Recent efforts toward an HIV vaccine focus on inducing broadly neutralizing antibodies, but eliciting both neutralizing antibodies (nAbs) and cellular responses may be superior. Here, we immunized macaques with an HIV envelope trimer, either alone to induce nAbs, or together with a heterologous viral vector regimen to elicit nAbs and cellular immunity, including CD8 + tissue-resident memory T cells. After ten vaginal challenges with autologous virus, protection was observed in both vaccine groups at 53.3% and 66.7%, respectively. A nAb titer >300 was generally associated with protection but in the heterologous viral vector + nAb group, titers <300 were sufficient. In this group, protection was durable as the animals resisted six more challenges 5 months later. Antigen stimulation of T cells in ex vivo vaginal tissue cultures triggered antiviral responses in myeloid and CD4 + T cells. We propose that cellular immune responses reduce the threshold of nAbs required to confer superior and durable protection.

Our previous work has shown that antigens adjuvanted with ligands specific for Toll-like receptor 4 (TLR4) and TLR7/8 encapsulated in poly(lactic-co-glycolic) acid (PLGA)-based nanoparticles (NPs) induce robust and durable immune responses in mice and macaques. We investigated the efficacy of these NP adjuvants in inducing protective immunity against simian immunodeficiency virus (SIV). Rhesus macaques (RMs) were immunized with NPs containing TLR4 and TLR7/8 agonists mixed with soluble recombinant SIVmac239-derived envelope (Env) gp140 and Gag p55 (protein) or with virus-like particles (VLPs) containing SIVmac239 Env and Gag. NPadjuvanted vaccines induced robust innate responses, antigen-specific antibody responses of a greater magnitude and persistence, and enhanced plasmablast responses compared to those achieved with alum-adjuvanted vaccines. NP-adjuvanted vaccines induced antigen-specific, long-lived plasma cells (LLPCs), which persisted in the bone marrow for several months after vaccination. NP-adjuvanted vaccines induced immune responses that were associated with enhanced protection against repeated low-dose, intravaginal challenges with heterologous SIVsmE660 in animals that carried TRIM5␣ restrictive alleles. The protection induced by immunization with protein-NP correlated with the prechallenge titers of Env-specific IgG antibodies in serum and vaginal secretions. However, no such correlate was apparent for immunization with VLP-NP or alum as the adjuvant. Transcriptional profiling of peripheral blood mononuclear cells isolated within the first few hours to days after primary vaccination revealed that NP-adjuvanted vaccines induced a molecular signature similar to that induced by the live attenuated yellow fever viral vaccine. This systems approach identified early blood transcriptional signatures that correlate with Envspecific antibody responses in vaginal secretions and protection against infection. These results demonstrate the adjuvanticity of the NP adjuvant in inducing persis-

Stabilized HIV-1 envelope (Env) trimers elicit tier 2 autologous neutralizing antibody (nAb) responses in immunized animals. We previously demonstrated that BG505 SOSIP.664.T332N gp140 (BG505 SOSIP) immunization of rhesus macaques (RM) provided robust protection against autologous intra-vaginal simian-human immunodeficiency virus (SHIV) challenge that was predicted by high serum nAb titers. Here, we show that nAb in these protected RM targeted a glycan hole proximal to residue 465 in gp120 in all cases. nAb also targeted another glycan hole at residues 241/289 and an epitope in V1 at varying frequencies. Non-neutralizing antibodies directed at N611-shielded epitopes in gp41 were also present but were more prevalent in RM with low nAb titers. Longitudinal analysis demonstrated that nAb broadened in some RM during sequential immunization but remained focused in others, the latter being associated with increases in nAb titer. Thirty-eight monoclonal antibodies (mAbs) isolated from a protected RM with an exceptionally high serum neutralization titer bound to the trimer in ELISA, and four of the mAbs potently neutralized the BG505 Env pseudovirus (PV) and SHIV. The four neutralizing mAbs were clonally related and targeted the 465 glycan hole to varying degrees, mimicking the serum. The data demonstrate that the C3/465 glycan hole cluster was the dominant neutralization target in high titer protected RM, despite other co-circulating neutralizing and non-neutralizing specificities. The isolation of a neutralizing mAb family argues that clonotype expansion occurred during BG505 SOSIP immunization, leading to high titer, protective nAb and setting a desirable benchmark for HIV vaccines.

