Stabilized trimeric peptide immunogens of the complete HIV-1 gp41 N-heptad repeat and their use as HIV-1 vaccine candidates.

Efforts to develop an HIV-1 vaccine include those focusing on conserved structural elements as the target of broadly neutralizing monoclonal antibodies. MAb D5 binds to a highly conserved hydrophobic pocket on the gp41 N-heptad repeat (NHR) coiled coil and neutralizes through prevention of viral fusion and entry. Assessment of 17-mer and 36-mer NHR peptides presenting the D5 epitope in rodent immunogenicity studies showed that the longer peptide elicited higher titers of neutralizing antibodies, suggesting that neutralizing epitopes outside of the D5 pocket may exist. Although the magnitude and breadth of neutralization elicited by NHR-targeting antigens are lower than that observed for antibodies directed to other epitopes on the envelope glycoprotein complex, it has been shown that NHR-directed antibodies are potentiated in TZM-bl cells containing the FcγRI receptor. Herein, we report the design and evaluation of covalently stabilized trimeric 51-mer peptides encompassing the complete gp41 NHR. We demonstrate that these peptide trimers function as effective antiviral entry inhibitors and retain the ability to present the D5 epitope. We further demonstrate in rodent and nonhuman primate immunization studies that our 51-mer constructs elicit a broader repertoire of neutralizing antibody and improved cross-clade neutralization of primary HIV-1 isolates relative to 17-mer and 36-mer NHR peptides in A3R5 and FcγR1-enhanced TZM-bl assays. These results demonstrate that sensitive neutralization assays can be used for structural enhancement of moderately potent neutralizing epitopes. Finally, we present expanded trimeric peptide designs which include unique low-molecular-weight scaffolds that provide versatility in our immunogen presentation strategy.

Structure-guided stabilization improves the ability of the HIV-1 gp41 hydrophobic pocket to elicit neutralizing antibodies.

The hydrophobic pocket found in the N-heptad repeat (NHR) region of HIV-1 gp41 is a highly conserved epitope that is the target of various HIV-1-neutralizing monoclonal antibodies. Although the high conservation of the pocket makes it an attractive vaccine candidate, it has been challenging to elicit potent anti-NHR antibodies via immunization. Here, we solved a high-resolution structure of the NHR mimetic IQN17, and, consistent with previous ligand-bound gp41 pocket structures, we observed remarkable conformational plasticity of the pocket. The high malleability of this pocket led us to test whether we could improve the immunogenicity of the gp41 pocket by stabilizing its conformation. We show that the addition of five amino acids at the C terminus of IQN17, to generate IQN22, introduces a stabilizing salt bridge at the base of the peptide that rigidifies the pocket. Mice immunized with IQN22 elicited higher avidity antibodies against the gp41 pocket and a more potent, albeit still weak, neutralizing response against HIV-1 compared with IQN17. Stabilized epitope-focused immunogens could serve as the basis for future HIV-1 fusion-inhibiting vaccines.

Commonly Elicited Antibodies against the Base of the HIV-1 Env Trimer Guide the Population-Level Evolution of a Structure-Regulating Region in gp41.

The antibody response against the HIV-1 envelope glycoproteins (Envs) guides evolution of this protein within each host. Whether antibodies with similar target specificities are elicited in different individuals and affect the population-level evolution of Env is poorly understood. To address this question, we analyzed properties of emerging variants in the gp41 fusion peptide-proximal region (FPPR) that exhibit distinct evolutionary patterns in HIV-1 clade B. For positions 534, 536, and 539 in the FPPR, alanine was the major emerging variant. However, 534A and 536A show a constant frequency in the population between 1979 and 2016, whereas 539A is gradually increasing. To understand the basis for these differences, we introduced alanine substitutions in the FPPR of primary HIV-1 strains and examined their functional and antigenic properties. Evolutionary patterns could not be explained by fusion competence or structural stability of the emerging variants. Instead, 534A and 536A exhibited modest but significant increases in sensitivity to antibodies against the membrane-proximal external region (MPER) and gp120-gp41 interface. These Envs were also more sensitive to poorly neutralizing sera from HIV-1-infected individuals than the clade ancestral form or 539A variant. Competition binding assays confirmed for all sera tested the presence of antibodies against the base of the Env trimer that compete with monoclonal antibodies targeting the MPER and gp120-gp41 interface. Our findings suggest that weakly neutralizing antibodies against the trimer base are commonly elicited; they do not exert catastrophic population size reduction effects on emerging variants but, instead, determine their set point frequencies in the population and historical patterns of change. IMPORTANCE Infection by HIV-1 elicits formation of antibodies that target the viral Env proteins and can inactivate the virus. The specific targets of these antibodies vary among infected individuals. It is unclear whether some target specificities are shared among the antibody responses of different individuals. We observed that antibodies against the base of the Env protein are commonly elicited during infection. The selective pressure applied by such antibodies is weak. As a result, they do not completely eliminate the sensitive forms of the virus from the population, but maintain their frequency at a low level that has not increased since the beginning of the AIDS pandemic. Interestingly, the changes in Env do not occur at the sites targeted by the antibodies, but at a distinct region of Env, the fusion peptide-proximal region, which regulates their exposure.

