Search

Introduction: HIV-1 envelope (Env) is the key target for antibodies (Abs) against the virus and thus an important HIV-1 vaccine component. Env is synthesized from a gp160 precursor with a signal peptide (SP) at its N-terminus. This study investigated the influence of the SP on Env antigenicity and immunogenicity., Methods: Env proteins from two HIV-1 isolates, AA05 and AC02, were analyzed as gp120 and gp160 in their native wild-type (WT) forms and as chimeras with swapped SPs (AA05-02 and AC02-05). The WT and chimeric Env were assessed for antigenicity and glycosylation using monoclonal antibodies (mAbs) and glycan probes. Immunogenicity was tested in mice using three vaccine types: gp120 protein, gp120 DNA+gp120 protein, and gp120 DNA+gp160 DNA., Results: The recombinant AC02 gp120 protein was antigenically superior to AA05 as indicated by higher reactivity with most mAbs tested. When SPs were swapped, the antigenicity of the chimeric gp120s (AA05-02 and AC02-05) resembled that of the gp120s from which the SPs were derived; AA05-02 was similar to AC02 and vice versa. Glycan probe reactivity followed a similar pattern: AA05-02 and AC02 showed similar affinity to high-mannose specific mAbs and lectins. Interestingly, the antigenicity of gp160s showed an opposite pattern; membrane-bound gp160 expressed with the AA05 SP (AA05 and AC02-05) showed greater mAb binding than gp160 with the AC02 SP (AC02 and AA05-02). Mice immunized with gp120 protein showed that AA05-02 induced stronger cross-reactive binding Ab responses than AA05 WT, and AC02 elicited stronger responses than AC02-05, indicating AC02 SP enhanced gp120 immunogenicity. However, when DNA vaccines were included (gp120 DNA+gp120 protein and gp120 DNA+gp160 DNA), the use of heterologous SPs diminished the immunogenicity of the WT immunogens. Among the three vaccine regimens tested, only gp120 DNA+gp160 DNA immunization elicited low-level Tier 2 neutralizing Abs, with AA05 WT inducing Abs with greater neutralization capabilities than AA05-02., Conclusion: These data demonstrate that the SP can significantly impact the antigenicity and immunogenicity of HIV-1 Env proteins. Hence, while SP swapping is a common practice in constructing Env immunogens, this study highlights the importance of careful consideration of the effects of replacing native SPs on the immunogenicity of Env vaccines., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Upadhyay, Rao, Behzadi, Feyznezhad, Lambert, Kumar, Kumar, Yang, Jiang, Luo, Nadas, Arthos, Kong, Zhang, Hioe and Duty.)

Induction of broadly neutralizing antibodies (bNAbs) is a major goal for HIV vaccine development. HIV envelope glycoprotein (Env)-specific bNAbs isolated from HIV-infected individuals exhibit substantial somatic hypermutation and correlate with T follicular helper (Tfh) responses. Using the VC10014 DNA-protein co-immunization vaccine platform consisting of gp160 plasmids and gp140 trimeric proteins derived from an HIV-1 infected subject that developed bNAbs, we determined the characteristics of the Env-specific humoral response in vaccinated rhesus macaques in the context of CD4+ T cell depletion. Unexpectedly, both CD4+ depleted and non-depleted animals developed comparable Tier 1 and 2 heterologous HIV-1 neutralizing plasma antibody titers. There was no deficit in protection from SHIV challenge, no diminution of titers of HIV Env-specific cross-clade binding antibodies, antibody dependent cellular phagocytosis, or antibody-dependent complement deposition in the CD4+ depleted animals. These collective results suggest that in the presence of diminished CD4+ T cell help, HIV neutralizing antibodies were still generated, which may have implications for developing effective HIV vaccine strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sarkar, Spencer, Barnette, Pandey, Sutton, Basu, Burch, Cleveland, Rosenberg, Rangel-Moreno, Keefer, Hessell, Haigwood and Kobie.)

The presence of pathogen-specific antibodies in an individual's blood-sample is used as an indication of previous exposure and infection to that specific pathogen (e.g., virus or bacterium). Measurement of the diagnostic antibodies is routinely achieved using solid phase immuno-assays such as ELISA tests and western blots. Here, we describe a sero-diagnostic approach based on phage-display of epitope arrays we term "Domain-Scan". We harness Next-generation sequencing (NGS) to measure the serum binding to dozens of epitopes derived from HIV-1 and HCV simultaneously. The distinction of healthy individuals from those infected with either HIV-1 or HCV, is modeled as a machine-learning classification problem, in which each determinant ("domain") is considered as a feature, and its NGS read-out provides values that correspond to the level of determinant-specific antibodies in the sample. We show that following training of a machine-learning model on labeled examples, we can very accurately classify unlabeled samples and pinpoint the domains that contribute most to the classification. Our experimental/computational Domain-Scan approach is general and can be adapted to other pathogens as long as sufficient training samples are provided., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hada-Neeman, Weiss-Ottolenghi, Wagner, Avram, Ashkenazy, Maor, Sklan, Shcherbakov, Pupko and Gershoni.)

