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.

The Immune Response to Mycobacterium tuberculosis in HIV-1-Coinfected Persons.

Globally, about 36.7 million people were living with HIV infection at the end of 2015. The most frequent infection co-occurring with HIV-1 is Mycobacterium tuberculosis-374,000 deaths per annum are attributable to HIV-tuberculosis, 75% of those occurring in Africa. HIV-1 infection increases the risk of tuberculosis by a factor of up to 26 and alters its clinical presentation, complicates diagnosis and treatment, and worsens outcome. Although HIV-1-induced depletion of CD4+ T cells underlies all these effects, more widespread immune deficits also contribute to susceptibility and pathogenesis. These defects present a challenge to understand and ameliorate, but also an opportunity to learn and optimize mechanisms that normally protect people against tuberculosis. The most effective means to prevent and ameliorate tuberculosis in HIV-1-infected people is antiretroviral therapy, but this may be complicated by pathological immune deterioration that in turn requires more effective host-directed anti-inflammatory therapies to be derived.

Selection of epigenetically privileged HIV-1 proviruses during treatment with panobinostat and interferon-α2a.

CD4+ T cells with latent HIV-1 infection persist despite treatment with antiretroviral agents and represent the main barrier to a cure of HIV-1 infection. Pharmacological disruption of viral latency may expose HIV-1-infected cells to host immune activity, but the clinical efficacy of latency-reversing agents for reducing HIV-1 persistence remains to be proven. Here, we show in a randomized-controlled human clinical trial that the histone deacetylase inhibitor panobinostat, when administered in combination with pegylated interferon-α2a, induces a structural transformation of the HIV-1 reservoir cell pool, characterized by a disproportionate overrepresentation of HIV-1 proviruses integrated in ZNF genes and in chromatin regions with reduced H3K27ac marks, the molecular target sites for panobinostat. By contrast, proviruses near H3K27ac marks were actively selected against, likely due to increased susceptibility to panobinostat. These data suggest that latency-reversing treatment can increase the immunological vulnerability of HIV-1 reservoir cells and accelerate the selection of epigenetically privileged HIV-1 proviruses.

Exploiting Mucosal Immunity for Antiviral Vaccines.

Mucosal surfaces provide a remarkably effective barrier against potentially dangerous pathogens. Therefore, enhancing mucosal immunity through vaccines-strengthening that first line of defense-holds significant promise for reducing the burden of viral diseases. The large and varied class of viral pathogens, however, continues to present thorny challenges to vaccine development. Two primary difficulties exist: Viruses exhibit a stunning diversity of strategies for evading the host immune response, and even when we understand the nature of effective immune protection against a given virus, eliciting that protection is technically challenging. Only a few mucosal vaccines have surmounted these obstacles thus far. Recent developments, however, could greatly improve vaccine design. In this review, we first sketch out our understanding of mucosal immunity and then compare the herpes simplex virus, human immunodeficiency virus, and influenza virus to illustrate the distinct challenges of developing successful vaccines and to outline potential solutions.

RetroCHMP3 blocks budding of enveloped viruses without blocking cytokinesis.

Many enveloped viruses require the endosomal sorting complexes required for transport (ESCRT) pathway to exit infected cells. This highly conserved pathway mediates essential cellular membrane fission events, which restricts the acquisition of adaptive mutations to counteract viral co-option. Here, we describe duplicated and truncated copies of the ESCRT-III factor CHMP3 that block ESCRT-dependent virus budding and arose independently in New World monkeys and mice. When expressed in human cells, these retroCHMP3 proteins potently inhibit release of retroviruses, paramyxoviruses, and filoviruses. Remarkably, retroCHMP3 proteins have evolved to reduce interactions with other ESCRT-III factors and have little effect on cellular ESCRT processes, revealing routes for decoupling cellular ESCRT functions from viral exploitation. The repurposing of duplicated ESCRT-III proteins thus provides a mechanism to generate broad-spectrum viral budding inhibitors without blocking highly conserved essential cellular ESCRT functions.

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.

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.

Regulation of HIV-1 Gag-Pol Expression by Shiftless, an Inhibitor of Programmed -1 Ribosomal Frameshifting.

Programmed -1 ribosomal frameshifting (-1PRF) is a widely used translation recoding mechanism. HIV-1 expresses Gag-Pol protein from the Gag-coding mRNA through -1PRF, and the ratio of Gag to Gag-Pol is strictly maintained for efficient viral replication. Here, we report that the interferon-stimulated gene product C19orf66 (herein named Shiftless) is a host factor that inhibits the -1PRF of HIV-1. Shiftless (SFL) also inhibited the -1PRF of a variety of mRNAs from both viruses and cellular genes. SFL interacted with the -1PRF signal of target mRNA and translating ribosomes and caused premature translation termination at the frameshifting site. Downregulation of translation release factor eRF3 or eRF1 reduced SFL-mediated premature translation termination. We propose that SFL binding to target mRNA and the translating ribosome interferes with the frameshifting process. These findings identify SFL as a broad-spectrum inhibitor of -1PRF and help to further elucidate the mechanisms of -1PRF.

