Vaccines and Broadly Neutralizing Antibodies for HIV-1 Prevention.

Development of improved approaches for HIV-1 prevention will likely be required for a durable end to the global AIDS pandemic. Recent advances in preclinical studies and early phase clinical trials offer renewed promise for immunologic strategies for blocking acquisition of HIV-1 infection. Clinical trials are currently underway to evaluate the efficacy of two vaccine candidates and a broadly neutralizing antibody (bNAb) to prevent HIV-1 infection in humans. However, the vast diversity of HIV-1 is a major challenge for both active and passive immunization. Here we review current immunologic strategies for HIV-1 prevention, with a focus on current and next-generation vaccines and bNAbs.

Vaccine induction of heterologous HIV-1-neutralizing antibody B cell lineages in humans.

A critical roadblock to HIV vaccine development is the inability to induce B cell lineages of broadly neutralizing antibodies (bnAbs) in humans. In people living with HIV-1, bnAbs take years to develop. The HVTN 133 clinical trial studied a peptide/liposome immunogen targeting B cell lineages of HIV-1 envelope (Env) membrane-proximal external region (MPER) bnAbs (NCT03934541). Here, we report MPER peptide-liposome induction of polyclonal HIV-1 B cell lineages of mature bnAbs and their precursors, the most potent of which neutralized 15% of global tier 2 HIV-1 strains and 35% of clade B strains with lineage initiation after the second immunization. Neutralization was enhanced by vaccine selection of improbable mutations that increased antibody binding to gp41 and lipids. This study demonstrates proof of concept for rapid vaccine induction of human B cell lineages with heterologous neutralizing activity and selection of antibody improbable mutations and outlines a path for successful HIV-1 vaccine development.

Broadly Neutralizing Antibodies to HIV and Their Role in Vaccine Design.

HIV employs multiple means to evade the humoral immune response, particularly the elicitation of and recognition by broadly neutralizing antibodies (bnAbs). Such antibodies can act antivirally against a wide spectrum of viruses by targeting relatively conserved regions on the surface HIV envelope trimer spike. Elicitation of and recognition by bnAbs are hindered by the arrangement of spikes on virions and the relatively difficult access to bnAb epitopes on spikes, including the proximity of variable regions and a high density of glycans. Yet, in a small proportion of HIV-infected individuals, potent bnAb responses do develop, and isolation of the corresponding monoclonal antibodies has been facilitated by identification of favorable donors with potent bnAb sera and by development of improved methods for human antibody generation. Molecular studies of recombinant Env trimers, alone and in interaction with bnAbs, are providing new insights that are fueling the development and testing of promising immunogens aimed at the elicitation of bnAbs.

Affinity gaps among B cells in germinal centers drive the selection of MPER precursors.

Current prophylactic human immunodeficiency virus 1 (HIV-1) vaccine research aims to elicit broadly neutralizing antibodies (bnAbs). Membrane-proximal external region (MPER)-targeting bnAbs, such as 10E8, provide exceptionally broad neutralization, but some are autoreactive. Here, we generated humanized B cell antigen receptor knock-in mouse models to test whether a series of germline-targeting immunogens could drive MPER-specific precursors toward bnAbs. We found that recruitment of 10E8 precursors to germinal centers (GCs) required a minimum affinity for germline-targeting immunogens, but the GC residency of MPER precursors was brief due to displacement by higher-affinity endogenous B cell competitors. Higher-affinity germline-targeting immunogens extended the GC residency of MPER precursors, but robust long-term GC residency and maturation were only observed for MPER-HuGL18, an MPER precursor clonotype able to close the affinity gap with endogenous B cell competitors in the GC. Thus, germline-targeting immunogens could induce MPER-targeting antibodies, and B cell residency in the GC may be regulated by a precursor-competitor affinity gap.

Vaccination induces broadly neutralizing antibody precursors to HIV gp41.

A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features.

Fab-dimerized glycan-reactive antibodies are a structural category of natural antibodies.