Human immunodeficiency virus (HIV) is subject to immune selective pressure soon after it establishes infection at the founder stage. As an individual progresses from the founder to chronic stage of infection, immune pressure forces a history of mutations that are embedded in envelope sequences. Determining this pathway of coevolving mutations can assist in understanding what is different with the founder virus and the essential pathways it takes to maintain infection. We have combined operations research and bioinformatics methods to extract key networks of mutations that differentiate founder and chronic stages for 156 subtype B and 107 subtype C envelope (gp160) sequences. The chronic networks for both subtypes revealed strikingly different hub-and-spoke topologies compared to the less structured transmission networks. This suggests that the hub nodes are impacted by the immune response and the resulting loss of fitness is compensated by mutations at the spoke positions. The major hubs in the chronic C network occur at positions 12, 137 (within the N136 glycan), and 822, and at position 306 for subtype B. While both founder networks had a more heterogeneous connected network structure, interestingly founder B subnetworks around positions 640 and 837 preferentially contained CD4 and coreceptor binding domains. Finally, we observed a differential effect of glycosylation between founder and chronic subtype B where the latter had mutational pathways significantly driven by N-glycosylation. Our study provides insights into the mutational pathways HIV takes to evade the immune response, and presents features more likely to establish founder infection, valuable for effective vaccine design., Competing Interests: The authors have declared that no competing interests exist.

The number, intensity and order of emergence of HIV-1 specific antibodies in serum or plasma were associated with the stage of HIV-1 infection. In this study, we retrospectively analyzed the HIV-1 confirmatory results tested by western blot (WB) or recombination immunoblot assay (RIBA) in Wuhan, 2012-2018, to access the profiles of HIV-1 specific antibodies. A total of 14432 HIV-suspected serum or plasma samples collected from local hospitals and other HIV screening laboratories were further screened by two 4th generation enzyme-linked immunosorbent assay (ELISA) kits in our laboratory, of which 11068 specimens (76.69%) had at least one positive ELISA result and thereby were finally confirmed with WB or RIBA. RIBA had identified 652 (81.09%) positive and 13 (1.62%) indeterminate cases from July 1, 2014 to January 7, 2015, while WB had identified 8358 (81.43%) positive and 643 (6.26%) indeterminate cases in the other times during 2012-2018. The indeterminate rate of WB was significant higher than that of RIBA (p<0.001). Although the number of HIV-1 infected subjects increased significantly from 2012 (n = 911) to 2018 (n = 1578), the positive rate of HIV-1 antibodies decreased markedly from 70.08% in 2012 to 58.79% in 2018 (p<0.001). The most commonly observed antibody profile was gp160+gp120+p66+(p55+)p51+gp41+p31+p24+p17+ (4131, 49.43%) for WB-MP and gp160+gp120+gp41+p31+p24+p17+ (382, 58.59%) for RIBA-WANTAI, and the absence of reactivity to three possible serologic markers for recent HIV-1 infection, p31, p66, and p51, increased significantly from 2012 to 2018, with the overall rate of 17.03%, 9.40%, and 15.15%, respectively. The suspected acute HIV-1 infection was also observed to be increased in recent years, with an overall rate of 1.00%. Our results indicated the detection rate had decreased for HIV-1 infection, but increased for suspected recent and acute HIV-1 infection during 2012-2018, reflecting the efforts of intervention among high risk population., Competing Interests: The authors have declared that no competing interests exist.

Engineering T cells and natural killer (NK) cells with anti-HIV chimeric antigen receptors (CAR) has emerged as a promising strategy to eradicate HIV-infected cells. However, current anti-HIV CARs are limited by targeting a single epitope of the HIV envelope glycoprotein gp160, which cannot counter the enormous diversity and mutability of viruses. Here, we report the development of a universal CAR-NK cell, which recognizes 2,4-dinitrophenyl (DNP) and can subsequently be redirected to target various epitopes of gp160 using DNP-conjugated antibodies as adaptor molecules. We show that this CAR-NK cell can recognize and kill mimic HIV-infected cell lines expressing subtypes B and C gp160. We additionally find that anti-gp160 antibodies targeting membrane-distal epitopes (including V1/V2, V3, and CD4bs) are more likely to activate universal CAR-NK cells against gp160 + target cells, compared with those targeting membrane-proximal epitopes located in the gp41 MPER. Finally, we confirm that HIV-infected primary human CD4 + T cells can be effectively killed using the same approach. Given that numerous anti-gp160 antibodies with different antigen specificities are readily available, this modular universal CAR-NK cell platform can potentially overcome HIV diversity, thus providing a promising strategy to eradicate HIV-infected cells.

