Development of a V1/V2-targeting Quaternary-specific Broadly ...

Background: Following the RV144 trial demonstration of 31% vaccine efficacy in preventing HIV infection in Thailand, HVTN 097 was designed to evaluate the same regimen in South Africans to ascertain whether their immune response profiles were similar to Thais. This study was conducted in preparation for evaluating a similar clade C HIV vaccine regimen in South Africa. Our study was critical as previous studies have demonstrated that age, gender and BMI impact vaccine-induced immune responses. Methods: ALVAC-HIV (vCP1521) expressing HIV-1 Env (clade E, gp120 from strain 92TH023 and clade B TM gp41 from strain HIVLai, and Gag and protease (clade B) was administered at baseline then 1, 3 and 6 months later with AIDSVAX B/E, bivalent HIV gp120 subtypes B (MN) and E (A244) adsorbed to alum at the last 2 vaccinations. Immune responses were measured 2 weeks after the last immunization. Intracellular Cytokine Staining identified response rates and frequencies of HIV-specific T cells expressing IFN-c and/or IL-2. Binding antibodies to HIV-1 gp120 and V1V2, IgG subclass, and antibody functions (ADCC, avidity index, nAb, and virion capture) are ongoing. Immune responses were stratified by age, gender and BMI. Results: In 68 participants, overall peak response rates of Envspecific CD4 + T cells expressing IFN-c and/or IL-2 was 70.6% (95% CI 58.9%, 80.1%), similar to or greater than responses in RV144. Although not significant, participants < 25 years old had higher response rates observed than those ‡ 26 (76.0% vs. 55.6%, p = 0.108). Response rates in females (75%; 95%CI 56.6%, 87.3%) were similar to males (67.5%; 95%CI 52.0%, 79.9%). Response rates stratified by BMI categories of < 25, 25– 30, > 30 were 63.6%, 82.4% and 85.7%, respectively (p < 0.1 for BMI ‡ 25). Conclusions: Response rates and magnitudes of Env-specific CD4 + T cells in South Africans induced by the same vaccine regimen used in RV144 were at least comparable to or better than those induced in RV144. Age/gender or BMI did not affect CD4 + T-cell response rates.

Towards Broadly Neutralizing Antibody Induction OA12.01 Maturation of Broadly Neutralizing V1V2-directed Antibodies in the Context of Autologous Viral Escape 1,2

infection (w.p.i.) with individual mAbs isolated at different time-points showing varying levels of maturation and neutralization activity. Mutations R166S or K169E became fixed at 176 w.p.i. and resulted in complete viral escape from all mAbs. Here we investigated the effect of earlier mutations at positions 166 and 169 on the maturation of the CAP256-VRC26 lineage. Methods: Potential escape mutations were identified using single genome envelope amplification at 11 time-points from 6– 94 w.p.i. Mutations were introduced into the sensitive superinfecting virus and tested in neutralization assays against the 12 mAbs to assess their role in escape. Results: By 42 w.p.i., just prior to the development of breadth, the wildtype 169K was replaced with either a 169I/R/Q/T in all sequences, indicating early immune pressure at this site. Introduction of K169I/Q mutations into the superinfecting virus abrogated neutralization by CAP256-VRC26.01 and 12, the least broad members of the family, with less effect on other lineage members. Similarly, at position 166, we observed a K166R mutation in 14/15 sequences at 94 w.p.i. that abrogated neutralization by all but the four broadest mAbs, CAP256VRC26.03, 04, 08 and 09. As 166R and 166K are the two most prevalent immunotypes at this position (in 70% and 14% of all global viruses, respectively), the increased breadth of these four mAbs is consistent with their ability to tolerate both residues. Conclusions: Mutations that arose in the viral population soon after the expansion and subsequent maturation of the CAP256VRC26 family exposed later lineage members to multiple immunotypes, thereby increasing their neutralization breadth. These data illustrate how a gradual process of autologous viral escape shaped the maturation of this mAb response and provides insights for future sequential vaccine regimens.