Natural antibodies (Abs) can target host glycans on the surface of pathogens. We studied the evolution of glycan-reactive B cells of rhesus macaques and humans using glycosylated HIV-1 envelope (Env) as a model antigen. 2G12 is a broadly neutralizing Ab (bnAb) that targets a conserved glycan patch on Env of geographically diverse HIV-1 strains using a unique heavy-chain (VH) domain-swapped architecture that results in fragment antigen-binding (Fab) dimerization. Here, we describe HIV-1 Env Fab-dimerized glycan (FDG)-reactive bnAbs without VH-swapped domains from simian-human immunodeficiency virus (SHIV)-infected macaques. FDG Abs also recognized cell-surface glycans on diverse pathogens, including yeast and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike. FDG precursors were expanded by glycan-bearing immunogens in macaques and were abundant in HIV-1-naive humans. Moreover, FDG precursors were predominately mutated IgM+IgD+CD27+, thus suggesting that they originated from a pool of antigen-experienced IgM+ or marginal zone B cells.

Broadly neutralizing antiviral antibodies.

A fascinating aspect of viral evolution relates to the ability of viruses to escape the adaptive immune response. The widely held view has been that the great variability of viral glycoproteins would be an absolute obstacle to the development of antibody-based therapies or vaccines that could confer broad and long-lasting protection. In the past five years, new approaches have been developed to interrogate human memory B cells and plasma cells with high efficiency and to isolate several broadly neutralizing antiviral antibodies against highly variable pathogens such as HIV-1 and influenza virus. These antibodies not only provide new tools for prophylaxis and therapy for viral diseases but also identify conserved epitopes that may be used to design new vaccines capable of conferring broader protection.

Restriction of HIV-1 Escape by a Highly Broad and Potent Neutralizing Antibody.

Broadly neutralizing antibodies (bNAbs) represent a promising approach to prevent and treat HIV-1 infection. However, viral escape through mutation of the HIV-1 envelope glycoprotein (Env) limits clinical applications. Here we describe 1-18, a new VH1-46-encoded CD4 binding site (CD4bs) bNAb with outstanding breadth (97%) and potency (GeoMean IC50 = 0.048 µg/mL). Notably, 1-18 is not susceptible to typical CD4bs escape mutations and effectively overcomes HIV-1 resistance to other CD4bs bNAbs. Moreover, mutational antigenic profiling uncovered restricted pathways of HIV-1 escape. Of most promise for therapeutic use, even 1-18 alone fully suppressed viremia in HIV-1-infected humanized mice without selecting for resistant viral variants. A 2.5-Å cryo-EM structure of a 1-18-BG505SOSIP.664 Env complex revealed that these characteristics are likely facilitated by a heavy-chain insertion and increased inter-protomer contacts. The ability of 1-18 to effectively restrict HIV-1 escape pathways provides a new option to successfully prevent and treat HIV-1 infection.

Antibody Lineages with Vaccine-Induced Antigen-Binding Hotspots Develop Broad HIV Neutralization.

The vaccine-mediated elicitation of antibodies (Abs) capable of neutralizing diverse HIV-1 strains has been a long-standing goal. To understand how broadly neutralizing antibodies (bNAbs) can be elicited, we identified, characterized, and tracked five neutralizing Ab lineages targeting the HIV-1-fusion peptide (FP) in vaccinated macaques over time. Genetic and structural analyses revealed two of these lineages to belong to a reproducible class capable of neutralizing up to 59% of 208 diverse viral strains. B cell analysis indicated each of the five lineages to have been initiated and expanded by FP-carrier priming, with envelope (Env)-trimer boosts inducing cross-reactive neutralization. These Abs had binding-energy hotspots focused on FP, whereas several FP-directed Abs induced by immunization with Env trimer-only were less FP-focused and less broadly neutralizing. Priming with a conserved subregion, such as FP, can thus induce Abs with binding-energy hotspots coincident with the target subregion and capable of broad neutralization.

High-Throughput Mapping of B Cell Receptor Sequences to Antigen Specificity.

