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Passive transfer of broadly neutralizing anti-HIV-1 antibodies (bNAbs) protects against infection, and therefore, eliciting bNAbs by vaccination is a major goal of HIV-1 vaccine efforts. bNAbs that target the CD4 binding site (CD4bs) on HIV-1 Env are among the most broadly active, but to date, responses elicited against this epitope in vaccinated animals have lacked potency and breadth. We hypothesized that CD4bs bNAbs resembling the antibody IOMA might be easier to elicit than other CD4bs antibodies that exhibit higher somatic mutation rates, a difficult-to-achieve mechanism to accommodate Env's N276 gp120 N-glycan, and rare five-residue light chain complementarity-determining region 3. As an initial test of this idea, we developed IOMA germline-targeting Env immunogens and evaluated a sequential immunization regimen in transgenic mice expressing germline-reverted IOMA. These mice developed CD4bs epitope-specific responses with heterologous neutralization, and cloned antibodies overcame neutralization roadblocks, including accommodating the N276 gp120 glycan, with some neutralizing selected HIV-1 strains more potently than IOMA. The immunization regimen also elicited CD4bs-specific responses in mice containing polyclonal antibody repertoires as well as rabbits and rhesus macaques. Thus, germline targeting of IOMA-class antibody precursors represents a potential vaccine strategy to induce CD4bs bNAbs.

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 N448 gp120 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., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Broadly neutralizing monoclonal antibodies protect against infection with HIV-1 in animal models, suggesting that a vaccine that elicits these antibodies would be protective in humans. However, it has not yet been possible to induce adequate serological responses by vaccination. Here, to activate B cells that express precursors of broadly neutralizing antibodies within polyclonal repertoires, we developed an immunogen, RC1, that facilitates the recognition of the variable loop 3 (V3)-glycan patch on the envelope protein of HIV-1. RC1 conceals non-conserved immunodominant regions by the addition of glycans and/or multimerization on virus-like particles. Immunization of mice, rabbits and rhesus macaques with RC1 elicited serological responses that targeted the V3-glycan patch. Antibody cloning and cryo-electron microscopy structures of antibody-envelope complexes confirmed that immunization with RC1 expands clones of B cells that carry the anti-V3-glycan patch antibodies, which resemble precursors of human broadly neutralizing antibodies. Thus, RC1 may be a suitable priming immunogen for sequential vaccination strategies in the context of polyclonal repertoires.

Individuals infected with HIV-1 require lifelong antiretroviral therapy, because interruption of treatment leads to rapid rebound viraemia. Here we report on a phase 1b clinical trial in which a combination of 3BNC117 and 10-1074, two potent monoclonal anti-HIV-1 broadly neutralizing antibodies that target independent sites on the HIV-1 envelope spike, was administered during analytical treatment interruption. Participants received three infusions of 30 mg kg -1 of each antibody at 0, 3 and 6 weeks. Infusions of the two antibodies were generally well-tolerated. The nine enrolled individuals with antibody-sensitive latent viral reservoirs maintained suppression for between 15 and more than 30 weeks (median of 21 weeks), and none developed viruses that were resistant to both antibodies. We conclude that the combination of the anti-HIV-1 monoclonal antibodies 3BNC117 and 10-1074 can maintain long-term suppression in the absence of antiretroviral therapy in individuals with antibody-sensitive viral reservoirs.

Some HIV-1-infected patients develop broad and potent HIV-1 neutralizing antibodies (bNAbs) that when passively transferred to mice or macaques can treat or prevent infection. However, bNAbs typically fail to neutralize coexisting autologous viruses due to antibody-mediated selection against sensitive viral strains. We describe an HIV-1 controller expressing HLA-B57*01 and HLA-B27*05 who maintained low viral loads for 30 years after infection and developed broad and potent serologic activity against HIV-1. Neutralization was attributed to three different bNAbs targeting nonoverlapping sites on the HIV-1 envelope trimer (Env). One of the three, BG18, an antibody directed against the glycan-V3 portion of Env, is the most potent member of this class reported to date and, as revealed by crystallography and electron microscopy, recognizes HIV-1 Env in a manner that is distinct from other bNAbs in this class. Single-genome sequencing of HIV-1 from serum samples obtained over a period of 9 years showed a diverse group of circulating viruses, 88.5% (31 of 35) of which remained sensitive to at least one of the temporally coincident autologous bNAbs and the individual's serum. Thus, bNAb-sensitive strains of HIV-1 coexist with potent neutralizing antibodies that target the virus and may contribute to control in this individual. When administered as a mix, the three bNAbs controlled viremia in HIV-1 YU2 -infected humanized mice. Our finding suggests that combinations of bNAbs may contribute to control of HIV-1 infection., (Copyright © 2017, American Association for the Advancement of Science.)