Global Increases in Human Immunodeficiency Virus Neutralization Sensitivity Due to Alterations in the Membrane-Proximal External Region of the Envelope Glycoprotein Can Be Minimized by Distant State 1-Stabilizing Changes.

Binding to the receptor, CD4, drives the pretriggered, "closed" (State-1) conformation of the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer ([gp120/gp41]3) into more "open" conformations. HIV-1 Env on the viral membrane is maintained in a State-1 conformation that resists binding and neutralization by commonly elicited antibodies. Premature triggering of Env before the virus engages a target cell typically leads to increased susceptibility to spontaneous inactivation or ligand-induced neutralization. Here, we showed that single amino acid substitutions in the gp41 membrane-proximal external region (MPER) of a primary HIV-1 strain resulted in viral phenotypes indicative of premature triggering of Env to downstream conformations. Specifically, the MPER changes reduced viral infectivity and globally increased virus sensitivity to poorly neutralizing antibodies, soluble CD4, a CD4-mimetic compound, and exposure to cold. In contrast, the MPER mutants exhibited decreased sensitivity to the State 1-preferring inhibitor, BMS-806, and to the PGT151 broadly neutralizing antibody. Depletion of cholesterol from virus particles did not produce the same State 1-destabilizing phenotypes as MPER alterations. Notably, State 1-stabilizing changes in Env distant from the MPER could minimize the phenotypic effects of MPER alteration but did not affect virus sensitivity to cholesterol depletion. Thus, membrane-proximal gp41 elements contribute to the maintenance of the pretriggered Env conformation. The conformationally disruptive effects of MPER changes can be minimized by distant State 1-stabilizing Env modifications, a strategy that may be useful in preserving the native pretriggered state of Env. IMPORTANCE The pretriggered shape of the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) is a major target for antibodies that can neutralize many strains of the virus. An effective HIV-1 vaccine may need to raise these types of antibodies, but this goal has proven difficult. One reason is that the pretriggered shape of Env is unstable and dependent on interactions near the viral membrane. Here, we showed that the membrane-proximal external region (MPER) of Env plays an important role in maintaining Env in a pretriggered shape. Alterations in the MPER resulted in global changes in Env conformation that disrupted its pretriggered shape. We also found that these disruptive effects of MPER changes could be minimized by distant Env modifications that stabilized the pretriggered shape. These modifications may be useful for preserving the native shape of Env for structural and vaccine studies.

The TZM-bl Reporter Cell Line Expresses Kynureninase That Can Neutralize 2F5-like Antibodies in the HIV-1 Neutralization Assay.

Induction of broadly neutralizing antibodies targeting ectodomain of the transmembrane (TM) glycoprotein gp41 HIV-1 provides a basis for the development of a universal anti-viral vaccine. The HeLa cell-derived TZM-bl reporter cell line is widely used for the estimation of lentiviruses neutralization by immune sera. The cell line is highly permissive to infection by most strains of HIV, SIV, and SHIV. Here we demonstrated that TZM-bl cells express a 48 kDa non-glycosylated protein (p48) recognized by broadly neutralizing monoclonal antibody (mAb) 2F5 targeting the ELDKWA (aa 669-674) epitope of gp41TM of HIV-1. A significant amount of p48 was found in the cell supernatant. The protein was identified as human kynureninase (KYNU), which has the ELDKWA epitope. The protein is further called "p48 KYNU". The HIV-1 neutralization by mAb 2F5 and 4E10 in the presence of p48KYNU was tested on Jurkat and TZM-bl cells. It was demonstrated that p48KYNU reduces neutralization by 2F5-like antibodies, but it has almost no effect on mAb 4E10. Therefore, p48KYNU can attenuate HIV-1 neutralization by 2F5-like antibodies and hence create false-negative results. Thus, previously tested immune sera that recognized the ELDKWA-epitope and demonstrated a "weak neutralization" of HIV-1 in TZM-bl assay should be reevaluated.