B cell receptor (BCR) sequencing is a powerful tool for interrogating immune responses to infection and vaccination, but it provides limited information about the antigen specificity of the sequenced BCRs. Here, we present LIBRA-seq (linking B cell receptor to antigen specificity through sequencing), a technology for high-throughput mapping of paired heavy- and light-chain BCR sequences to their cognate antigen specificities. B cells are mixed with a panel of DNA-barcoded antigens so that both the antigen barcode(s) and BCR sequence are recovered via single-cell next-generation sequencing. Using LIBRA-seq, we mapped the antigen specificity of thousands of B cells from two HIV-infected subjects. The predicted specificities were confirmed for a number of HIV- and influenza-specific antibodies, including known and novel broadly neutralizing antibodies. LIBRA-seq will be an integral tool for antibody discovery and vaccine development efforts against a wide range of antigen targets.

Slow Delivery Immunization Enhances HIV Neutralizing Antibody and Germinal Center Responses via Modulation of Immunodominance.

Conventional immunization strategies will likely be insufficient for the development of a broadly neutralizing antibody (bnAb) vaccine for HIV or other difficult pathogens because of the immunological hurdles posed, including B cell immunodominance and germinal center (GC) quantity and quality. We found that two independent methods of slow delivery immunization of rhesus monkeys (RMs) resulted in more robust T follicular helper (TFH) cell responses and GC B cells with improved Env-binding, tracked by longitudinal fine needle aspirates. Improved GCs correlated with the development of >20-fold higher titers of autologous nAbs. Using a new RM genomic immunoglobulin locus reference, we identified differential IgV gene use between immunization modalities. Ab mapping demonstrated targeting of immunodominant non-neutralizing epitopes by conventional bolus-immunized animals, whereas slow delivery-immunized animals targeted a more diverse set of epitopes. Thus, alternative immunization strategies can enhance nAb development by altering GCs and modulating the immunodominance of non-neutralizing epitopes.

The transcription factor CREB1 is a mechanistic driver of immunogenicity and reduced HIV-1 acquisition following ALVAC vaccination.

Development of effective human immunodeficiency virus 1 (HIV-1) vaccines requires synergy between innate and adaptive immune cells. Here we show that induction of the transcription factor CREB1 and its target genes by the recombinant canarypox vector ALVAC + Alum augments immunogenicity in non-human primates (NHPs) and predicts reduced HIV-1 acquisition in the RV144 trial. These target genes include those encoding cytokines/chemokines associated with heightened protection from simian immunodeficiency virus challenge in NHPs. Expression of CREB1 target genes probably results from direct cGAMP (STING agonist)-modulated p-CREB1 activity that drives the recruitment of CD4+ T cells and B cells to the site of antigen presentation. Importantly, unlike NHPs immunized with ALVAC + Alum, those immunized with ALVAC + MF59, the regimen in the HVTN702 trial that showed no protection from HIV infection, exhibited significantly reduced CREB1 target gene expression. Our integrated systems biology approach has validated CREB1 as a critical driver of vaccine efficacy and highlights that adjuvants that trigger CREB1 signaling may be critical for efficacious HIV-1 vaccines.

Identification of Near-Pan-neutralizing Antibodies against HIV-1 by Deconvolution of Plasma Humoral Responses.

Anti-HIV-1 envelope broadly neutralizing monoclonal antibodies (bNAbs) isolated from memory B cells may not fully represent HIV-1-neutralizing profiles measured in plasma. Accordingly, we characterized near-pan-neutralizing antibodies extracted directly from the plasma of two "elite neutralizers." Circulating anti-gp120 polyclonal antibodies were deconvoluted using proteomics to guide lineage analysis of bone marrow plasma cells. In both subjects, a single lineage of anti-CD4-binding site (CD4bs) antibodies explained the plasma-neutralizing activity. Importantly, members of these lineages potently neutralized 89%-100% of a multi-tier 117 pseudovirus panel, closely matching the specificity and breadth of the circulating antibodies. X-ray crystallographic analysis of one monoclonal, N49P7, suggested a unique ability to bypass the CD4bs Phe43 cavity, while reaching deep into highly conserved residues of Layer 3 of the gp120 inner domain, likely explaining its extreme potency and breadth. Further direct analyses of plasma anti-HIV-1 bNAbs should provide new insights for developing antibody-based antiviral agents and vaccines.