HIV-1 vaccine design is informed by structural studies elucidating mechanisms by which broadly neutralizing antibodies (bNAbs) recognize and/or accommodate N-glycans on the trimeric envelope glycoprotein (Env). Variability in high-mannose and complex-type Env glycoforms leads to heterogeneity that usually precludes visualization of the native glycan shield. We present 3.5-Å- and 3.9-Å-resolution crystal structures of the HIV-1 Env trimer with fully processed and native glycosylation, revealing a glycan shield of high-mannose and complex-type N-glycans, which we used to define complete epitopes of two bNAbs. Env trimer was complexed with 10-1074 (against the V3-loop) and IOMA, a new CD4-binding site (CD4bs) antibody. Although IOMA derives from VH1-2*02, the germline gene of CD4bs-targeting VRC01-class bNAbs, its light chain lacks the short CDRL3 that defines VRC01-class bNAbs. Thus IOMA resembles 8ANC131-class/VH1-46-derived CD4bs bNAbs, which have normal-length CDRL3s. The existence of bNAbs that combine features of VRC01-class and 8ANC131-class antibodies has implications for immunization strategies targeting VRC01-like bNAbs.

Interruption of combination antiretroviral therapy in HIV-1-infected individuals leads to rapid viral rebound. Here we report the results of a phase IIa open label clinical trial evaluating 3BNC117,a broad and potent neutralizing antibody against the CD4 binding site of the HIV-1 Env protein, during analytical treatment interruption in 13 HIV-1-infected individuals. Participants with 3BNC117-sensitive virus outgrowth cultures were enrolled. Results show that two or four 30 mg kg(-1) 3BNC117 infusions,separated by 3 or 2 weeks, respectively, are generally well tolerated.Infusions are associated with a delay in viral rebound of 5-9 weeks after two infusions, and up to 19 weeks after four infusions, or an average of 6.7 and 9.9 weeks, respectively, compared with 2.6 weeks for historical controls (P < 0.00001). Rebound viruses arise predominantly from a single provirus. In most individuals,emerging viruses show increased resistance, indicating escape.However, 30% of participants remained suppressed until antibody concentrations waned below 20 μg ml(-1), and the viruses emerging in all but one of these individuals showed no apparent resistance to 3BCN117, suggesting failure to escape over a period of 9-19 weeks.We conclude that the administration of 3BNC117 exerts strong selective pressure on HIV-1 emerging from latent reservoirs during analytical treatment interruption in humans.

3BNC117 is a broad and potent neutralizing antibody to HIV-1 that targets the CD4 binding site on the viral envelope spike. When administered passively, this antibody can prevent infection in animal models and suppress viremia in HIV-1-infected individuals. Here we report that HIV-1 immunotherapy with a single injection of 3BNC117 affects host antibody responses in viremic individuals. In comparison to untreated controls that showed little change in their neutralizing activity over a 6-month period, 3BNC117 infusion significantly improved neutralizing responses to heterologous tier 2 viruses in nearly all study participants. We conclude that 3BNC117-mediated immunotherapy enhances host humoral immunity to HIV-1., (Copyright © 2016, American Association for the Advancement of Science.)

HIV-1 immunotherapy with a combination of first generation monoclonal antibodies was largely ineffective in pre-clinical and clinical settings and was therefore abandoned. However, recently developed single-cell-based antibody cloning methods have uncovered a new generation of far more potent broadly neutralizing antibodies to HIV-1 (refs 4, 5). These antibodies can prevent infection and suppress viraemia in humanized mice and nonhuman primates, but their potential for human HIV-1 immunotherapy has not been evaluated. Here we report the results of a first-in-man dose escalation phase 1 clinical trial of 3BNC117, a potent human CD4 binding site antibody, in uninfected and HIV-1-infected individuals. 3BNC117 infusion was well tolerated and demonstrated favourable pharmacokinetics. A single 30 mg kg(-1) infusion of 3BNC117 reduced the viral load in HIV-1-infected individuals by 0.8-2.5 log10 and viraemia remained significantly reduced for 28 days. Emergence of resistant viral strains was variable, with some individuals remaining sensitive to 3BNC117 for a period of 28 days. We conclude that, as a single agent, 3BNC117 is safe and effective in reducing HIV-1 viraemia, and that immunotherapy should be explored as a new modality for HIV-1 prevention, therapy and cure.