OA12.02 Development of a V1/V2-targeting Quaternary-specific Broadly Neutralizing Lineage Elise Landais1, Bryan S. Briney2, Sergei L. Kosakovsky-Pond3, Daniel T. MacLeod1, Yolanda Lie4, Paul Algate5, Dennis R. Burton2, Terri Wrin4, Po-Ying Chan-Hui5, Pascal Poignard1,2, The IAVI Protocol C Investigators & The IAVI African HIV Research Network 1

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Jinal N. Bhiman , Nicole A. Doria-Rose , Constantinos Kurt Wibmer1,2, Daniel J. Sheward4, Carolyn Williamson4,5, Salim S. Abdool Karim5, Peter D. Kwong3, John R. Mascola3, Lynn Morris1,2,5, Penny L. Moore1,2,5 1

Centre for HIV and STIs, National Institute for Communicable Diseases of the National Health Laboratory Services, Johannesburg, South Africa, 2School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa, 3Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, South Africa, 4Institute of Infectious Diseases and Molecular Medicine, Division of Medical Virology, University of Cape Town and NHLS, Cape Town, South Africa, 5 Centre for the AIDS Programme of Research in South Africa (CAPRISA), University of KwaZulu Natal, Durban, South Africa Background: A family of 12 anti-V1V2 monoclonal antibodies (mAbs) was previously isolated from an HIV-1 superinfected individual, CAP256. This lineage emerged 30–34 weeks post-

The International AIDS Vaccine Initiative, Neutralizing Antibody Center, La Jolla, CA, United States, 2The Scripps Research Institute, Department of Immunology and Microbial Sciences, La Jolla, CA, United States, 3University of California San Diego, Department of Medicine, La Jolla, CA, United States, 4Monogram Biosciences Inc, San Francisco, CA, United States, 5Theraclone Sciences Inc., Seattle, WA, United States Background: Designing a vaccine capable of eliciting broadly neutralizing antibodies (bNAbs) to HIV requires a better understanding of these Ab maturation pathways as they occur by interplay with HIV envelope glycoprotein evolution. IAVI Protocol C is a large longitudinal cohort of primary HIV-1 infection in sub-Saharan Africa. The development of bNAb responses was evaluated in 385 Protocol C donors and individuals with bNAb specificities targeting diverse highly conserved epitopes were selected for mAb isolation and maturation studies. Methods: Donor PC64 serum neutralizing activity was mapped to a N160-glycan-dependent, kifunensine-sensitive V1V2 quarternary epitope. The PG9-like neutralizing activity was detectable as early as 18 months post infection (mpi), peaked at 36 mpi and waned slightly during 2 years of additional follow-up. Envelope

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glycoprotein genes from PC64 were cloned at 10 different time points between 1 and 48 mpi. High throughput B-cell activation and functional screening were used to isolate mAbs with PG9-like neutralizing activity at 6 time points post infection. Deep sequencing of memory B-cells from longitudinal PBMCs samples was performed using the MiSeq (Illumina) platform. Results: Ten related mAbs with PG9-like neutralizing activity were isolated from the month-36 sample. The Abs use the VH315*01 and VK3-20*01 genes and possess long CDRH3s (25 amino acids) that contain tyrosine residues. Deep sequencing of 12 longitudinal samples spanning 4 years of infection suggested the emergence of the lineage around 9 mpi, identifying a sequence with 99.66% nucleotide identity to germline. Analysis of Env sequences showed evidence of escape mutations and an increase in diversity after 12 mpi, likely a consequence of selection pressure from the PG9-like lineage. Conclusions: Additional Ab isolated from different time points are currently being characterized. The study will provide important insight regarding the maturation pathways of bNAbs and critical information for vaccine design.

