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An important approach to stopping the AIDS epidemic is the development of a vaccine that elicits antibodies that block virus capture, the initial interactions of HIV-1 with the target cells, and replication. We utilized a previously developed qRT-PCR-based assay to examine the effects of broadly neutralizing antibodies (bNAbs), plasma from vaccine trials, and monoclonal antibodies (mAbs) on virus capture and replication. A panel of bNAbs inhibited primary HIV-1 replication in PBMCs but not virus capture. Plasma from RV144 and RV305 trial vaccinees demonstrated inhibition of virus capture with the HIV-1 subtype prevalent in Thailand. Several RV305 derived V2-specific mAbs inhibited virus replication. One of these RV305 derived V2-specific mAbs inhibited both virus capture and replication, demonstrating that it is possible to elicit antibodies by vaccination that inhibit virus capture and replication. Induction of a combination of such antibodies may be the key to protection from HIV-1 acquisition. • RV144 and RV305 vaccinee plasma inhibited primary HIV-1 capture by PBMCs. • One of the RV305 elicited mAb inhibited both virus capture and replication. • Vaccination can induce antibodies that inhibit HIV-1 capture and replication. [ABSTRACT FROM AUTHOR]

The sequence diversity of HIV-1 is the biggest hurdle for the design of a prophylactic vaccine. Mosaic (Mos) antigens consisting of synthetically shuffled epitopes from various HIV-1 strains are currently tested in the clinical vaccine trial Mosaico (NCT03964415). Besides adenovirus vectors encoding variants of Mos.Gag-Pol and soluble Mos.Env proteins, the Mosaico vaccine entails vectors mediating gene transfer and expression of the membrane-anchored Env-variant Mos2S.Env. We thus examined whether the expression of mosaic Gag mediates the formation of virus-like particles (VLPs). Mos1.Gag- and Mos2.Gag-VLP-formation was readily detected using Western blot- and electron microscopic-analysis. Upon co-expression of both mosaic Gag variants with Mos2S.Env, incorporation of Env into Gag-formed VLPs was observed. The display of the respective neutralization-sensitive target epitopes on Mos2S.Env-decorated VLPs was demonstrated employing a panel of broadly neutralizing antibodies (bNAbs) in a VLP-capture assay. This opens new perspectives for future HIV vaccine designs. • Mosaic Gag proteins as components of a HIV vaccine form VLPs. • Mosaic Env proteins efficiently decorate mosaic Gag-formed VLPs. • Broadly neutralizing antibodies (bNAbs) recognize target epitopes in mosaic Env proteins displayed on mosaic Gag-formed VLPs. [ABSTRACT FROM AUTHOR]

Antibodies (Abs) recognizing the Dengue virus (DENV) E dimer epitope (EDE) that potently neutralize all DENV serotypes are promising templates for vaccine design. As an important feature for some Abs is their bivalency, we sought to define the role avidity plays in neutralization by EDE Abs. We compared neutralization activity between bivalent IgGs and monovalent Ab fragments (Fabs) for two EDE Abs, A11 and C10. IgG forms of both Abs exhibited more potent neutralization activity than their counterpart Fabs, yet only for C10 was this enhanced activity associated with bivalent binding. A11 and C10 also exhibited differential binding profiles to DENV virus-like particles under acidic conditions mimicking the environment that triggers viral membrane fusion, suggesting that EDE Abs employ diverse neutralization mechanisms despite sharing an epitope. Delineating the full range of Ab binding modes and neutralization mechanisms against a single epitope may inform therapeutic approaches and refine vaccine design. • Broadly-neutralizing antibodies target the Dengue virus E dimer epitope. • Monovalent antibody fragments neutralize Dengue less potently than whole antibodies. • Not all antibodies are capable of binding the Dengue E dimer epitope bivalently. • Antibodies targeting the same epitope may exhibit diverse neutralization mechanisms. [ABSTRACT FROM AUTHOR]