The site on the HIV-1 gp120 glycoprotein that binds the CD4 receptor is recognized by broadly reactive antibodies, several of which neutralize over 90% of HIV-1 strains. To understand how antibodies achieve such neutralization, we isolated CD4-binding-site (CD4bs) antibodies and analyzed 16 co-crystal structures -8 determined here- of CD4bs antibodies from 14 donors. The 16 antibodies segregated by recognition mode and developmental ontogeny into two types: CDR H3-dominated and VH-gene-restricted. Both could achieve greater than 80% neutralization breadth, and both could develop in the same donor. Although paratope chemistries differed, all 16 gp120-CD4bs antibody complexes showed geometric similarity, with antibody-neutralization breadth correlating with antibody-angle of approach relative to the most effective antibody of each type. The repertoire for effective recognition of the CD4 supersite thus comprises antibodies with distinct paratopes arrayed about two optimal geometric orientations, one achieved by CDR H3 ontogenies and the other achieved by VH-gene-restricted ontogenies., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Antibodies developed during HIV-1 infection lose efficacy as the viral spike mutates. We postulated that anti-HIV-1 antibodies primarily bind monovalently because HIV's low spike density impedes bivalent binding through inter-spike crosslinking, and the spike structure prohibits bivalent binding through intra-spike crosslinking. Monovalent binding reduces avidity and potency, thus expanding the range of mutations permitting antibody evasion. To test this idea, we engineered antibody-based molecules capable of bivalent binding through intra-spike crosslinking. We used DNA as a "molecular ruler" to measure intra-epitope distances on virion-bound spikes and construct intra-spike crosslinking molecules. Optimal bivalent reagents exhibited up to 2.5 orders of magnitude increased potency (>100-fold average increases across virus panels) and identified conformational states of virion-bound spikes. The demonstration that intra-spike crosslinking lowers the concentration of antibodies required for neutralization supports the hypothesis that low spike densities facilitate antibody evasion and the use of molecules capable of intra-spike crosslinking for therapy or passive protection., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Antibody-mediated immunotherapy is effective in humanized mice when combinations of broadly neutralizing antibodies (bNAbs) are used that target nonoverlapping sites on the human immunodeficiency virus type 1 (HIV-1) envelope. In contrast, single bNAbs can control simian-human immunodeficiency virus (SHIV) infection in immune-competent macaques, suggesting that the host immune response might also contribute to the control of viremia. Here, we investigate how the autologous antibody response in intact hosts can contribute to the success of immunotherapy. We find that frequently arising antibodies that normally fail to control HIV-1 infection can synergize with passively administered bNAbs by preventing the emergence of bNAb viral escape variants., (© 2014 Klein et al.)

Broadly neutralizing antibodies (bNAbs) to HIV-1 envelope glycoprotein (Env) can prevent infection in animal models. Characterized bNAb targets, although key to vaccine and therapeutic strategies, are currently limited. We defined a new site of vulnerability by solving structures of bNAb 8ANC195 complexed with monomeric gp120 by X-ray crystallography and trimeric Env by electron microscopy. The site includes portions of gp41 and N-linked glycans adjacent to the CD4-binding site on gp120, making 8ANC195 the first donor-derived anti-HIV-1 bNAb with an epitope spanning both Env subunits. Rather than penetrating the glycan shield by using a single variable-region CDR loop, 8ANC195 inserted its entire heavy-chain variable domain into a gap to form a large interface with gp120 glycans and regions of the gp120 inner domain not contacted by other bNAbs. By isolating additional 8ANC195 clonal variants, we identified a more potent variant, which may be valuable for therapeutic approaches using bNAb combinations with nonoverlapping epitopes., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Despite 30 years of effort, there is no effective vaccine for HIV-1. However, antibodies can prevent HIV-1 infection in humanized mice and macaques when passively transferred. New single-cell-based methods have uncovered many broad and potent donor-derived antibodies, and structural studies have revealed the molecular bases for their activities. The new data suggest why such antibodies are difficult to elicit and inform HIV-1 vaccine development efforts. In addition to protecting against infection, the newly identified antibodies can suppress active infections in mice and macaques, suggesting they could be valuable additions to anti-HIV-1 therapies and to strategies to eradicate HIV-1 infection., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Advances in single-cell antibody cloning methods have led to the identification of a variety of broadly neutralizing anti-HIV-1 antibodies. We developed a computational tool (Antibody Database) to help identify critical residues on the HIV-1 envelope protein whose natural variation affects antibody activity. Our simplifying assumption was that, for a given antibody, a significant portion of the dispersion of neutralization activity across a panel of HIV-1 strains is due to the amino acid identity or glycosylation state at a small number of specific sites, each acting independently. A model of an antibody's neutralization IC50 was developed in which each site contributes a term to the logarithm of the modeled IC50. The analysis program attempts to determine the set of rules that minimizes the sum of the residuals between observed and modeled IC50 values. The predictive quality of the identified rules may be assessed in part by whether there is support for rules within individual viral clades. As a test case, we analyzed antibody 8ANC195, an anti-glycoprotein gp120 antibody of unknown specificity. The model for this antibody indicated that several glycosylation sites were critical for neutralization. We evaluated this prediction by measuring neutralization potencies of 8ANC195 against HIV-1 in vitro and in an antibody therapy experiment in humanized mice. These experiments confirmed that 8ANC195 represents a distinct class of glycan-dependent anti-HIV-1 antibody and validated the utility of computational analysis of neutralization panel data.