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.

NONO Detects the Nuclear HIV Capsid to Promote cGAS-Mediated Innate Immune Activation.

Detection of viruses by innate immune sensors induces protective antiviral immunity. The viral DNA sensor cyclic GMP-AMP synthase (cGAS) is necessary for detection of HIV by human dendritic cells and macrophages. However, synthesis of HIV DNA during infection is not sufficient for immune activation. The capsid protein, which associates with viral DNA, has a pivotal role in enabling cGAS-mediated immune activation. We now find that NONO is an essential sensor of the HIV capsid in the nucleus. NONO protein directly binds capsid with higher affinity for weakly pathogenic HIV-2 than highly pathogenic HIV-1. Upon infection, NONO is essential for cGAS activation by HIV and cGAS association with HIV DNA in the nucleus. NONO recognizes a conserved region in HIV capsid with limited tolerance for escape mutations. Detection of nuclear viral capsid by NONO to promote DNA sensing by cGAS reveals an innate strategy to achieve distinction of viruses from self in the nucleus.

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.

Single-cell multiomics reveals persistence of HIV-1 in expanded cytotoxic T cell clones.

Understanding the drivers and markers of clonally expanding HIV-1-infected CD4+ T cells is essential for HIV-1 eradication. We used single-cell ECCITE-seq, which captures surface protein expression, cellular transcriptome, HIV-1 RNA, and TCR sequences within the same single cell to track clonal expansion dynamics in longitudinally archived samples from six HIV-1-infected individuals (during viremia and after suppressive antiretroviral therapy) and two uninfected individuals, in unstimulated conditions and after CMV and HIV-1 antigen stimulation. Despite antiretroviral therapy, persistent antigen and TNF responses shaped T cell clonal expansion. HIV-1 resided in Th1-polarized, antigen-responding T cells expressing BCL2 and SERPINB9 that may resist cell death. HIV-1 RNA+ T cell clones were larger in clone size, established during viremia, persistent after viral suppression, and enriched in GZMB+ cytotoxic effector memory Th1 cells. Targeting HIV-1-infected cytotoxic CD4+ T cells and drivers of clonal expansion provides another direction for HIV-1 eradication.

Vaccination induces maturation in a mouse model of diverse unmutated VRC01-class precursors to HIV-neutralizing antibodies with >50% breadth.

Vaccine elicitation of broadly neutralizing antibodies (bnAbs) is a key HIV-research goal. The VRC01 class of bnAbs targets the CD4-binding site on the HIV-envelope trimer and requires extensive somatic hypermutation (SHM) to neutralize effectively. Despite substantial progress, vaccine-induced VRC01-class antibodies starting from unmutated precursors have exhibited limited neutralization breadth, particularly against viruses bearing glycan on loop D residue N276 (glycan276), present on most circulating strains. Here, using sequential immunization of immunoglobulin (Ig)-humanized mice expressing diverse unmutated VRC01-class antibody precursors, we elicited serum responses capable of neutralizing viruses bearing glycan276 and isolated multiple lineages of VRC01-class bnAbs, including two with >50% breadth on a 208-strain panel. Crystal structures of representative bnAbs revealed the same mode of recognition as known VRC01-class bnAbs. Structure-function studies further pinpointed key mutations and correlated their induction with specific immunizations. VRC01-class bnAbs can thus be matured by sequential immunization from unmutated ancestors to >50% breadth, and we delineate immunogens and regimens inducing key SHM.

Recognition of HIV-1 capsid by PQBP1 licenses an innate immune sensing of nascent HIV-1 DNA.

We have previously described polyglutamine-binding protein 1 (PQBP1) as an adapter required for the cyclic GMP-AMP synthase (cGAS)-mediated innate response to the human immunodeficiency virus 1 (HIV-1) and other lentiviruses. Cytoplasmic HIV-1 DNA is a transient and low-abundance pathogen-associated molecular pattern (PAMP), and the mechanism for its detection and verification is not fully understood. Here, we show a two-factor authentication strategy by the innate surveillance machinery to selectively respond to the low concentration of HIV-1 DNA, while distinguishing these species from extranuclear DNA molecules. We find that, upon HIV-1 infection, PQBP1 decorates the intact viral capsid, and this serves as a primary verification step for the viral nucleic acid cargo. As reverse transcription and capsid disassembly initiate, cGAS is recruited to the capsid in a PQBP1-dependent manner. This positions cGAS at the site of PAMP generation and sanctions its response to a low-abundance DNA PAMP.