We report here on HIV-1 immunization results in rabbits and macaques co-immunized with clade C gp160 DNA and gp140 trimeric envelope vaccines, a strategy similar to a recent clinical trial that showed improved speed and magnitude of humoral responses. Clade C envelopes were isolated from CAP257, an individual who developed a unique temporal pattern of neutralization breadth development, comprising three separate "Waves" targeting distinct Env epitopes and different HIV clades. We used phylogeny and neutralization criteria to down-select envelope vaccine candidates, and confirmed antigenicity of our antigens by interaction with well-characterized broadly neutralizing monoclonal antibodies. Using these envelopes, we performed rabbit studies that screened for immunogenicity of CAP257 Envs from timepoints preceding peak neutralization breadth in each Wave. Selected CAP257 envelopes from Waves 1 and 2, during the first 2 years of infection that were highly immunogenic in rabbits were then tested in macaques. We found that in rabbits and macaques, co-immunization of DNA, and protein envelope-based vaccines induced maximum binding and neutralizing antibody titers with three immunizations. No further benefit was obtained with additional immunizations. The vaccine strategies recapitulated the Wave-specific epitope targeting observed in the CAP257 participant, and elicited Tier 1A, 1B, and Tier 2 heterologous neutralization. CAP257 envelope immunogens also induced the development of ADCC and T FH responses in macaques, and these responses positively correlated with heterologous neutralization. Together, the results from two animal models in this study have implications for identifying effective vaccine immunogens. We used a multi-step strategy to (1) select an Env donor with well-characterized neutralization breadth development; (2) study Env phylogeny for potential immunogens circulating near peak breadth timepoints during the first 2 years of infection; (3) test down-selected Envs for antigenicity; (4) screen down-selected Envs in an effective vaccine regimen in rabbits; and (5) advance the most immunogenic Envs to NHP studies. The results were an induction of high titers of HIV-1 envelope-specific antibodies with increasing avidity and cross-clade neutralizing antibodies with effector functions that together may improve the potential for protection in a pre-clinical SHIV model., (Copyright © 2020 Malherbe, Wibmer, Nonyane, Reed, Sather, Spencer, Schuman, Guo, Pandey, Robins, Park, Fuller, Sacha, Moore, Hessell and Haigwood.)

The HIV-1 envelope protein (Env) is the target of neutralizing antibodies and the template for vaccine immunogen design. The dynamic conformational equilibrium of trimeric Env influences its antigenicity and potential immunogenicity. Antibodies that bind at the trimer apex stabilize a "closed" conformation characteristic of the most difficult to neutralize isolates. A goal of vaccine development is therefore to mimic the closed conformation in a designed immunogen. A disulfide-stabilized, trimeric Env ectodomain-the "SOSIP" construct-has many of the relevant properties; it is also particularly suitable for structure determination. Some single-molecule studies have, however, suggested that the SOSIP trimer is not a good representation of Env on the surface of a virion or an infected cell. We isolated Env (fully cleaved to gp120 and gp41) from the surface of expressing cells using tagged, apex-binding Fab PG16 and determined the structure of the PG16-Env complex by cryo-EM to an overall resolution of 4.6 Å. Placing the only purification tag on the Fab ensured that the isolated Env was continuously stabilized in its closed, native conformation. The Env structure in this complex corresponds closely to the SOSIP structures determined by both x-ray crystallography and cryo-EM. Although the membrane-interacting elements are not resolved in our reconstruction, we can make inferences about the connection between ectodomain and membrane-proximal external region (MPER) by reference to the published cryo-tomography structure of an Env "spike" and the NMR structure of the MPER-transmembrane segment. We discuss these results in view of the conflicting interpretations in the literature., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Passive administration of HIV-directed broadly neutralizing antibodies (bNAbs) can prevent infection in animal models, and human efficacy trials are under way. Single-chain variable fragments (scFv), comprised of only the variable regions of antibody heavy and light chains, are smaller molecules that may offer advantages over full-length IgG. We designed and expressed scFv of HIV bNAbs prioritized for clinical testing that target the V2-apex (CAP256-VRC26.25), V3-glycan supersite (PGT121), CD4 binding site (3BNC117), and MPER (10E8v4). The use of either a 15- or 18-amino-acid glycine-serine linker between the heavy- and light-chain fragments provided adequate levels of scFv expression. When tested against a 45-multisubtype virus panel, all four scFv retained good neutralizing activity, although there was variable loss of function compared to the parental IgG antibodies. For CAP256-VRC26.25, there was a significant 138-fold loss of potency that was in part related to differential interaction with charged amino acids at positions 169 and 170 in the V2 epitope. Potency was reduced for the 3BNC117 (13-fold) and PGT121 (4-fold) scFv among viruses lacking the N276 and N332 glycans, respectively, and in viruses with a longer V1 loop for PGT121. This suggested that scFv interacted with their epitopes in subtly different ways, with variation at key residues affecting scFv neutralization more than the matched IgGs. Remarkably, the scFv of 10E8v4 maintained breadth of 100% with only a minor reduction in potency. Overall, scFv of clinically relevant bNAbs had significant neutralizing activity, indicating that they are suitable for passive immunization to prevent HIV-1 infection. IMPORTANCE Monoclonal antibodies have been isolated against conserved epitopes on the HIV trimer and are being investigated for passive immunization. Some of the challenges associated with full-sized antibody proteins may be overcome by using single-chain variable fragments (scFv). These smaller forms of antibodies can be produced more efficiently, may show fewer off-target effects with increased tissue penetration, and are more adaptable to vectored-mediated expression than IgG. Here, we demonstrate that scFv of four HIV-directed bNAbs (CAP256-VRC26.25, PGT121, 3BNC117, and 10E8v4) had significant neutralizing activity against diverse global strains of HIV. Loss of potency and/or breadth was shown to be due to increased dependence of the scFv on key residues within the epitope. These smaller antibody molecules with functional activity in the therapeutic range may be suitable for further development as passive immunity for HIV prevention., (Copyright © 2020 American Society for Microbiology.)