A recent study of plasma neutralization breadth in HIV-1 infected individuals at nine International AIDS Vaccine Initiative (IAVI) sites reported that viral load, HLA-A*03 genotype, and subtype C infection were strongly associated with the development of neutralization breadth. Here, we refine the findings of that study by analyzing the impact of the transmitted/founder (T/F) envelope (Env), early Env diversification, and autologous neutralization on the development of plasma neutralization breadth in 21 participants identified during recent infection at two of those sites: Kigali, Rwanda (n = 9) and Lusaka, Zambia (n = 12). Single-genome analysis of full-length T/F Env sequences revealed that all 21 individuals were infected with a highly homogeneous population of viral variants, which were categorized as subtype C (n = 12), A1 (n = 7), or recombinant AC (n = 2). An extensive amino acid sequence-based analysis of variable loop lengths and glycosylation patterns in the T/F Envs revealed that a lower ratio of NXS to NXT-encoded glycan motifs correlated with neutralization breadth. Further analysis comparing amino acid sequence changes, insertions/deletions, and glycan motif alterations between the T/F Env and autologous early Env variants revealed that extensive diversification focused in the V2, V4, and V5 regions of gp120, accompanied by contemporaneous viral escape, significantly favored the development of breadth. These results suggest that more efficient glycosylation of subtype A and C T/F Envs through fewer NXS-encoded glycan sites is more likely to elicit antibodies that can transition from autologous to heterologous neutralizing activity following exposure to gp120 diversification. This initiates an Env-antibody co-evolution cycle that increases neutralization breadth, and is further augmented over time by additional viral and host factors. These findings suggest that understanding how variation in the efficiency of site-specific glycosylation influences neutralizing antibody elicitation and targeting could advance the design of immunogens aimed at inducing antibodies that can transition from autologous to heterologous neutralizing activity.

Although the correlates of immunological protection from human immunodeficiency virus or simian immunodeficiency virus infection remain incompletely understood, it is generally believed that medium to high titers of serum neutralizing antibodies (nAbs) against the challenge virus will prevent infection. This paradigm is based on a series of studies in which passive transfer of HIV-specific nAbs protected rhesus macaques (RMs) from subsequent mucosal challenge with a chimeric human/simian immunodeficiency virus. However, it is unknown whether nAb titers define protection in the setting of active immunization. Here we determined serum nAb titers against breakthrough transmitted/founder (T/F) SIVsmE660-derived envelope glycoprotein (Env) variants from 14 RMs immunized with SIVmac239-based DNA-prime/modified vaccinia virus Ankara-boost vaccine regimens that included GM-CSF or CD40L adjuvants and conferred significant but incomplete protection against repeated low-dose intrarectal challenge. A single Env variant established infection in all RMs except one, with no identifiable genetic signature associated with vaccination breakthrough compared with T/F Envs from four unvaccinated monkeys. Breakthrough T/F Env pseudoviruses were potently neutralized in vitro by heterologous pooled serum from chronically SIVsmE660-infected monkeys at IC50 titers exceeding 1:1,000,000. Remarkably, the T/F Env pseudoviruses from 13 of 14 monkeys were also susceptible to neutralization by autologous prechallenge serum at in vitro IC50 titers ranging from 1:742–1:10,832. These titers were similar to those observed in vaccinated RMs that remained uninfected. These data suggest that the relationship between serum nAb titers and protection from mucosal SIV challenge in the setting of active immunization is more complex than previously recognized, warranting further studies into the balance between immune activation, target cell availability, and protective antibody responses.

Multiple antibody effector functions arise in HIV-1 infection that could be harnessed to protect against infection or clear the persistent reservoir. Here, we have investigated the genetic and functional memory B cell and antibody landscape present during early infection in six individuals infected with either subtype A, C, or an A/C recombinant HIV-1. These individuals demonstrated varying levels of plasma autologous neutralization (nAb) against the transmitted/founder envelope (T/F Env) pseudovirus and non-neutralizing Fc-mediated effector function (nnFc) antibody-dependent cell-mediated cytotoxicity (ADCC) against the T/F Env gp120 protein at ~7 months after infection. Genetic analysis of the immunoglobulin heavy (VH) and light (VL) chain variable domain gene segments from 352 autologous T/F Env gp120-specific single B cells recovered at this same 7-month time-point revealed an over-representation of the VH1-69 germline in five of six individuals. A defining feature of the VH1-69 utilizing gp120-specific antibodies was their significantly more hydrophobic complementarity-determining region-2 (CDRH2) regions compared to other VH CDRH2 sequences from each individual. While none of the VH1-69 antibodies possessed strong neutralizing activity against virions pseudotyped with the autologous T/F Env, almost a third were capable of mediating high ADCC activity, as assayed by intracellular granzyme B activity in CEM.NKr.CCR5 target cells coated with autologous T/F Env gp120. High ADCC mediating VH1-69 antibodies exhibited shorter complementarity-determining region-3 (CDRH3) lengths and a more neutral isoelectric point than antibodies lacking this function. In the individual that developed the highest autologous ADCC responses, the high granzyme B producing antibodies bound to surface expressed envelope in the absence of CD4 and were not enhanced by the addition of soluble CD4. Overall, VH1-69 utilizing antibodies are commonly induced against gp120 in diverse HIV-1 infections and a subset of these antibodies can mediate ADCC functions, serving as a bridge between the innate and adaptive immune response to HIV-1.