Internalization of HIV-1 by Phagocytes Is Increased When Virions Are Opsonized with Multimeric Antibody in the Presence of Complement.

The low abundance of envelope spikes and the inability of IgG to aggregate virions render HIV-1 an inadequate target for antibody-mediated clearance by phagocytes. In an attempt to improve the ability of antibody to mediate the internalization of HIV-1 virions, we generated multimers of the broadly neutralizing HIV-1-specific monoclonal antibody (MAb) VRC01 using site-directed mutagenesis of the Fc segment. We then measured virion internalization using primary human monocytes and neutrophils. We found that, in the absence of complement, immune complexes consisting of HIV-1 virions and VRC01 multimers were slightly more efficiently internalized than were complexes formed with monomeric VRC01. The presence of complement, however, greatly augmented internalization of immune complexes formed with the multimeric MAb but had little impact on monomeric MAb-mediated internalization. Multimerization and the presence of complement overcome the limited ability of monomeric antibody to mediate internalization of HIV-1 virions and may thus provide a therapeutic approach to clearing virus. IMPORTANCE Antibody-mediated internalization of HIV-1 by phagocytes, a potential mechanism for clearing virus, is very inefficient. In an effort to improve viral clearance, we produced a multimeric form of the broadly neutralizing monoclonal antibody VRC01. We found that VRC01 antibody multimers (primarily hexamers) were only slightly more efficient in mediating HIV-1 internalization than was monomeric VRC01. However, the addition of complement resulted in substantially greater internalization of multimer-opsonized virus. In contrast, complement had little if any impact on internalization of monomer-opsonized virus. Therefore, antibody multimerization in combination with complement may overcome the limited ability of monomeric antibody to mediate internalization of HIV-1 virions. Our findings may provide a therapeutic approach to clearing virus.

SOSIP Trimer-Specific Antibodies Isolated from a Simian-Human Immunodeficiency Virus-Infected Monkey with versus without a Pre-blocking Step with gp41.

BG505 SOSIP.664 (hereafter referred to as SOSIP), a stabilized trimeric mimic of the HIV-1 envelope spike resembling the native viral spike, is a useful tool for isolating anti-HIV-1 neutralizing antibodies. We screened long-term SHIV-AD8 infected rhesus monkeys for potency and breadth of serum neutralizing activity against autologous and heterologous viruses: SHIV-AD8, HIV-1 YU2, HIV-1 JR-CSF, and HIV-1 NL4-3. Monkey rh2436 neutralized all viruses tested and showed strong reactivity to the SOSIP trimer, suggesting this was a promising candidate for attempts at monoclonal antibody (MAb) isolation. MAbs were isolated by performing single B-cell sorts from peripheral blood mononuclear cells (PBMC) by FACS using the SOSIP trimer as a probe. An initial round of sorted cells revealed the majority of isolated MAbs were directed to the gp41 external domain portion of the SOSIP trimer and were mostly non-neutralizing against tested isolates. A second sort was performed, introducing a gp41 blocking step prior to PBMC staining and FACS sorting. These isolated MAbs bound SOSIP trimer but were no longer directed to the gp41 external domain portion. A significantly higher proportion of MAbs with neutralizing activity were obtained with this strategy. Our data show this pre-blocking step with gp41 greatly increases the yield of non-gp41-reactive, SOSIP-specific MAbs and increases the likelihood of isolating MAbs with neutralizing activity. IMPORTANCE Recent advancements in the field have focused on the isolation and use of broadly neutralizing antibodies for both prophylaxis and therapy. Finding a useful probe to isolate broad potent neutralizing antibodies while avoiding non-neutralizing antibodies is important. The SOSIP trimer has been shown to be a great tool for this purpose because it binds known broadly neutralizing antibodies. However, the SOSIP trimer can isolate non-neutralizing antibodies as well, including gp41-specific MAbs. Introducing a pre-blocking step with gp41 recombinant protein decreased the percent of gp41-specific antibodies isolated with SOSIP probe, as well as increased the number of neutralizing antibodies isolated. This method can be used as a tool to increase the chances of isolating neutralizing antibodies.