Epitranscriptomic addition of m6A regulates HIV-1 RNA stability and alternative splicing.

Previous work has demonstrated that the epitranscriptomic addition of m6A to viral transcripts can promote the replication and pathogenicity of a wide range of DNA and RNA viruses, including HIV-1, yet the underlying mechanisms responsible for this effect have remained unclear. It is known that m6A function is largely mediated by cellular m6A binding proteins or readers, yet how these regulate viral gene expression in general, and HIV-1 gene expression in particular, has been controversial. Here, we confirm that m6A addition indeed regulates HIV-1 RNA expression and demonstrate that this effect is largely mediated by the nuclear m6A reader YTHDC1 and the cytoplasmic m6A reader YTHDF2. Both YTHDC1 and YTHDF2 bind to multiple distinct and overlapping sites on the HIV-1 RNA genome, with YTHDC1 recruitment serving to regulate the alternative splicing of HIV-1 RNAs. Unexpectedly, while YTHDF2 binding to m6A residues present on cellular mRNAs resulted in their destabilization as previously reported, YTHDF2 binding to m6A sites on HIV-1 transcripts resulted in a marked increase in the stability of these viral RNAs. Thus, YTHDF2 binding can exert diametrically opposite effects on RNA stability, depending on RNA sequence context.

Broad and Potent Neutralizing Antibodies Recognize the Silent Face of the HIV Envelope.

Broadly neutralizing antibodies (bNAbs) against HIV-1 envelope (Env) inform vaccine design and are potential therapeutic agents. We identified SF12 and related bNAbs with up to 62% neutralization breadth from an HIV-infected donor. SF12 recognized a glycan-dominated epitope on Env's silent face and was potent against clade AE viruses, which are poorly covered by V3-glycan bNAbs. A 3.3Å cryo-EM structure of a SF12-Env trimer complex showed additional contacts to Env protein residues by SF12 compared with VRC-PG05, the only other known donor-derived silentface antibody, explaining SF12's increased neutralization breadth, potency, and resistance to Env mutation routes. Asymmetric binding of SF12 was associated with distinct N-glycan conformations across Env protomers, demonstrating intra-Env glycan heterogeneity. Administrating SF12 to HIV-1-infected humanized mice suppressed viremia and selected for viruses lacking the N448gp120 glycan. Effective bNAbs can therefore be raised against HIV-1 Env's silent face, suggesting their potential for HIV-1 prevention, therapy, and vaccine development.

Vaccination with Glycan-Modified HIV NFL Envelope Trimer-Liposomes Elicits Broadly Neutralizing Antibodies to Multiple Sites of Vulnerability.

The elicitation of broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope glycoprotein (Env) trimer remains a major vaccine challenge. Most cross-conserved protein determinants are occluded by self-N-glycan shielding, limiting B cell recognition of the underlying polypeptide surface. The exceptions to the contiguous glycan shield include the conserved receptor CD4 binding site (CD4bs) and glycoprotein (gp)41 elements proximal to the furin cleavage site. Accordingly, we performed heterologous trimer-liposome prime:boosting in rabbits to drive B cells specific for cross-conserved sites. To preferentially expose the CD4bs to B cells, we eliminated proximal N-glycans while maintaining the native-like state of the cleavage-independent NFL trimers, followed by gradual N-glycan restoration coupled with heterologous boosting. This approach successfully elicited CD4bs-directed, cross-neutralizing Abs, including one targeting a unique glycan-protein epitope and a bNAb (87% breadth) directed to the gp120:gp41 interface, both resolved by high-resolution cryoelectron microscopy. This study provides proof-of-principle immunogenicity toward eliciting bNAbs by vaccination.

Isolation and Structure of an Antibody that Fully Neutralizes Isolate SIVmac239 Reveals Functional Similarity of SIV and HIV Glycan Shields.

HIV- and SIV-envelope (Env) trimers are both extensively glycosylated, and antibodies identified to date have been unable to fully neutralize SIVmac239. Here, we report the isolation, structure, and glycan interactions of antibody ITS90.03, a monoclonal antibody that completely neutralized the highly neutralization-resistant isolate, SIVmac239. The co-crystal structure of a fully glycosylated SIVmac239-gp120 core in complex with rhesus CD4 and the antigen-binding fragment of ITS90.03 at 2.5-Å resolution revealed that ITS90 recognized an epitope comprised of 45% glycan. SIV-gp120 core, rhesus CD4, and their complex could each be aligned structurally to their human counterparts. The structure revealed that glycans masked most of the SIV Env protein surface, with ITS90 targeting a glycan hole, which is occupied in ∼83% of SIV strains by glycan N238. Overall, the SIV glycan shield appears to functionally resemble its HIV counterpart in coverage of spike, shielding from antibody, and modulation of receptor accessibility.