To develop an effective therapeutic vaccine against HIV-1, prediction of the most conserved epitopes derived from major proteins using bioinformatics tools is an alternative achievement. The epitope-driven vaccines against variable pathogens represented successful results. Hence, to overcome this hyper-variable virus, we designed the highly conserved and immunodominant peptide epitopes. Two servers were used to predict peptide-MHC-I binding affinity including NetMHCpan4.0 and Syfpeithi servers. The NetMHCIIpan3.2 server was utilized for MHC-II binding affinity. Then, we determined immunogenicity scores and allergenicity by the IEDB immunogenicity predictor and Algpred, respectively. Next, for estimation of toxicity and population coverage, ToxinPred server and IEDB population coverage tool were applied. After that, the MHC-peptide binding was investigated by GalexyPepDock peptide-protein flexible docking server. Finally, two different DNA and peptide constructs containing Nef-Vif-Gp160-P24 and Nef-Vpu-Gp160-P24 were prepared and complexed with four various cell penetrating peptides (CPPs) for delivery into mammalian cells (MPG and HR9 CPPs for DNA delivery, and CyLoP-1 and LDP-NLS CPPs for protein delivery). Our results indicated that the designed DNA and peptide constructs could form non-covalent stable nanoparticles at certain ratios as observed by scanning electron microscope (SEM) and Zetasizer. The flow cytometry results obtained from in vitro transfection of the nanoparticles into HEK-293T cell lines showed that the percentage of GFP expressing cells was about 38.38 ± 1.34%, 25.36% ± 0.30, 54.95% ± 0.84, and 25.11% ± 0.36 for MPG/pEGFP-nef-vif-gp160-p24, MPG/pEGFP-nef-vpu-gp160-p24, HR9/pEGFP-nef-vif-gp160-p24 and HR9/pEGFP-nef-vpu-gp160-p24, respectively. Thus, these data showed that the DNA construct harboring nef-vif-gp160-p24 multi-epitope gene had higher efficiency than the DNA construct harboring nef-vpu-gp160-p24 multi-epitope gene to penetrate into the cells. Moreover, delivery of the recombinant Nef-Vif-Gp160-P24 and Nef-Vpu-Gp160-P24 polyepitope peptides in HEK-293T cells was confirmed as a single band about 32 kDa using western blot analysis. Although, both DNA and peptide constructs could be successfully transported by a variety of CPPs into the cells, but the difference between them in transfection rate will influence the levels of immune responses for development of therapeutic vaccines., Competing Interests: The authors have declared that no competing interests exist.

Human anti-HIV-1 broadly neutralizing antibodies (bNAbs) protect against infection in animal models. However, bNAbs have not been elicited by vaccination in diverse wild-type animals or humans, in part because B cells expressing the precursors of these antibodies do not recognize most HIV-1 envelopes (Envs). Immunogens have been designed that activate these B cell precursors in vivo, but they also activate competing off-target responses. Here we report on a complementary approach to expand specific B cells using an anti-idiotypic antibody, iv8, that selects for naive human B cells expressing immunoglobulin light chains with 5-amino acid complementarity determining region 3s, a key feature of anti-CD4 binding site (CD4bs)-specific VRC01-class antibodies. In mice, iv8 induced target cells to expand and mature in the context of a polyclonal immune system and produced serologic responses targeting the CD4bs on Env. In summary, the results demonstrate that an anti-idiotypic antibody can specifically recognize and expand rare B cells that express VRC01-class antibodies against HIV-1., (© 2019 Dosenovic et al.)

Background: Laboratory assays for determining recent HIV-1 infection are an important public health tool for aiding in the estimation of HIV incidence. Some incidence assay analytes are remarkably predictive of time since seroconversion and may be useful for additional applications, such as predicting recent transmission events during HIV outbreaks and informing prevention strategies., Methods: Plasma samples (n = 154) from a recent HIV-1 outbreak in a rural community in Indiana were tested with the customized HIV-1 Multiplex assay, based on the Bio-Rad Bio-Plex platform, which measures antibody response to HIV envelope antigens, gp120, gp160, and gp41. Assay cutoffs for each analyte were established to determine whether an individual seroconverted within 30, 60, or 90 days of the sample collection date. In addition, a novel bioinformatics method was implemented to infer infection dates of persons newly diagnosed with HIV during the outbreak., Results: Sensitivity/specificity of the HIV-1 Multiplex assay for predicting seroconversion within 30, 60, and 90 days, based on a training data set, was 90.5%/95.4%, 94.1%/90%, and 89.4%/82.9%, respectively. Of 154 new diagnoses in Indiana between December 2014 and August 2016, the majority (71%) of recent infections (≤3 months since seroconversion) were identified between February and May 2016. The epidemiologic curve derived from the bioinformatics analysis indicated HIV transmission began as early as 2010, grew exponentially in 2014, and leveled off in April 2015., Conclusions: The HIV-1 Multiplex assay has the potential to identify and monitor trends in recent infection during an epidemic to assess the efficacy of programmatic or treatment interventions.