Broadly neutralizing antibodies (bNAbs) may constitute an essential component of a protective vaccine against HIV-1, yet no immunogen has been able to elicit them. To characterize the development of bNAbs in HIV-1 subtype C infected individuals, a panel of 18 Env-pseudotyped viruses was used to screen 18 study participants. The specificity of plasma neutralization was mapped against Env mutants and MPER chimeras. Envelope (env) gene sequence evolution was characterized by single genome amplification and sequencing. Three out of eighteen individuals developed broad plasma neutralizing activity (>60% breadth). Two of the three participants may target epitopes comprising glycans at position 276 of the D loop in the CD4 binding site and 332 glycan supersite, respectively. Deletion of these glycans was associated with neutralization resistance. Our study describes the kinetics of the development of plasma neutralizing activity and identified amino acid residue changes suggestive of immune pressure on putative epitopes. The study enhances our understanding of how neutralization breadth develops in the course of HIV-1 subtype C infection. [ABSTRACT FROM AUTHOR]

Efficient cleavage of HIV-1 Env gp160 into its constituent subunits correlates with selective binding to neutralizing antibodies and are the closest mimetic of native, functional Envs. This was first demonstrated with the clade B Env, JRFL. The correlation between efficient cleavage and selective binding to neutralizing antibodies is the guiding principle for immunogen design for HIV vaccine. We have recently reported that Envs 4-2.J41 (clade C) and JRCSF (clade B) are also efficiently cleaved and show similar properties. However, an efficiently cleaved, membrane-bound clade A Env suitable for genetic vaccination has not been directly demonstrated. Here we report that BG505 and a new clade A Env, QB726.70M.ENV.C4 (or A5) are efficiently cleaved on cell membrane. A5 shows desirable antigenic properties comparable with BG505 on cell surface. A5SOSIP in supernatant displays majority of bNAb binding epitopes. Thus, both BG505 and A5 Envs can be used in DNA prime-protein boost vaccination studies. [ABSTRACT FROM AUTHOR]

Development of vaccines capable of eliciting broadly neutralizing antibodies (bNAbs) is a key goal to controlling the global AIDS epidemic. To be effective, bNAbs must block the capture of HIV-1 to prevent viral acquisition and establishment of reservoirs. However, the role of bNAbs, particularly during initial exposure of primary viruses to host cells, has not been fully examined. Using a sensitive, quantitative, and high-throughput qRT-PCR assay, we found that primary viruses were captured by host cells and converted into a trypsin-resistant form in less than five minutes. We discovered, unexpectedly, that bNAbs did not block primary virus capture, although they inhibited the capture of pseudoviruses/IMCs and production of progeny viruses at 48 h. Further, viruses escaped bNAb inhibition unless the bNAbs were present in the initial minutes of exposure of virus to host cells. These findings will have important implications for HIV-1 vaccine design and determination of vaccine efficacy. [ABSTRACT FROM AUTHOR]

HIV-1 compartmentalisation is likely to have important implications for a preventative vaccine as well as eradication strategies. We genetically characterised HIV-1 subtype C variants in lymph nodes, peripheral blood mononuclear cells and plasma of six antiretroviral (ART) naïve individuals and four individuals on ART. Full-length env (n = 171) and gag (n = 250) sequences were generated from participants using single genome amplification. Phylogenetic relatedness of sequences was assessed, and compartmentalisation was determined using both distance and tree-based methods implemented in HyPhy. Additionally, potential associations between compartmentalisation and immune escape mutations were assessed. Partial viral compartmentalisation was present in nine of the ten participants. Broadly neutralising antibody (bnAb) escape was found to be associated with partial env compartmentalisation in some individuals, while cytotoxic T lymphocyte escape mutations in Gag were limited and did not differ between compartments. Viral compartmentalisation may be an important consideration for bnAb use in viral eradication., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Anti-HIV-1 broadly neutralizing antibodies (bnAbs) have been extensively tested against pesudoviruses of diverse strains. We generated and characterized HIV-1 primary isolates from antiretroviral naïve infected Indian children, and determined their susceptibility to known NAbs. All the 8 isolates belonged to subtype-C and were R5 tropic. Majority of these viruses were resistant to neutralization by NAbs, suggesting that the bnAbs, known to efficiently neutralize pseudoviruses (adult and pediatric) of different strains, are less effective against pediatric primary isolates. Interestingly, AIIMS_329 isolate displayed high susceptibility to neutralization by PG9 and PG16bnAbs, with IC 50 titer of 1.3 and 0.97 μg/ml, suggesting exposure of this epitope on this virus. All isolates except AIIMS_506 were neutralized by contemporaneous plasma antibodies. Our findings suggest that primary isolates, due to close resemblance to viruses in natural infection, should be used to evaluate NAbs as effective vaccine candidates in both children and adults. [ABSTRACT FROM AUTHOR]