RAB11FIP5 Expression and Altered Natural Killer Cell Function Are Associated with Induction of HIV Broadly Neutralizing Antibody Responses.

HIV-1 broadly neutralizing antibodies (bnAbs) are difficult to induce with vaccines but are generated in ∼50% of HIV-1-infected individuals. Understanding the molecular mechanisms of host control of bnAb induction is critical to vaccine design. Here, we performed a transcriptome analysis of blood mononuclear cells from 47 HIV-1-infected individuals who made bnAbs and 46 HIV-1-infected individuals who did not and identified in bnAb individuals upregulation of RAB11FIP5, encoding a Rab effector protein associated with recycling endosomes. Natural killer (NK) cells had the highest differential expression of RAB11FIP5, which was associated with greater dysregulation of NK cell subsets in bnAb subjects. NK cells from bnAb individuals had a more adaptive/dysfunctional phenotype and exhibited impaired degranulation and cytokine production that correlated with RAB11FIP5 transcript levels. Moreover, RAB11FIP5 overexpression modulated the function of NK cells. These data suggest that NK cells and Rab11 recycling endosomal transport are involved in regulation of HIV-1 bnAb development.

Aberrant B cell repertoire selection associated with HIV neutralizing antibody breadth.

A goal of HIV vaccine development is to elicit antibodies with neutralizing breadth. Broadly neutralizing antibodies (bNAbs) to HIV often have unusual sequences with long heavy-chain complementarity-determining region loops, high somatic mutation rates and polyreactivity. A subset of HIV-infected individuals develops such antibodies, but it is unclear whether this reflects systematic differences in their antibody repertoires or is a consequence of rare stochastic events involving individual clones. We sequenced antibody heavy-chain repertoires in a large cohort of HIV-infected individuals with bNAb responses or no neutralization breadth and uninfected controls, identifying consistent features of bNAb repertoires, encompassing thousands of B cell clones per individual, with correlated T cell phenotypes. These repertoire features were not observed during chronic cytomegalovirus infection in an independent cohort. Our data indicate that the development of numerous B cell lineages with antibody features associated with autoreactivity may be a key aspect in the development of HIV neutralizing antibody breadth.

The role of antibodies in HIV vaccines.

Licensed vaccines against viral diseases generate antibodies that neutralize the infecting virus and protect against infection or disease. Similarly, an effective vaccine against HIV-1 will likely need to induce antibodies that prevent initial infection of host cells or that limit early events of viral dissemination. Such antibodies must target the surface envelope glycoproteins of HIV-1, which are highly variable in sequence and structure. The first subunit vaccines to enter clinical trails were safe and immunogenic but unable to elicit antibodies that neutralized most circulating strains of HIV-1. However, potent virus neutralizing antibodies (NAbs) can develop during the course of HIV-1 infection, and this is the type of antibody response that researchers seek to generate with a vaccine. Thus, current vaccine design efforts have focused on a more detailed understanding of these broadly neutralizing antibodies and their epitopes to inform the design of improved vaccines.

Heavy-chain CDR3-engineered B cells facilitate in vivo evaluation of HIV-1 vaccine candidates.