Cell membrane-anchored anti-HIV single-chain antibodies and bifunctional inhibitors targeting the gp41 fusion protein: new strategies for HIV gene therapy.

Emerging studies indicate that infusion of HIV-resistant cells could be an effective strategy to achieve a sterilizing or functional cure. We recently reported that glycosylphosphatidylinositol (GPI)-anchored nanobody or a fusion inhibitory peptide can render modified cells resistant to HIV-1 infection. In this study, we comprehensively characterized a panel of newly isolated HIV-1-neutralizing antibodies as GPI-anchored inhibitors. Fusion genes encoding the single-chain variable fragment (scFv) of 3BNC117, N6, PGT126, PGT128, 10E8, or 35O22 were constructed with a self-inactivating lentiviral vector, and they were efficiently expressed in the lipid raft sites of target cell membrane without affecting the expression of HIV-1 receptors (CD4, CCR5 and CXCR4). Significantly, transduced cells exhibited various degrees of resistance to cell-free HIV-1 infection and cell-associated HIV-1 transmission, as well as viral Env-mediated cell-cell fusion, with the cells modified by GPI-10E8 showing the most potent and broad anti-HIV activity. In mechanism, GPI-10E8 also interfered with the processing of viral Env in transduced cells and attenuated the infectivity of progeny viruses. By genetically linking 10E8 with a fusion inhibitor peptide, we subsequently designed a group of eight bifunctional constructs as cell membrane-based inhibitors, designated CMI01∼CMI08, which rendered cells completely resistant to HIV-1, HIV-2, and simian immunodeficiency virus (SIV). In human CD4+ T cells, GPI-10E8 and its bifunctional derivatives blocked both CCR5- and CXCR4-tropic HIV-1 isolates efficiently, and the modified cells displayed robust survival selection under HIV-1 infection. Therefore, our studies provide new strategies for generating HIV-resistant cells, which can be used alone or with other gene therapy approaches.

The SARS CoV-2 spike directed non-neutralizing polyclonal antibodies cross-react with Human immunodeficiency virus (HIV-1) gp41.

Cross-reactivity among the two diverse viruses is believed to originate from the concept of antibodies recognizing similar epitopes on the two viral surfaces. Cross-reactive antibody responses have been seen in previous variants of SARS and SARS-CoV-2, but little is known about the cross reactivity with other similar RNA viruses like HIV-1. In the present study, we examined the reactivity the SARS-CoV-2 directed antibodies, via spike, immunized mice sera and demonstrated whether they conferred any cross-reactive neutralization against HIV-1. Our findings show that SARS-CoV-2 spike immunized mice antibodies cross-react with the HIV-1 Env protein. Cross-neutralization among the two viruses is uncommon, suggesting the presence of a non-neutralizing antibody response to conserved epitopes amongst the two viruses. Our results indicate, that SARS-CoV-2 spike antibody cross reactivity is targeted towards the gp41 region of the HIV-1 Env (gp160) protein. Overall, our investigation not only answers a crucial question about the understanding of cross-reactive epitopes of antibodies generated in different viral infections, but also provides critical evidence for developing vaccine immunogens and novel treatment strategies with enhanced efficacy capable of recognising diverse pathogens with similar antigenic features.

Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens.

HIV-1 vaccine immunofocusing strategies may be able to induce broadly-reactive neutralizing antibodies (NAbs). Here, we engineered a panel of diverse, membrane-resident native HIV-1 trimers vulnerable to two broad targets-the V2 apex and fusion peptide (FP). Selection criteria included i) high expression and ii) infectious function, so that trimer neutralization sensitivity can be profiled in pseudovirus (PV) assays. Initially, we boosted the expression of 17 candidate trimers by truncating gp41 and introducing a gp120-gp41 SOS disulfide to prevent gp120 shedding. "Repairs" were made to fill glycan holes and eliminate other strain-specific aberrations. A new neutralization assay allowed PV infection when our standard assay was insufficient. Trimers with exposed V3 loops, a target of non-NAbs, were discarded. To try to increase V2-sensitivity, we removed clashing glycans and modified the C-strand. Notably, a D167N mutation improved V2-sensitivity in several cases. Glycopeptide analysis of JR-FL trimers revealed near complete sequon occupation and that filling the N197 glycan hole was well-tolerated. In contrast, sequon optimization and inserting/removing glycans at other positions frequently had global "ripple" effects on glycan maturation and sequon occupation throughout the gp120 outer domain and gp41. V2 MAb CH01 selectively bound to trimers with small high mannose glycans near the base of the V1 loop, thereby avoiding clashes. Knocking in a rare N49 glycan was found to perturb gp41 glycans, increasing FP NAb sensitivity-and sometimes improving expression. Finally, a biophysical analysis of VLPs revealed that i) ~25% of particles bear Env spikes, ii) spontaneous particle budding is high and only increases 4-fold upon Gag transfection, and iii) Env+ particles express ~30-40 spikes. Taken together, we identified 7 diverse trimers with a range of sensitivities to two targets to allow rigorous testing of immunofocusing vaccine concepts.

