabs present prior to HIV exposure have the capacity to reduce HIV infection rates in human populations. Passive immunization with the new exceptionally potent and broadly reactive HIV-specific antibodies provides a means to test this concept in human clinical trials. The goal of the initial studies will be to determine the safety of the bnab products. Additional aims of early studies with direct bnab administration will be to define the pharmacokinetic profiles and identify appropriate dosing regimens. For vector-mediated delivery, vector safety and persistence will be primary aims, as well as antibody expression levels and durability. These initial studies will establish a foundation to enable subsequent studies to define bnab properties required for efficacy -- for example, the necessary bnab concentrations in sera and mucosal sites, and essential Fc mediated effector functions. Identifying these properties will provide critical immunogenicity targets for the field, aiding in the prioritization of vaccine products more likely to achieve efficacy, and facilitating identification of immune correlates of protection. The HVTN is well-suited to conduct these clinical trials with clinic sites experienced in the necessary safety evaluations and studies employing frequent sampling. In addition, the HVTN Laboratory Program has extensive expertise in antibody evaluations in sera and mucosal specimens. The discovery of ever more potent and broadly reactive antibodies allows investigators to evaluate these bnabs in clinical trials. Such trials can provide the critical proof of concept for the utilization of bnabs in preventing HIV acquisition in humans. These trials can also sup- 16 FEBRUARY 2013 VOLUME 4:2 HVTNEWS