The broadly neutralizing antibody (bnAb) VRC01 is being evaluated for its efficacy to prevent HIV-1 infection in the Antibody Mediated Prevention (AMP) trials. A secondary objective of AMP utilizes sieve analysis to investigate how VRC01 prevention efficacy (PE) varies with HIV-1 envelope (Env) amino acid (AA) sequence features. An exhaustive analysis that tests how PE depends on every AA feature with sufficient variation would have low statistical power. To design an adequately powered primary sieve analysis for AMP, we modeled VRC01 neutralization as a function of Env AA sequence features of 611 HIV-1 gp160 pseudoviruses from the CATNAP database, with objectives: (1) to develop models that best predict the neutralization readouts; and (2) to rank AA features by their predictive importance with classification and regression methods. The dataset was split in half, and machine learning algorithms were applied to each half, each analyzed separately using cross-validation and hold-out validation. We selected Super Learner, a nonparametric ensemble-based cross-validated learning method, for advancement to the primary sieve analysis. This method predicted the dichotomous resistance outcome of whether the IC50 neutralization titer of VRC01 for a given Env pseudovirus is right-censored (indicating resistance) with an average validated AUC of 0.868 across the two hold-out datasets. Quantitative log IC50 was predicted with an average validated R2 of 0.355. Features predicting neutralization sensitivity or resistance included 26 surface-accessible residues in the VRC01 and CD4 binding footprints, the length of gp120, the length of Env, the number of cysteines in gp120, the number of cysteines in Env, and 4 potential N-linked glycosylation sites; the top features will be advanced to the primary sieve analysis. This modeling framework may also inform the study of VRC01 in the treatment of HIV-infected persons., Competing Interests: The authors have declared that no competing interests exist.

Several broadly neutralizing antibodies (bNAbs) that can target HIV strains with large degrees of variability have recently been identified. However, efforts to induce synthesis of such bNAbs that can protect against HIV infection have not met with much success. Identification of specific epitopes encoded in the HIV-1 envelope (Env) that can direct the host to synthesize bNAbs remains a challenge. In a previous study, we identified 12 antiretroviral therapy-naive HIV-1-infected individuals whose plasma exhibited broad cross-clade neutralization property against different clades of HIV-1. In this study, we sequenced the full-length HIV-1 gp160 from 11 of these individuals and analyzed the sequences to identify bNAb epitopes. We identified critical residues in the viral envelopes that contribute to the formation of conformational epitopes and possibly induce the production of bNAbs, using in silico methods. We found that many of the sequences had conserved glycans at positions N160 (10/11) and N332 (9/11), which are known to be critical for the binding of PG9/PG16-like and PGT128-like bNAbs, respectively. We also observed conservation of critical glycans at positions N234 and N276 critical for the interaction with CD4 binding site bNAbs in 8/11 and 11/11 sequences, respectively. We modeled the three-dimensional structure of the 11 HIV-1 envelopes and found that though each had structural differences, the critical residues were mostly present on the surface of the Env structures. The identified critical residues are proposed as candidates for further evaluation as bNAb epitopes.

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.

Vaccine-elicited immunoglobulin G (IgG) has been shown to be important for protection against simian-human immunodeficiency virus (SHIV) infection in rhesus monkeys. However, it remains unclear whether vaccine-elicited IgA responses are beneficial or detrimental for protection. In this study, we evaluated the kinetics, magnitude, breadth, and linear epitope specificities of vaccine-elicited IgG and IgA responses in serum and mucosal secretions following intramuscular immunization with adenovirus 26 (Ad26) prime, Env protein boost vaccination regimens. The systemic and mucosal antibody responses exhibited kinetics similar to those of the serum antibody responses but lower titers than the serum antibody responses. Moreover, the IgG and IgA responses were correlated, both in terms of the magnitude of the responses and in terms of the antibody specificities against linear human immunodeficiency virus type 1 (HIV-1) Env, Gag, and Pol epitopes. These data suggest that IgG and IgA responses are highly coordinated in both peripheral blood and mucosal compartments following Ad26/Env vaccination in rhesus monkeys. IMPORTANCE Vaccine-elicited IgG responses are important for protection against simian-human immunodeficiency virus (SHIV) infection in nonhuman primates. However, much less is known about the role and function of IgA, despite it being the predominant antibody in mucosal sites. There is debate as to whether HIV-1-specific IgA responses are beneficial or detrimental, since serum anti-Env IgA titers were shown to be inversely correlated with protection in the RV144 clinical trial. We thus assessed vaccine-elicited IgG and IgA antibody responses in peripheral blood and mucosal secretions following vaccination with the Ad26/Env vaccine., (Copyright © 2018 Kang et al.)

The HIV-1 envelope (Env) spike is a trimer of gp120/gp41 heterodimers that mediates viral entry. Binding to CD4 on the host cell membrane is the first essential step for infection but disrupts the native antigenic state of Env, posing a key obstacle to vaccine development. We locked the HIV-1 Env trimer in a pre-fusion configuration, resulting in impaired CD4 binding and enhanced binding to broadly neutralizing antibodies. This design was achieved via structure-guided introduction of neo-disulfide bonds bridging the gp120 inner and outer domains and was successfully applied to soluble trimers and native gp160 from different HIV-1 clades. Crystallization illustrated the structural basis for CD4-binding impairment. Immunization of rabbits with locked trimers from two different clades elicited neutralizing antibodies against tier-2 viruses with a repaired glycan shield regardless of treatment with a functional CD4 mimic. Thus, interdomain stabilization provides a widely applicable template for the design of Env-based HIV-1 vaccines., (Published by Elsevier Inc.)