Antibody Conjugates for Targeted Therapy Against HIV-1 as an Emerging Tool for HIV-1 Cure.

Although advances in antiretroviral therapy (ART) have significantly improved the life expectancy of people living with HIV-1 (PLWH) by suppressing HIV-1 replication, a cure for HIV/AIDS remains elusive. Recent findings of the emergence of drug resistance against various ART have resulted in an increased number of treatment failures, thus the development of novel strategies for HIV-1 cure is of immediate need. Antibody-based therapy is a well-established tool in the treatment of various diseases and the engineering of new antibody derivatives is expanding the realms of its application. An antibody-based carrier of anti-HIV-1 molecules, or antibody conjugates (ACs), could address the limitations of current HIV-1 ART by decreasing possible off-target effects, reduce toxicity, increasing the therapeutic index, and lowering production costs. Broadly neutralizing antibodies (bNAbs) with exceptional breadth and potency against HIV-1 are currently being explored to prevent or treat HIV-1 infection in the clinic. Moreover, bNAbs can be engineered to deliver cytotoxic or immune regulating molecules as ACs, further increasing its therapeutic potential for HIV-1 cure. ACs are currently an important component of anticancer treatment with several FDA-approved constructs, however, to date, no ACs are approved to treat viral infections. This review aims to outline the development of AC for HIV-1 cure, examine the variety of carriers and payloads used, and discuss the potential of ACs in the current HIV-1 cure landscape.

HIV-1 Envelope Conformation, Allostery, and Dynamics.

The HIV-1 envelope glycoprotein (Env) mediates host cell fusion and is the primary target for HIV-1 vaccine design. The Env undergoes a series of functionally important conformational rearrangements upon engagement of its host cell receptor, CD4. As the sole target for broadly neutralizing antibodies, our understanding of these transitions plays a critical role in vaccine immunogen design. Here, we review available experimental data interrogating the HIV-1 Env conformation and detail computational efforts aimed at delineating the series of conformational changes connecting these rearrangements. These studies have provided a structural mapping of prefusion closed, open, and transition intermediate structures, the allosteric elements controlling rearrangements, and state-to-state transition dynamics. The combination of these investigations and innovations in molecular modeling set the stage for advanced studies examining rearrangements at greater spatial and temporal resolution.

Neutralizing Antibodies Induced by First-Generation gp41-Stabilized HIV-1 Envelope Trimers and Nanoparticles.

The immunogenicity of gp41-stabilized HIV-1 BG505 envelope (Env) trimers and nanoparticles (NPs) was recently assessed in mice and rabbits. Here, we combined Env-specific B-cell sorting and repertoire sequencing to identify neutralizing antibodies (NAbs) from immunized animals. A panel of mouse NAbs was isolated from mice immunized with a 60-meric I3-01 NP presenting 20 stabilized trimers. Three mouse NAbs potently neutralized BG505.T332N by recognizing a glycan epitope centered in the C3/V4 region on BG505 Env, as revealed by electron microscopy (EM), X-ray crystallography, and epitope mapping. A set of rabbit NAbs was isolated from rabbits immunized with a soluble trimer and a 24-meric ferritin NP presenting 8 trimers. Neutralization assays against BG505.T332N variants confirmed that potent rabbit NAbs targeted previously described glycan holes on BG505 Env and accounted for a significant portion of the autologous NAb response in both the trimer and ferritin NP groups. Last, we examined NAb responses that were induced by non-BG505 Env immunogens. We determined a 3.4-Å-resolution crystal structure for the clade C transmitted/founder (T/F) Du172.17 Env with a redesigned heptad repeat 1 (HR1) bend in gp41. This clade C Env, in a soluble trimer form and in a multivalent form with 8 trimers attached to ferritin NP, and the gp41-stabilized clade A Q482-d12 Env trimer elicited distinct NAb responses in rabbits, with notable differences in neutralization breadth. Although eliciting a broad NAb response remains a major challenge, our study provides valuable information on an HIV-1 vaccine design strategy that combines gp41 stabilization and NP display. IMPORTANCE Self-assembling protein nanoparticles (NPs) presenting BG505 envelope (Env) trimers can elicit tier 2 HIV-1-neutralizing antibody (NAb) responses more effectively than soluble trimers. In the present study, monoclonal NAbs were isolated from previously immunized mice and rabbits for structural and functional analyses, which revealed that potent mouse NAbs recognize the C3/V4 region and small NP-elicited rabbit NAbs primarily target known glycan holes on BG505 Env. This study validates the gp41 stabilization strategy for HIV-1 Env vaccine design and highlights the challenge in eliciting a broad NAb response.