HIV-1 envelope glycoproteins (Env) and Env-based immunogens usually do not interact efficiently with the inferred germline precursors of known broadly neutralizing antibodies (bNAbs). This deficiency may be one reason why Env and Env-based immunogens are not efficient at inducing bNAbs. We evaluated the binding of 15 inferred germline precursors of bNAbs directed to different epitope clusters to three soluble native-like SOSIP.664 Env trimers. We found that native-like SOSIP.664 trimers bind to some inferred germline precursors of bNAbs, particularly ones involving the V1/V2 loops at the apex of the trimer. The data imply that native-like SOSIP.664 trimers will be an appropriate platform for structure-guided design improvements intended to create immunogens able to target the germline precursors of bNAbs. [ABSTRACT FROM AUTHOR]

Here we investigated the binding of Dengue virus envelope glycoprotein domain III (DIII) by two broadly neutralizing antibodies (bNAbs), 4E11 and 4E5A. There are four serotypes of Dengue virus (DENV-1 to -4), whose DIII sequences vary by up to 49%. We used combinatorial alanine scanning mutagenesis, a phage display approach, to map functional epitopes (those residues that contribute most significantly to the energetics of antibody–antigen interaction) on these four serotypes. Our results showed that 4E11, which binds strongly to DENV-1, -2, and -3, and moderately to DENV-4, recognized a common conserved core functional epitope involving DIII residues K310, L/I387, L389, and W391. There were also unique recognition features for each serotype, suggesting that 4E11 has flexible recognition requirements. Similar scanning studies for the related bNAb 4E5A, which binds more tightly to DENV-4, identified broader functional epitopes on DENV-1. These results provide useful information for immunogen and therapeutic antibody design. [ABSTRACT FROM AUTHOR]

Abstract: The need for an effective vaccine to prevent the global spread of human immunodeficiency virus type 1 (HIV-1) is well recognized. Passive immunization and challenge studies in non-human primates testify that broadly neutralizing antibodies (BrNAbs) can accomplish protection against infection. In recent years, the introduction of new techniques has facilitated the discovery of an unprecedented number of new human BrNAbs that target and delineate diverse conserved epitopes on the envelope glycoprotein spike (Env). The epitopes of these BrNAbs can serve as templates for immunogen design aimed to induce similar antibodies. Here we will review the characteristics of the different classes of BrNAbs and their target epitopes, as well as factors associated with their development and implications for vaccine design. [Copyright &y& Elsevier]

Abstract: Previously we reported a broadly HIV-1 neutralizing mini-antibody (Fab 3674) of modest potency that was derived from a human non-immune phage library by panning against the chimeric gp41-derived construct NCCG-gp41. This construct presents the N-heptad repeat of the gp41 ectodomain as a stable, helical, disulfide-linked trimer that extends in helical phase from the six-helix bundle of gp41. In this paper, Fab 3674 was subjected to affinity maturation against the NCCG-gp41 antigen by targeted diversification of the CDR-H2 loop to generate a panel of Fabs with diverse neutralization activity. Three affinity-matured Fabs selected for further study, Fabs 8060, 8066 and 8068, showed significant increases in both potency and breadth of neutralization against HIV-1 pseudotyped with envelopes of primary isolates from the standard subtype B and C HIV-1 reference panels. The parental Fab 3674 is 10–20-fold less potent in monovalent than bivalent format over the entire B and C panels of HIV-1 pseudotypes. Of note is that the improved neutralization activity of the affinity-matured Fabs relative to the parental Fab 3674 was, on average, significantly greater for the Fabs in monovalent than bivalent format. This suggests that the increased avidity of the Fabs for the target antigen in bivalent format can be partially offset by kinetic and/or steric advantages afforded by the smaller monovalent Fabs. Indeed, the best affinity-matured Fab (8066) in monovalent format (∼50 kDa) was comparable in HIV-1 neutralization potency to the parental Fab 3674 in bivalent format (∼120 kDa) across the subtype B and C reference panels. [Copyright &y& Elsevier]