Traditional restriction endonuclease-based cloning has been routinely used to generate replication-competent simian-human immunodeficiency viruses (SHIV) and simian tropic HIV (stHIV). This approach requires the existence of suitable restriction sites or the introduction of nucleotide changes to create them. Here, using an In-Fusion cloning technique that involves homologous recombination, we generated SHIVs and stHIVs based on epidemiologically linked clade C transmitted/founder HIV molecular clones from Zambia. Replacing vif from these HIV molecular clones with vif of SIVmac239 resulted in chimeric genomes used to generate infectious stHIV viruses. Likewise, exchanging HIV env genes and introducing N375 mutations to enhance macaque CD4 binding site and cloned into a SHIVAD8-EO backbone. The generated SHIVs and stHIV were infectious in TZMbl and ZB5 cells, as well as macaque PBMCs. Therefore, this method can replace traditional methods and be a valuable tool for the rapid generation and testing of molecular clones of stHIV and SHIV based on primary clinical isolates will be valuable to generate rapid novel challenge viruses for HIV vaccine/cure studies.

The subtype C HIV-1 isolate MW965.26 is a highly neutralization-sensitive tier 1a primary isolate that is widely used in vaccine studies, but the basis for the sensitive neutralization phenotype of this isolate is not known. Substituting the MW965.26 V1/V2 domain into a neutralization-sensitive SF162 Env clone resulted in high resistance to standard anti-V3 monoclonal antibodies, demonstrating that this region possesses strong masking activity in a standard Env backbone and indicating that determinants elsewhere in MW965.26 Env are responsible for its unusual neutralization sensitivity. Key determinants for this phenotype were mapped by generating chimeric Envs between MW965.26 Env and a typical resistant Env clone, the consensus C (ConC) clone, and localized to two residues, Cys384 in the C3 domain and Asn502 in the C5 domain. Substituting the sensitizing mutations Y384C and K502N at these positions into several resistant primary Envs resulted in conversion to neutralization-sensitive phenotypes, demonstrating the generalizability of this effect. In contrast to the sensitizing effects of these substitutions on normally masked epitopes, these mutations reduced the sensitivity of VRC01-like epitopes overlapping the CD4-binding domain, while they had no effect on several other classes of broadly neutralizing epitopes, including members of several lineages of V2-dependent quaternary epitopes and representatives of N332 glycan-dependent epitopes (PGT121) and quaternary, cleavage-dependent epitopes centered at the gp41-gp120 interface on intact HIV-1 Env trimers (PGT151). These results identify novel substitutions in gp120 that regulate the expression of alternative conformations of Env and differentially affect the exposure of different classes of epitopes, thereby influencing the neutralization phenotype of primary HIV-1 isolates. IMPORTANCE A better understanding of the mechanisms that determine the wide range of neutralization sensitivity of circulating primary HIV-1 isolates would provide important information about the natural structural and conformational diversity of HIV-1 Env and how this affects the neutralization phenotype. A useful way of studying this is to determine the molecular basis for the unusually high neutralization sensitivities of the limited number of available tier 1a viruses. This study localized the neutralization sensitivity of MW965.26, an extremely sensitive subtype C-derived primary isolate, to two rare substitutions in the C3 and C5 domains and demonstrated that the sequences at these positions differentially affect the presentation of epitopes recognized by different classes of standard and conformation-dependent broadly neutralizing antibodies. These results provide novel insight into how these regions regulate the neutralization phenotype and provide tools for controlling the Env conformation that could have applications both for structural studies and in vaccine design., (Copyright © 2018 American Society for Microbiology.)

HIV is a highly mutable virus, and over 30 years after its discovery, a vaccine or cure is still not available. The isolation of broadly neutralizing antibodies (bnAbs) from HIV-infected patients has led to renewed hope for a prophylactic vaccine capable of combating the scourge of HIV. A major challenge is the design of immunogens and vaccination protocols that can elicit bnAbs that target regions of the virus's spike proteins where the likelihood of mutational escape is low due to the high fitness cost of mutations. Related challenges include the choice of combinations of bnAbs for therapy. An accurate representation of viral fitness as a function of its protein sequences (a fitness landscape), with explicit accounting of the effects of coupling between mutations, could help address these challenges. We describe a computational approach that has allowed us to infer a fitness landscape for gp160, the HIV polyprotein that comprises the viral spike that is targeted by antibodies. We validate the inferred landscape through comparisons with experimental fitness measurements, and various other metrics. We show that an effective antibody that prevents immune escape must selectively bind to high escape cost residues that are surrounded by those where mutations incur a low fitness cost, motivating future applications of our landscape for immunogen design., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Soluble envelope glycoprotein (Env) trimers (SOSIP.664 gp140) are attractive HIV-1 vaccine candidates, with structures that mimic the native membrane-bound Env spike (gp160). Since engineering trimers can be limited by the difficulty of rationally predicting beneficial mutations, here we used a more comprehensive mutagenesis approach with the goal of identifying trimer variants with improved antigenic and stability properties. We created 341 cysteine pairs at predicted points of stabilization throughout gp140, 149 proline residue substitutions at every residue of the gp41 ectodomain, and 362 space-filling residue substitutions at every hydrophobic and aromatic residue in gp140. The parental protein target, the clade B strain B41 SOSIP.664 gp140, does not bind the broadly neutralizing antibody PGT151 and so was used here to identify improved variants that also provide insight into the structural basis for Env antigenicity. Each of the 852 mutants was expressed in human cells and screened for antigenicity using four different monoclonal antibodies (MAbs), including PGT151. We identified 29 trimer variants with antigenic improvements derived from each of the three mutagenesis strategies. We selected four variants (Q203F, T538F, I548F, and M629P) for more comprehensive biochemical, structural, and antigenicity analyses. The T538F substitution had the most beneficial effect overall, including restoration of the PGT151 epitope. The improved B41 SOSIP.664 trimer variants identified here may be useful for vaccine and structural studies. IMPORTANCE Soluble Env trimers have become attractive HIV-1 vaccine candidates, but the prototype designs are capable of further improvement through protein engineering. Using a high-throughput screening technology (shotgun mutagenesis) to create and evaluate 852 variants, we were able to identify sequence changes that were beneficial to the antigenicity and stability of soluble trimers based on the clade B B41 env gene. The strategies described here may be useful for identifying a wider range of antigenically and structurally improved soluble trimers based on multiple genotypes for use in programs intended to create a broadly protective HIV-1 vaccine., (Copyright © 2017 American Society for Microbiology.)