Recently identified broadly neutralizing antibodies (bNAbs) that potently neutralize most HIV-1 strains are key to potential antibody-based therapeutic approaches to combat HIV/AIDS in the absence of an effective vaccine. Increasing bNAb potencies and resistance to common routes of HIV-1 escape through mutation would facilitate their use as therapeutics. We previously used structure-based design to create the bNAb NIH45-46(G54W), which exhibits superior potency and/or breadth compared with other bNAbs. We report new, more effective NIH45-46(G54W) variants designed using analyses of the NIH45-46-gp120 complex structure and sequences of NIH45-46(G54W)-resistant HIV-1 strains. One variant, 45-46m2, neutralizes 96% of HIV-1 strains in a cross-clade panel and viruses isolated from an HIV-infected individual that are resistant to all other known bNAbs, making it the single most broad and potent anti-HIV-1 antibody to date. A description of its mechanism is presented based on a 45-46m2-gp120 crystal structure. A second variant, 45-46m7, designed to thwart HIV-1 resistance to NIH45-46(G54W) arising from mutations in a gp120 consensus sequence, targets a common route of HIV-1 escape. In combination, 45-46m2 and 45-46m7 reduce the possible routes for the evolution of fit viral escape mutants in HIV-1YU-2-infected humanized mice, with viremic control exhibited when a third antibody, 10-1074, was added to the combination.

Defining the virus-host interactions responsible for HIV-1 transmission, including the phenotypic requirements of viruses capable of establishing de novo infections, could be important for AIDS vaccine development. Previous analyses have failed to identify phenotypic properties other than chemokine receptor 5 (CCR5) and CD4+ T-cell tropism that are preferentially associated with viral transmission. However, most of these studies were limited to examining envelope (Env) function in the context of pseudoviruses. Here, we generated infectious molecular clones of transmitted founder (TF; n = 27) and chronic control (CC; n = 14) viruses of subtypes B (n = 18) and C (n = 23) and compared their phenotypic properties in assays specifically designed to probe the earliest stages of HIV-1 infection. We found that TF virions were 1.7-fold more infectious (P = 0.049) and contained 1.9-fold more Env per particle (P = 0.048) compared with CC viruses. TF viruses were also captured by monocyte-derived dendritic cells 1.7-fold more efficiently (P = 0.035) and more readily transferred to CD4+ T cells (P = 0.025). In primary CD4+ T cells, TF and CC viruses replicated with comparable kinetics; however, when propagated in the presence of IFN-α, TF viruses replicated to higher titers than CC viruses. This difference was significant for subtype B (P = 0.000013) but not subtype C (P = 0.53) viruses, possibly reflecting demographic differences of the respective patient cohorts. Together, these data indicate that TF viruses are enriched for higher Env content, enhanced cell-free infectivity, improved dendritic cell interaction, and relative IFN-α resistance. These viral properties, which likely act in concert, should be considered in the development and testing of AIDS vaccines.

The existence of very potent, broadly neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) offers the potential for prophylaxis against HIV-1 infection by passive immunization or gene therapy. Both routes permit the delivery of modified forms of IgGs. Smaller reagents are favored when considering ease of tissue penetration and the limited capacities of gene therapy vectors. Immunoadhesin (single-chain fragment variable [scFv]-Fc) forms of IgGs are one class of relatively small reagent that has been explored for delivery by adeno-associated virus. Here we investigated the neutralization potencies of immunoadhesins compared to those of their parent IgGs. For the antibodies VRC01, PG9, and PG16, the immunoadhesins showed modestly reduced potencies, likely reflecting reduced affinities compared to those of the parent IgG, and the VRC01 immunoadhesin formed dimers and multimers with reduced neutralization potencies. Although scFv forms of neutralizing antibodies may exhibit affinity reductions, they provide a means of building reagents with multiple activities. Attachment of the VRC01 scFv to PG16 IgG yielded a bispecific reagent whose neutralization activity combined activities from both parent antibodies. Although the neutralization activity due to each component was partially reduced, the combined reagent is attractive since fewer strains escaped neutralization.