OA12.03 Development of Broadly Neutralizing Anti-HIV-1 Antibodies during Natural Infection through Early Epitope Acquisition and Subsequent Maturation D Noah Sather1, Sara Carbonetti1, Delphine Malherbe2, Franco Pissani3, Andrew B. Stuart1, Ann J. Hessell2, Spyros Kalams4, Nancy L. Haigwood2, Leonidas Stamatatos1 1 Seattle Biomedical Research Institute, Seattle, WA, United States, 2Oregon National Primate Research Center, Beaverton, OR, United States, 3Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, MD, United States, 4 Vanderbilt University, Nashville, TN, United States

Background: Approximately 20–30% of HIV-1 infected subjects develop broadly neutralizing antibodies (bNAbs), which are prototypes for the types of antibodies that will likely need to be elicited by a successful HIV-1 vaccine. Delineating the key early events that lead to the development of bNAbs during infection may help guide the development of immunogens and vaccine regimens to prevent HIV-1 infection. Methods: We followed two HIV-1 positive subjects from before they developed bNAb activity until several years after breadth was detected in the plasma. We studies viral Envelope evolution over the course of infection and studied its relationship to the development of bNAbs in early infection. Results: Both subjects developed bNAb activity one year post infection, which ultimately mapped to the membrane proximal external region (MPER) and the CD4 receptor binding site. For one subject, we were able to identify anti-MPER activity in the earliest plasma sample that exhibited no bNAb activity, indicating that this epitope specificity was acquired very early on, but that these antibodies initially were not able to mediate neutralization. Escape mutations within the bNAb epitopes did not arise in the circulating envelopes until bNAb activity was detectable in the plasma, indicating that this early response was not sufficient to drive viral escape. Finally, we found that global anti-Envelope binding avidity significantly increased at the time bNAbs developed, indicating that antibody maturation was a key step. Conclusions: We report one potential mechanism by which bNAbs develop during natural infection in which an epitope target is acquired very early on during the course of infection.

After sufficient time and antibody maturation, the response matures to acquire broadly neutralizing activity, forcing the virus to escape. These findings reinforce a key role for antibody maturation in the development of bNAbs, and highlight the necessity of developing vaccine regimens that are able to drive somatic hypermutation.

OA12.04 Investigating Epitope Exposure on Native Trimers Sergey Menis1, Chris Carrico2, Dan Kulp1, William Schief 1,3 1

The Scripps Research Institute, Department of Immunology and Microbial Sciences, La Jolla, CA, United States, 2Buck Institute for Research on Aging, Novato, CA, United States, 3 Ragon Institute of MGH, MIT and Harvard, Boston, MA, United States Background: HIV infection typically generates autologous but not broadly neutralizing antibodies, presumably due to immunodominance of strain-specific epitopes on the native spike. Consistent with this view, it has been reported that rabbit immunization with the BG505 SOSIP soluble trimer, a faithful mimic of the native spike, induces potent BG505-neutralizing but not broadly neutralizing responses. We sought to employ the known structure of the BG505 SOSIP trimer to investigate the exposure of variable and conserved epitopes on the native spike. Methods: We computationally built a large ensemble of glycosylated BG505 SOSIP trimers with a variety of backbone and glycan conformations to approximate the motions of the native trimer. We then identified potential antibody epitopes in each member of the ensemble in an unbiased and exhaustive manner. Finally, we computed the frequency at which each position in the trimer is observed in an epitope. This frequency represents a quantitative estimate of the exposure to antibody for any particular position. Results: Antibodies with common CDR lengths access epitopes in the variable regions (V1, V2, V4, V5) considerably more frequently (220 times more frequently) than in the conserved CD4binding-site (CD4bs). Glycosylation is the primary culprit: in the absence of glycans, the variable regions are accessed only 7 times more frequently than the CD4bs. Dominant V5-directed responses may further reduce responses to conserved CD4bs epitopes, by competition. Increased CDRH3 length allows antibody access to hidden epitopes but also increases access to variable epitopes. Conclusions: Variable epitopes in V1, V2, V4, V5 are predicted to be immunodominant on native spikes, purely on the basis of exposure. Antibodies against these epitopes likely inhibit induction of broadly neutralizing antibodies by trimer immunogens. Our analysis may guide design of trimers and immunization regimens for more favorable immune responses.

OA12.05 An Inflammatory Profile that Predicts the Development of Neutralizing Antibody Breadth Anne Sophie Dugast1, Kelly Arnold2, Michelle Hoffner1, Florencia Pereyra1, Michael Seaman3, Bruce Walker1, Douglas Lauffenburger2, Galit Alter1 1

Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, United States, 2MIT, Department of Biological Engineering, Cambridge, MA, United States, 3Center for Virology and Vaccine Research, Boston, MA, United States

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