V2-glycan/apex broadly neutralizing antibodies (bnAbs) recognize a closed quaternary epitope of the HIV-1 envelope glycoprotein (Env). This closed structure is necessary to elicit apex antibodies and useful to guide the maturation of other bnAb classes. To compare antigens designed to maintain this conformation, we evaluated apex-specific responses in mice engrafted with a diverse repertoire of B cells expressing the HCDR3 of the apex bnAb VRC26.25. Engineered B cells affinity matured, guiding the improvement of VRC26.25 itself. We found that soluble Env (SOSIP) variants differed significantly in their ability to raise anti-apex responses. A transmembrane SOSIP (SOSIP-TM) delivered as an mRNA-lipid nanoparticle elicited more potent neutralizing responses than multimerized SOSIP proteins. Importantly, SOSIP-TM elicited neutralizing sera from B cells engineered with the predicted VRC26.25-HCDR3 progenitor, which also affinity matured. Our data show that HCDR3-edited B cells facilitate efficient in vivo comparisons of Env antigens and highlight the potential of an HCDR3-focused vaccine approach.

Non-neutralizing Antibodies Alter the Course of HIV-1 Infection In Vivo.

Non-neutralizing antibodies (nnAbs) to HIV-1 show little measurable activity in prevention or therapy in animal models yet were the only correlate of protection in the RV144 vaccine trial. To investigate the role of nnAbs on HIV-1 infection in vivo, we devised a replication-competent HIV-1 reporter virus that expresses a heterologous HA-tag on the surface of infected cells and virions. Anti-HA antibodies bind to, but do not neutralize, the reporter virus in vitro. However, anti-HA protects against infection in humanized mice and strongly selects for nnAb-resistant viruses in an entirely Fc-dependent manner. Similar results were also obtained with tier 2 HIV-1 viruses using a human anti-gp41 nnAb, 246D. While nnAbs are demonstrably less effective than broadly neutralizing antibodies (bNAbs) against HIV-1 in vitro and in vivo, the data show that nnAbs can protect against and alter the course of HIV-1 infection in vivo. PAPERCLIP.

Membrane-bound mRNA immunogens lower the threshold to activate HIV Env V2 apex-directed broadly neutralizing B cell precursors in humanized mice.

Eliciting broadly neutralizing antibodies (bnAbs) is the core of HIV vaccine design. bnAbs specific to the V2-apex region of the HIV envelope acquire breadth and potency with modest somatic hypermutation, making them attractive vaccination targets. To evaluate Apex germline-targeting (ApexGT) vaccine candidates, we engineered knockin (KI) mouse models expressing the germline B cell receptor (BCR) of the bnAb PCT64. We found that high affinity of the ApexGT immunogen for PCT64-germline BCRs was necessary to specifically activate KI B cells at human physiological frequencies, recruit them to germinal centers, and select for mature bnAb mutations. Relative to protein, mRNA-encoded membrane-bound ApexGT immunization significantly increased activation and recruitment of PCT64 precursors to germinal centers and lowered their affinity threshold. We have thus developed additional models for HIV vaccine research, validated ApexGT immunogens for priming V2-apex bnAb precursors, and identified mRNA-LNP as a suitable approach to substantially improve the B cell response.

Human immunoglobulin repertoire analysis guides design of vaccine priming immunogens targeting HIV V2-apex broadly neutralizing antibody precursors.

Broadly neutralizing antibodies (bnAbs) to the HIV envelope (Env) V2-apex region are important leads for HIV vaccine design. Most V2-apex bnAbs engage Env with an uncommonly long heavy-chain complementarity-determining region 3 (HCDR3), suggesting that the rarity of bnAb precursors poses a challenge for vaccine priming. We created precursor sequence definitions for V2-apex HCDR3-dependent bnAbs and searched for related precursors in human antibody heavy-chain ultradeep sequencing data from 14 HIV-unexposed donors. We found potential precursors in a majority of donors for only two long-HCDR3 V2-apex bnAbs, PCT64 and PG9, identifying these bnAbs as priority vaccine targets. We then engineered ApexGT Env trimers that bound inferred germlines for PCT64 and PG9 and had higher affinities for bnAbs, determined cryo-EM structures of ApexGT trimers complexed with inferred-germline and bnAb forms of PCT64 and PG9, and developed an mRNA-encoded cell-surface ApexGT trimer. These methods and immunogens have promise to assist HIV vaccine development.