A Derivative of the D5 Monoclonal Antibody That Targets the gp41 N-Heptad Repeat of HIV-1 with Broad Tier-2-Neutralizing Activity.

HIV-1 infection is initiated by the viral glycoprotein Env, which, after interaction with cellular coreceptors, adopts a transient conformation known as the prehairpin intermediate (PHI). The N-heptad repeat (NHR) is a highly conserved region of gp41 exposed in the PHI; it is the target of the FDA-approved drug enfuvirtide and of neutralizing monoclonal antibodies (mAbs). However, to date, these mAbs have only been weakly effective against tier-1 HIV-1 strains, which are most sensitive to neutralizing antibodies. Here, we engineered and tested 11 IgG variants of D5, an anti-NHR mAb, by recombining previously described mutations in four of D5's six antibody complementarity-determining regions. One variant, D5_AR, demonstrated 6-fold enhancement in the 50% inhibitory dose (ID50) against lentivirus pseudotyped with HXB2 Env. D5_AR exhibited weak cross-clade neutralizing activity against a diverse set of tier-2 HIV-1 viruses, which are less sensitive to neutralizing antibodies than tier-1 viruses and are the target of current antibody-based vaccine efforts. In addition, the neutralization potency of D5_AR IgG was greatly enhanced in target cells expressing FcγRI, with ID50 values of <0.1 µg/ml; this immunoglobulin receptor is expressed on macrophages and dendritic cells, which are implicated in the early stages of HIV-1 infection of mucosal surfaces. D5 and D5_AR have equivalent neutralization potency in IgG, Fab, and single-chain variable-fragment (scFv) formats, indicating that neutralization is not impacted by steric hindrance. Taken together, these results provide support for vaccine strategies that target the PHI by eliciting antibodies against the gp41 NHR and support investigation of anti-NHR mAbs in nonhuman primate passive immunization studies. IMPORTANCE Despite advances in antiretroviral therapy, HIV remains a global epidemic and has claimed more than 32 million lives. Accordingly, developing an effective HIV vaccine remains an urgent public health need. The gp41 N-heptad repeat (NHR) of the HIV-1 prehairpin intermediate (PHI) is highly conserved (>90%) and is inhibited by the FDA-approved drug enfuvirtide, making it an attractive vaccine target. However, to date, anti-NHR antibodies have not been potent. Here, we engineered D5_AR, a more potent variant of the anti-NHR antibody D5, and established its ability to inhibit HIV-1 strains that are more difficult to neutralize and are more representative of circulating strains (tier-2 strains). The neutralizing activity of D5_AR was greatly potentiated in cells expressing FcγRI; FcγRI is expressed on cells that are implicated at the earliest stages of sexual HIV-1 transmission. Taken together, these results bolster efforts to target the gp41 NHR and the PHI for vaccine development.

Membrane Env Liposomes Facilitate Immunization with Multivalent Full-Length HIV Spikes.