Antibodies against the CD4 binding site (CD4bs) on the HIV-1 spike protein gp120 can show exceptional potency and breadth. We determined structures of NIH45-46, a more potent clonal variant of VRC01, alone and bound to gp120. Comparisons with VRC01-gp120 revealed that a four-residue insertion in heavy chain complementarity-determining region 3 (CDRH3) contributed to increased interaction between NIH45-46 and the gp120 inner domain, which correlated with enhanced neutralization. We used structure-based design to create NIH45-46(G54W), a single substitution in CDRH2 that increases contact with the gp120 bridging sheet and improves breadth and potency, critical properties for potential clinical use, by an order of magnitude. Together with the NIH45-46-gp120 structure, these results indicate that gp120 inner domain and bridging sheet residues should be included in immunogens to elicit CD4bs antibodies.

We previously showed that broadly neutralizing anti-HIV-1 antibody 2G12 (human IgG1) naturally forms dimers that are more potent than monomeric 2G12 in in vitro neutralization of various strains of HIV-1. In this study, we have investigated the protective effects of monomeric versus dimeric 2G12 against HIV-1 infection in vivo using a humanized mouse model. Our results showed that passively transferred, purified 2G12 dimer is more potent than 2G12 monomer at preventing CD4 T cell loss and suppressing the increase of viral load following HIV-1 infection of humanized mice. Using humanized mice bearing IgG "backpack" tumors that provided 2G12 antibodies continuously, we found that a sustained dimer concentration of 5-25 µg/ml during the course of infection provides effective protection against HIV-1. Importantly, 2G12 dimer at this concentration does not favor mutations of the HIV-1 envelope that would cause the virus to completely escape 2G12 neutralization. We have therefore identified dimeric 2G12 as a potent prophylactic reagent against HIV-1 in vivo, which could be used as part of an antibody cocktail to prevent HIV-1 infection.

Monoclonal antibodies b12 and 4E10 are broadly neutralizing against a variety of strains of the human immunodeficiency virus type 1 (HIV-1). The epitope for b12 maps to the CD4-binding site in the gp120 subunit of HIV-1's trimeric gp120-gp41 envelope spike, whereas 4E10 recognizes the membrane-proximal external region (MPER) of gp41. Here, we constructed and compared a series of architectures for the b12 and 4E10 combining sites that differed in size, valency, and flexibility. In a comparative analysis of the ability of the b12 and 4E10 constructs to neutralize a panel of clade B HIV-1 strains, we observed that the ability of bivalent constructs to cross-link envelope spikes on the virion surface made a greater contribution to neutralization by b12 than by 4E10. Increased distance and flexibility between antibody combining sites correlated with enhanced neutralization for both antibodies, suggesting restricted mobility for the trimeric spikes embedded in the virion surface. The size of a construct did not appear to be correlated with neutralization potency for b12, but larger 4E10 constructs exhibited a steric occlusion effect, which we interpret as evidence for restricted access to its gp41 epitope. The combination of limited avidity and steric occlusion suggests a mechanism for evading neutralization by antibodies that target epitopes in the highly conserved MPER of gp41.

The antigen-binding fragment of the broadly neutralizing human immunodeficiency virus type 1 (HIV-1) antibody 2G12 has an unusual three-dimensional (3D) domain-swapped structure with two aligned combining sites that facilitates recognition of its carbohydrate epitope on gp120. When expressed as an intact immunoglobulin G (IgG), 2G12 formed typical IgG monomers containing two combining sites and a small fraction of a higher-molecular-weight species, which showed a significant increase in neutralization potency (50- to 80-fold compared to 2G12 monomer) across a range of clade A and B strains of HIV-1. Here we show that the higher-molecular-weight species corresponds to a 2G12 dimer containing four combining sites and present a model for how intermolecular 3D domain swapping could create a 2G12 dimer. Based on the structural model for a 3D domain-swapped 2G12 dimer, we designed and tested a series of 2G12 mutants predicted to increase the ratio of 2G12 dimer to monomer. We report a mutation that effectively increases the 2G12 dimer/monomer ratio without decreasing the expression yield. Increasing the proportion of 2G12 dimer compared to monomer could lead to a more potent reagent for gene therapy or passive immunization.