Immune control of human immunodeficiency virus type 1 (HIV) infection is typically associated with effective Gag-specific CD8 + T-cell responses. We here focus on HLA-B*14, which protects against HIV disease progression, but the immunodominant HLA-B*14-restricted anti-HIV response is Env specific (ERYLKDQQL, HLA-B*14-EL9). A subdominant HLA-B*14-restricted response targets Gag (DRYFKTLRA, HLA-B*14-DA9). Using HLA-B*14/peptide-saporin-conjugated tetramers, we show that HLA-B*14-EL9 is substantially more potent at inhibiting viral replication than HLA-B*14-DA9. HLA-B*14-EL9 also has significantly higher functional avidity ( P < 0.0001) and drives stronger selection pressure on the virus than HLA-B*14-DA9. However, these differences were HLA-B*14 subtype specific, applying only to HLA-B*14:02 and not to HLA-B*14:01. Furthermore, the HLA-B*14-associated protection against HIV disease progression is significantly greater for HLA-B*14:02 than for HLA-B*14:01, consistent with the superior antiviral efficacy of the HLA-B*14-EL9 response. Thus, although Gag-specific CD8 + T-cell responses may usually have greater anti-HIV efficacy, factors independent of protein specificity, including functional avidity of individual responses, are also critically important to immune control of HIV. IMPORTANCE In HIV infection, although cytotoxic T lymphocytes (CTL) play a potentially critical role in eradication of viral reservoirs, the features that constitute an effective response remain poorly defined. We focus on HLA-B*14, unique among HLAs associated with control of HIV in that the dominant CTL response is Env specific, not Gag specific. We demonstrate that Env-specific HLA-B*14-restricted activity is substantially more efficacious than the subdominant HLA-B*14-restricted Gag response. Env immunodominance over Gag and strong Env-mediated selection pressure on HIV are observed only in subjects expressing HLA-B*14:02, and not HLA-B*14:01. This reflects the increased functional avidity of the Env response over Gag, substantially more marked for HLA-B*14:02. Finally, we show that HLA-B*14:02 is significantly more strongly associated with viremic control than HLA-B*14:01. These findings indicate that, although Gag-specific CTL may usually have greater anti-HIV efficacy than Env responses, factors independent of protein specificity, including functional avidity, may carry greater weight in mediating effective control of HIV., (Copyright © 2017 Leitman et al.)

Induction of broadly neutralizing antibodies (bNAbs) by HIV-1 envelope glycoprotein immunogens would be a major advance toward an effective vaccine. A critical step in this process is the activation of naive B cells expressing germline (gl) antibody precursors that have the potential to evolve into bNAbs. Here, we reengineered the BG505 SOSIP.664 glycoprotein to engage gl precursors of bNAbs that target either the trimer apex or the CD4-binding site. The resulting BG505 SOSIP.v4.1-GT1 trimer binds multiple bNAb gl precursors in vitro. Immunization experiments in knock-in mice expressing gl-VRC01 or gl-PGT121 show that this trimer activates B cells in vivo, resulting in the secretion of specific antibodies into the sera. A crystal structure of the gl-targeting trimer at 3.2-Å resolution in complex with neutralizing antibodies 35O22 and 9H+109L reveals a native-like conformation and the successful incorporation of design features associated with binding of multiple gl-bNAb precursors., (© 2017 Medina-Ramírez et al.)