A major goal of HIV vaccine design is to elicit broadly neutralizing antibodies (bNAbs). Such bNAbs target HIV's trimeric, membrane-embedded envelope glycoprotein spikes (mEnv). Soluble Env (sEnv) trimers have been used as vaccines, but engineering sEnvs for stability, multivalency, and desired antigenicity is problematic and deletes key neutralizing epitopes on glycoprotein 41 (gp41) while creating neoepitopes that elicit unwanted antibodies. Meanwhile, multivalent mEnv vaccines are challenging to develop due to trimer instability and low mEnv copy number amid other extraneous proteins on virus-like particles. Here, we describe a multivalent mEnv vaccine platform that does not require protein engineering or extraneous proteins. mEnv trimers were fixed, purified, and combined with naked liposomes in mild detergent. On removal of detergent, mEnv spikes were observed embedded in liposome particles (mean diameter, 133 nm) in correct orientation. These particles were recognized by HIV bNAbs and not non-NAbs and are designated mEnv liposomes (MELs). Following a sequential immunization scheme in rabbits, MELs elicited antibodies that neutralized tier 2 HIV isolates. Analysis of serum antibody specificities, including those to epitopes involving a missing conserved N-glycosylation site at position 197 near the CD4 binding site on two of the immunogens, provides clues on how NAb responses can be improved with modified immunogens. In sum, MELs are a biochemically defined platform that enables rational immunization strategies to elicit HIV bNAbs using multimerized mEnv. IMPORTANCE A vaccine that induced broadly neutralizing antibodies against HIV would likely end the AIDS pandemic. Such antibodies target membrane-embedded envelope glycoprotein spikes (mEnv) that HIV uses to enter cells. Due to HIV Env's low expression and instability, soluble stabilized Env trimers have been used as vaccine candidates, but these have an altered base that disrupts targets of HIV broadly neutralizing antibodies that bind near the membrane and are not available for all HIV isolates. Here, we describe membrane Env liposomes (MELs) that display a multivalent array of stable mEnvs on liposome particles. MELs showed the expected antibody recognition properties, including targeting parts of mEnv missing on soluble Envs. Immunization with MELs elicited antibodies that neutralized diverse HIV isolates. The MEL platform facilitates vaccine development with potentially any HIV Env at high valency, and a similar approach may be useful for eliciting antibodies to membrane-embedded targets of therapeutic interest.

Structure of HIV-1 gp41 with its membrane anchors targeted by neutralizing antibodies.

The HIV-1 gp120/gp41 trimer undergoes a series of conformational changes in order to catalyze gp41-induced fusion of viral and cellular membranes. Here, we present the crystal structure of gp41 locked in a fusion intermediate state by an MPER-specific neutralizing antibody. The structure illustrates the conformational plasticity of the six membrane anchors arranged asymmetrically with the fusion peptides and the transmembrane regions pointing into different directions. Hinge regions located adjacent to the fusion peptide and the transmembrane region facilitate the conformational flexibility that allows high-affinity binding of broadly neutralizing anti-MPER antibodies. Molecular dynamics simulation of the MPER Ab-stabilized gp41 conformation reveals a possible transition pathway into the final post-fusion conformation with the central fusion peptides forming a hydrophobic core with flanking transmembrane regions. This suggests that MPER-specific broadly neutralizing antibodies can block final steps of refolding of the fusion peptide and the transmembrane region, which is required for completing membrane fusion.

Antibody response against selected epitopes in the HIV-1 envelope gp41 ectodomain contributes to reduce viral burden in HIV-1 infected patients.

The ectodomain of gp41 is the target of potent binding and neutralizing antibodies (NAbs) and is being explored in new strategies for antibody-based HIV vaccines. Previous studies have suggested that the W164A-3S (3S) and EC26-2A4 (EC26) peptides located in the gp41 ectodomain may be potential HIV vaccine candidates. We assessed 3S- and EC26-specific binding antibody responses and related neutralizing activity in a large panel of chronic HIV-1-infected Portuguese individuals on ART. A similar proportion of participants had antibodies binding to 3S (9.6%) and EC26 (9.9%) peptides but the level of reactivity against 3S was significantly higher compared to EC26, except in the rare patients with double peptide reactivity. The higher antigenicity of 3S was unrelated with disease stage, as assessed by CD4+ T cell counts, but it was directly related with plasma viral load. Most patients that were tested (89.9%, N = 268) showed tier 1 neutralizing activity, the potency being inversely associated with plasma viral load. In the subset of patients that were tested for neutralization of tier 2 isolates, neutralization breadth was inversely correlated with plasma viral load and directly correlated with CD4+ T cell counts. These results are consistent with a role for neutralizing antibodies in controlling viral replication and preventing the decline of CD4+ T lymphocytes. Importantly, in patients with 3S-specific antibodies, neutralizing titers were inversely correlated with viral RNA levels and proviral DNA levels. Moreover, patients with 3S and/or EC26-specific antibodies showed a 1.9-fold higher tier 2 neutralization score than patients without antibodies suggesting that 3S and/or EC26-specific antibodies contribute to neutralization breadth and potency in HIV-1 infected patients. Overall, these results suggest that antibodies targeting the S3 and EC26 epitopes may contribute to reduce viral burden and provide further support for the inclusion of 3S and EC26 epitopes in HIV-1 vaccine candidates.