Antibodies that can neutralize diverse viral strains are likely to be an important component of a protective human immunodeficiency virus type 1 (HIV-1) vaccine. To this end, preclinical simian immunodeficiency virus (SIV)-based nonhuman primate immunization regimens have been designed to evaluate and enhance antibody-mediated protection. However, these trials often rely on a limited selection of SIV strains with extreme neutralization phenotypes to assess vaccine-elicited antibody activity. To mirror the viral panels used to assess HIV-1 antibody breadth, we created and characterized a novel panel of 14 genetically and phenotypically diverse SIVsm envelope (Env) glycoproteins. To assess the utility of this panel, we characterized the neutralizing activity elicited by four SIVmac239 envelope-expressing DNA/modified vaccinia virus Ankara vector-and protein-based vaccination regimens that included the immunomodulatory adjuvants granulocyte-macrophage colony-stimulating factor, Toll-like receptor (TLR) ligands, and CD40 ligand. The SIVsm Env panel exhibited a spectrum of neutralization sensitivity to SIV-infected plasma pools and monoclonal antibodies, allowing categorization into three tiers. Pooled sera from 91 rhesus macaques immunized in the four trials consistently neutralized only the highly sensitive tier 1a SIVsm Envs, regardless of the immunization regimen. The inability of vaccine-mediated antibodies to neutralize the moderately resistant tier 1b and tier 2 SIVsm Envs defined here suggests that those antibodies were directed toward epitopes that are not accessible on most SIVsm Envs. To achieve a broader and more effective neutralization profile in preclinical vaccine studies that is relevant to known features of HIV-1 neutralization, more emphasis should be placed on optimizing the Env immunogen, as the neutralization profile achieved by the addition of adjuvants does not appear to supersede the neutralizing antibody profile determined by the immunogen. IMPORTANCEMany in the HIV/AIDS vaccine field believe that the ability to elicit broadly neutralizing antibodies capable of blocking genetically diverse HIV-1 variants is a critical component of a protective vaccine. Various SIV-based nonhuman primate vaccine studies have investigated ways to improve antibody-mediated protection against a heterologous SIV challenge, including administering adjuvants that might stimulate a greater neutralization breadth. Using a novel SIV neutralization panel and samples from four rhesus macaque vaccine trials designed for cross comparison, we show that different regimens expressing the same SIV envelope immunogen consistently elicit antibodies that neutralize only the very sensitive tier 1a SIV variants. The results argue that the neutralizing antibody profile elicited by a vaccine is primarily determined by the envelope immunogen and is not substantially broadened by including adjuvants, resulting in the conclusion that the envelope immunogen itself should be the primary consideration in efforts to elicit antibodi...

The goals of preclinical HIV vaccine studies in nonhuman primates are to develop and test different approaches for their ability to generate protective immunity. Here, we compared the impact of 7 different vaccine modalities, all expressing the HIV-1 1086.C clade C envelope (Env), on (i) the magnitude and durability of antigen-specific serum antibody responses and (ii) autologous and heterologous neutralizing antibody capacity. These vaccination regimens included immunization with different combinations of DNA, modified vaccinia virus Ankara (MVA), soluble gp140 protein, and different adjuvants. Serum samples collected from 130 immunized monkeys at two key time points were analyzed using the TZM-bl cell assay: at 2 weeks after the final immunization (week 40/41) and on the day of challenge (week 58). Key initial findings were that inclusion of a gp140 protein boost had a significant impact on the magnitude and durability of Env-specific IgG antibodies, and addition of 3M-052 adjuvant was associated with better neutralizing activity against the SHIV1157ipd3N4 challenge virus and a heterologous HIV-1 CRF01 Env, CNE8. We measured neutralization against a panel of 12 tier 2 Envs using a newly described computational tool to quantify serum neutralization potency by factoring in the predetermined neutralization tier of each reference Env. This analysis revealed modest neutralization breadth, with DNA/MVA immunization followed by gp140 protein boosts in 3M-052 adjuvant producing the best scores. This study highlights that protein-containing regimens provide a solid foundation for the further development of novel adjuvants and inclusion of trimeric Env immunogens that could eventually elicit a higher level of neutralizing antibody breadth. IMPORTANCE Despite much progress, we still do not have a clear understanding of how to elicit a protective neutralizing antibody response against HIV-1 through vaccination. There have been great strides in the development of envelope immunogens that mimic the virus particle, but less is known about how different vaccination modalities and adjuvants contribute to shaping the antibody response. We compared seven different vaccines that were administered to rhesus macaques and that delivered the same envelope protein through various modalities and with different adjuvants. The results demonstrate that some vaccine components are better than others at eliciting neutralizing antibodies with breadth.