Abstract: The conserved nature of the epitopes of the four broadly neutralizing antibodies (BNAbs), b12, 2G12, 2F5, and 4E10, may imply that the sensitivity of HIV-1 for these BNAbs remains fairly constant over the course of infection. Here, we demonstrate that viruses isolated early during the course of infection were mostly sensitive to HIVIg and antibody neutralization, although variation was observed in neutralization sensitivity of coexisting viruses to the different antibodies as well as between viruses from different patients. HIV-1 resistance to HIVIg developed relatively early during follow-up in three out of five patients, while early, b12 sensitive viruses in three out of five patients were replaced by b12 resistant variants relatively late in infection. In contrast, viruses generally remained sensitive to 2F5 and 4E10 neutralization over the course of infection, although 2F5 and/or 4E10 resistant variants did emerge later in infection in four out of five patients. In most patients, HIV-1 resistance to 2F5 or 4E10 did not correlate with mutations at critical amino acid positions in their defined epitopes. Viruses resistant to 2G12-mediated neutralization were present throughout the course of infection. As viral resistance against BNAb-mediated neutralization generally developed when autologous serum neutralizing activity had faded, it seems unlikely that these changes are driven by escape from autologous humoral immunity. [Copyright &y& Elsevier]

Abstract: Although the majority of primary HIV-1 variants can be neutralized by broadly neutralizing antibodies such as b12, 2G12, 2F5 and 4E10, resistance to these antibodies has been reported as well. The ability of the broadly neutralizing antibodies to inhibit a variety of viruses suggests that their epitopes are conserved and escape from these antibodies may thus come at a cost to viral fitness. Here we demonstrate that resistance to broadly neutralizing antibodies was in general not associated with a reduced replicative capacity of the virus in vitro. This indicates that loss of replicative capacity due to escape from broadly neutralizing antibodies may be limited. [Copyright &y& Elsevier]

The membrane proximal external region (MPER) of HIV-1 gp41 is targeted by broadly neutralizing antibodies (bnAbs) 4E10 and 10E8. In this proof-of-concept study, we evaluated a novel multi-immunogen vaccine strategy referred to as Incremental, Phased Antigenic Stimulation for Rapid Antibody Maturation (IPAS-RAM) to induce 4E10/10E8-like bnAbs. Rabbits were immunized sequentially, but in a phased manner, with three immunogens that are progressively more native (gp41-28×3, gp41-54CT, and rVV-gp160 DH12 ). Although nAbs were not induced, epitope-mapping analyses indicated that IPAS-RAM vaccination was better able to target antibodies towards the 4E10/10E8 epitopes than homologous prime-boost immunization using gp41-28×3 alone. MPER-specific rabbit monoclonal antibodies were generated, including 9F6. Although it lacked neutralizing activity, the target epitope profile of 9F6 closely resembled those of 4E10 and 10E8 ( 671 NWFDITNWLWYIK 683 ). B-cell repertoire analyses suggested the importance of co-immunizations for maturation of 9F6, which warrants further evaluation of our IPAS-RAM vaccine strategy using an improved priming immunogen., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Human immunodeficiency virus type 1 (HIV-1) transmission and infection occur mainly via the mucosal surfaces. The commensal bacteria residing in these surfaces can potentially be employed as a vehicle for delivering inhibitors to prevent HIV-1 infection. In this study, we have employed a bacteria-based strategy to display a broadly neutralizing antibody VRC01, which could potentially be used to prevent HIV-1 infection. The VRC01 antibody mimics CD4-binding to gp120 and has broadly neutralization activities against HIV-1. We have designed a construct that can express the fusion peptide of the scFv-VRC01 antibody together with the autotransporter β-barrel domain of IgAP gene from Neisseria gonorrhoeae, which enabled surface display of the antibody molecule. Our results indicate that the scFv-VRC01 antibody molecule was displayed on the surface of the bacteria as demonstrated by flow cytometry and immunofluorescence microscopy. The engineered bacteria can capture HIV-1 particles via surface-binding and inhibit HIV-1 infection in cell culture., (Copyright © 2014 Elsevier Inc. All rights reserved.)