The high-affinity immunoglobulin receptor FcγRI potentiates HIV-1 neutralization via antibodies against the gp41 N-heptad repeat.

The HIV-1 gp41 N-heptad repeat (NHR) region of the prehairpin intermediate, which is transiently exposed during HIV-1 viral membrane fusion, is a validated clinical target in humans and is inhibited by the Food and Drug Administration (FDA)-approved drug enfuvirtide. However, vaccine candidates targeting the NHR have yielded only modest neutralization activities in animals; this inhibition has been largely restricted to tier-1 viruses, which are most sensitive to neutralization by sera from HIV-1-infected individuals. Here, we show that the neutralization activity of the well-characterized NHR-targeting antibody D5 is potentiated >5,000-fold in TZM-bl cells expressing FcγRI compared with those without, resulting in neutralization of many tier-2 viruses (which are less susceptible to neutralization by sera from HIV-1-infected individuals and are the target of current antibody-based vaccine efforts). Further, antisera from guinea pigs immunized with the NHR-based vaccine candidate (ccIZN36)3 neutralized tier-2 viruses from multiple clades in an FcγRI-dependent manner. As FcγRI is expressed on macrophages and dendritic cells, which are present at mucosal surfaces and are implicated in the early establishment of HIV-1 infection following sexual transmission, these results may be important in the development of a prophylactic HIV-1 vaccine.

The CH1α domain of mucosal gp41 IgA contributes to antibody specificity and antiviral functions in HIV-1 highly exposed Sero-Negative individuals.

The antibody molecule comprises a variable domain conferring antigen specificity and affinity distinct from the heavy chain constant (CH) domains dictating effector functions. We here interrogate this paradigm by evaluating the unique influence of the CH1α domain on epitope specificity and functions using two mucosal gp41-specific Fab-IgAs (FabA) derived from HIV-1 highly-exposed but persistently seronegative individuals (HESN). These HESN develop selectively affinity-matured HIV-1-specific mucosal IgA that target the gp41 viral envelope and might provide protection although by unclear mechanisms. Isotype-switching FabAs into Fab-IgGs (FabGs) results in a >10-fold loss in affinity for HIV-1 clade A, B, and C gp41, together with reduced neutralization of HIV-1 cross-clade. The FabA conformational epitopes map selectively on gp41 in 6-Helix bundle and pre-fusion conformations cross-clade, unlike FabGs. Finally, we designed in silico, a 12 amino-acid peptide recapitulating one FabA conformational epitope that inhibits the FabA binding to gp41 cross-clade and its neutralizing activity. Altogether, our results reveal that the CH1α domain shapes the antibody paratope through an allosteric effect, thereby strengthening the antibody specificity and functional activities. Further, they clarify the mechanisms by which these HESN IgAs might confer protection against HIV-1-sexual acquisition. The IgA-specific epitope we characterized by reverse vaccinology could help designing a mucosal HIV-1 vaccine.

Neutralizing Antibodies Targeting HIV-1 gp41.

HIV-1 vaccine research has obtained an enormous boost since the discovery of many broadly neutralizing antibodies (bnAbs) targeting all accessible sites on the HIV-1 envelope glycoprotein (Env). This in turn facilitated high-resolution structures of the Env glycoprotein in complex with bnAbs. Here we focus on gp41, its highly conserved heptad repeat region 1 (HR1), the fusion peptide (FP) and the membrane-proximal external region (MPER). Notably, the broadest neutralizing antibodies target MPER. Both gp41 HR1 and MPER are only fully accessible once receptor-induced conformational changes have taken place, although some studies suggest access to MPER in the close to native Env conformation. We summarize the data on the structure and function of neutralizing antibodies targeting gp41 HR1, FP and MPER and we review their access to Env and their complex formation with gp41 HR1, MPER peptides and FP within native Env. We further discuss MPER bnAb binding to lipids and the role of somatic mutations in recognizing a bipartite epitope composed of the conserved MPER sequence and membrane components. The problematic of gp41 HR1 access and MPER bnAb auto- and polyreactivity is developed in the light of inducing such antibodies by vaccination.