SummaryThe development of stabilized recombinant HIV envelope trimers that mimic the virion surface molecule has increased enthusiasm for a neutralizing antibody (nAb)-based HIV vaccine. However, there is limited experience with recombinant trimers as immunogens in nonhuman primates, which are typically used as a model for humans. Here, we tested multiple immunogens and immunization strategies head-to-head to determine their impact on the quantity, quality, and kinetics of autologous tier 2 nAb development. A bilateral, adjuvanted, subcutaneous immunization protocol induced reproducible tier 2 nAb responses after only two immunizations 8 weeks apart, and these were further enhanced by a third immunization with BG505 SOSIP trimer. We identified immunogens that minimized non-neutralizing V3 responses and demonstrated that continuous immunogen delivery could enhance nAb responses. nAb responses were strongly associated with germinal center reactions, as assessed by lymph node fine needle aspiration. This study provides a framework for preclinical and clinical vaccine studies targeting nAb elicitation.

Highlights d Slow delivery immunization enhances HIV neutralizing antibody development in monkeys d Slow delivery immunization alters immunodominance of the responding B cells d Weekly longitudinal germinal center (GC) B and T FH analyses provides new GC insights d High-resolution rhesus immunoglobulin locus genomic reference sequence

SUMMARY Generating Tier 2 HIV neutralizing antibody (nAb) responses by immunization remains a challenging problem, and the immunological barriers to induction of such responses with Env immunogens remain unclear. Here, some rhesus monkeys developed autologous Tier 2 nAbs upon HIV Env trimer immunization (SOSIP.v5.2), while others did not. This was not because HIV Env trimers were immunologically silent, as all monkeys made similar ELISA-binding antibody responses; the key difference was nAb versus non-nAb responses. We explored immunological barriers to HIV nAb responses by combining a suite of techniques, including longitudinal lymph node fine needle aspirates. Unexpectedly, nAb development best correlated with booster immunization GC B cell magnitude and Tfh characteristics of the Env-specific CD4 T cells. Notably, these factors distinguished between successful and unsuccessful antibody responses, as GC B cell frequencies and stoichiometry to GC Tfh cells correlated with nAb development but did not correlate with total Env Ab binding titers.

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A range of current candidate AIDS vaccine regimens are focused on generating protective HIV neutralizing antibody responses. Many of these efforts rely on the rhesus macaque animal model. Understanding how protective antibody responses develop and how to increase their efficacy are both major knowledge gaps. Germinal centers (GC) are the engines of antibody affinity maturation. GC T follicular helper (GC Tfh) CD4 T cells are required for GCs. Studying vaccine-specific GC Tfh cells after protein immunizations has been challenging, as antigen-specific GC Tfh cells are difficult to identify by conventional intracellular cytokine staining (ICS). Cytokine production by GC Tfh cells may be intrinsically limited in comparison to other T helper effector cells, as the biological role of a GC Tfh cell is to provide help to individual B cells within the GC, rather than secreting large amounts of cytokines bathing a tissue. To test this idea, we developed a cytokine-independent method to identify antigen-specific GC Tfh cells. RNAseq was performed using TCR stimulated GC Tfh cells to identify candidate markers. Validation experiments determined CD25 (IL2Rα) and OX40 to be highly upregulated activation induced markers (AIM) on the surface of GC Tfh cells after stimulation. In comparison to ICS, the AIM assay identified > 10-fold more antigen-specific GC Tfh cells in HIV Env protein immunized macaques (BG505 SOSIP). CD4 T cells in blood were also studied. In sum, AIM demonstrates that antigen-specific GC Tfh cells are intrinsically stingy producers of cytokines, which is likely an essential part of their biological function.

34The induction of broad and potent immunity by vaccines is the key focus of research efforts 35 aimed at protecting against HIV-1 infection. Soluble native-like HIV-1 envelope glycoproteins 36 have shown promise as vaccine candidates as they can induce potent autologous neutralizing 37 responses in rabbits and non-human primates. In this study, monoclonal antibodies were isolated 38 and characterized from rhesus macaques immunized with the BG505 SOSIP.664 trimer to better 39 understand vaccine-induced antibody responses. Our studies reveal a diverse landscape of 40 antibodies recognizing immunodominant strain-specific epitopes and non-neutralizing neo-41 epitopes. Additionally, we isolated a subset of mAbs against an epitope cluster at the gp120-gp41 42 interface that recognize the highly conserved fusion peptide and the glycan at position 88 and 43 have characteristics akin to several human-derived broadly neutralizing antibodies. 44 (nucleotide level) was 6.4% (range: 2.1%-10.2%) with an average HC complementarity-126 determining region 3 (CDR-H3) length of 15 amino acids (aa) (range: 7-23) ( Table S2). The rh1987 127 KC mAbs utilized HC variable genes from the IGHV3 and IGHV4 families and predominantly used 128 KC V genes from the IGKV1 family (Table S2). All of the rh1987 KC mAbs had a CDR-L3 length of 9 129 aa and their average KC SHM (nucleotide level) was 4.7% (range: 2.6%-6.0%) (Table S2). A single 130 clonal family with 4 members (RM19A) was detected among rh1987 KC mAbs with members 131 isolated from both week 22 and week 25 samples (Table S2). The rh1987 LC mAbs used HC V 132 genes from the IGHV1, IGHV3 and IGHV4 families and LC V genes mainly from the IGLV2 gene 133 family ( Table S2). The rh1987 LC mAbs had an average CDR-L3 length of 10 aa (range: 9-11) with 134 an average LC SHM (nucleotide level) of 3.8% (range: 0.9%-10.6%) (Table S2). Two clonal families 135

Pre-existing immunity to adenovirus (Ad) reduces the efficacy of Ad-based vaccines. The goal of this study was to define the prevalence, magnitude, functionality and phenotype of Ad-specific human T cells directly ex vivo. To study the magnitude of T-cell responses to Ad, we developed a highly reproducible whole Ad vector stimulation assay for use with polychromatic flow cytometry. Ad-specific CD4 + and CD8 + T cell were detected in all 17 human subjects tested and were capable of proliferating upon restimulation. Ad-specific CD4 + T cells were primarily monofunctional CD4 + T cells that produced IL-2, IFN-γ or TNFα and expressed the memory markers CD27 and CD45RO. In contrast, Ad5-specific CD8 + T cells were more polyfunctional, expressing effector-like combinations of IFN-γ, MIP1α and perforin, and generally lacked CD27 and CD45RO expression. Ad-specific CD4 + and CD8 + T cell responses against chimpanzee-derived AdC6 and AdC7 were found in all subjects, indicating the commonality of cross-serotype reactivity of Ad-specific T cells. This cross-reactivity is due in part to extensive CD4 + and CD8 + T cell recognition of hexon regions conserved between multiple Ad serotypes. The prevalence, cross reactivity and effector like functions of Ad-specific T-cells in humans may affect the efficacy of Ad vector-based vaccines by eliminating vector infected cells even when rare serotype Ad vectors are employed.

The mechanisms underlying possible increased HIV-1 acquisition in adenovirus 5 (Ad5)-seropositive subjects vaccinated with Ad5-HIV-1 vectors in the Merck STEP trial remain unclear. We find Ad5 serostatus does not predict Ad5-specific CD4+ T-cell frequency, and no durable significant differences in Ad5-specific CD4+ T-cells between Ad5-seropositive and seronegative subjects were observed following vaccination. These findings indicate no causative role for Ad5-specific CD4+ T-cells in increasing HIV-1 susceptibility in the STEP trial.