No immunogen to date has reliably elicited broadly neutralizing antibodies to HIV in humans or animal models. Advances in the design of immunogens that antigenically mimic the HIV envelope glycoprotein (Env), such as the soluble cleaved trimer BG505 SOSIP, have improved the elicitation of potent isolate-specific antibody responses in rabbits and macaques, but so far failed to induce broadly neutralizing antibodies. One possible reason for this failure is that the relevant antibody repertoires are poorly suited to target the conserved epitope regions on Env, which are somewhat occluded relative to the exposed variable epitopes. Here, to test this hypothesis, we immunized four cows with BG505 SOSIP. The antibody repertoire of cows contains long third heavy chain complementary determining regions (HCDR3) with an ultralong subset that can reach more than 70 amino acids in length. Remarkably, BG505 SOSIP immunization resulted in rapid elicitation of broad and potent serum antibody responses in all four cows. Longitudinal serum analysis for one cow showed the development of neutralization breadth (20%, n = 117 cross-clade isolates) in 42 days and 96% breadth (n = 117) at 381 days. A monoclonal antibody isolated from this cow harboured an ultralong HCDR3 of 60 amino acids and neutralized 72% of cross-clade isolates (n = 117) with a potent median IC 50 of 0.028 μg ml -1 . Breadth was elicited with a single trimer immunogen and did not require additional envelope diversity. Immunization of cows may provide an avenue to rapidly generate antibody prophylactics and therapeutics to address disease agents that have evolved to avoid human antibody responses.

Previously, VLPs bearing JR-FL strain HIV-1 Envelope trimers elicited potent neutralizing antibodies (nAbs) in 2/8 rabbits (PLoS Pathog 11(5): e1004932) by taking advantage of a naturally absent glycan at position 197 that borders the CD4 binding site (CD4bs). In new immunizations, we attempted to improve nAb responses by removing the N362 glycan that also lines the CD4bs. All 4 rabbits developed nAbs. One targeted the N197 glycan hole like our previous sera. Two sera depended on the N463 glycan, again suggesting CD4bs overlap. Heterologous boosts appeared to reduce nAb clashes with the N362 glycan. The fourth serum targeted a N362 glycan-sensitive epitope. VLP manufacture challenges prevented us from immunizing larger rabbit numbers to empower a robust statistical analysis. Nevertheless, trends suggest that targeted glycan removal may improve nAb induction by exposing new epitopes and that it may be possible to modify nAb specificity using rational heterologous boosts., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The extraordinary genetic diversity of the HIV-1 envelope spike [Env; trimeric (gp160) 3 , cleaved to (gp120/gp41) 3 ] poses challenges for vaccine development. Envs of different clinical isolates exhibit different sensitivities to antibody-mediated neutralization. Envs of difficult-to-neutralize viruses are thought to be more stable and conformationally homogeneous trimers than those of easy-to-neutralize viruses, thereby providing more effective concealment of conserved, functionally critical sites. In this study we have characterized the antigenic properties of an Env derived from one of the most neutralization-resistant HIV-1 isolates, CH120.6. Sequence variation at neutralizing epitopes does not fully account for its exceptional resistance to antibodies. The full-length, membrane-bound CH120.6 Env is indeed stable and conformationally homogeneous. Its antigenicity correlates closely with its neutralization sensitivity, and major changes in antigenicity upon CD4 engagement appear to be restricted to the coreceptor site. The CH120.6 gp140 trimer, the soluble and uncleaved ectodomain of (gp160) 3 , retains many antigenic properties of the intact Env, consistent with a conformation close to that of Env spikes on a virion, whereas its monomeric gp120 exposes many nonneutralizing or strain-specific epitopes. Thus, trimer organization and stability are important determinants not only for occluding many epitopes but also for conferring resistance to neutralization by all but a small set of antibodies. Env preparations derived from neutralization-resistant viruses may induce irrelevant antibody responses less frequently than do other Envs and may be excellent templates for developing soluble immunogens., Competing Interests: Conflict of interest statement: S.K-.G., S.T., C.E.L., and A.C. were employees of Novartis Vaccines and Diagnostics at the time of the study. Following the acquisition of Novartis Vaccines and Diagnostics by the GlaxoSmithKline (GSK) group of companies in March 2015, S.K.-G., S.T., C.E.L., and A.C. became employees of the GSK group of companies. A.C. reports ownership of GSK shares. M.S. and W. Weissenhorn (reviewer) are coauthors of several papers (the last in 2014). The role of M.S. in the work in those papers was to provide neutralization analyses, and he does not have an active research collaboration with W. Weissenhorn.

HIV-1 envelope spike (Env) is a heavily glycosylated, type I membrane protein that mediates fusion of viral and cell membranes to initiate infection. It is also a primary target of neutralizing antibodies and thus an important candidate for vaccine development. We have recently reported a nuclear magnetic resonance structure of the transmembrane (TM) domain of HIV-1 Env reconstituted in a membrane-like environment. Taking HIV-1 as an example, we discuss here how a TM domain can anchor, stabilize, and modulate a viral envelope spike and how its high-resolution structure can contribute to understanding viral membrane fusion and to immunogen design., (© 2016 Federation of European Biochemical Societies.)

The HIV gp160 envelope fusion protein is situated in the viral membrane and mediates virus entry into its host cell. Increasing evidence suggests that virtually all parts of the HIV envelope are structurally and functionally dependent on membranes. Protein-lipid interactions and membrane properties influence the dynamics of a manifold of gp160 biological activities such as membrane fusion, immune suppression and gp160 incorporation into virions during HIV budding and assembly. In the following we will summarize our current understanding of this interdependence between membrane interaction, structural conformation and functionality of the different gp160 domains. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider., (Copyright © 2016. Published by Elsevier B.V.)

The envelope (Env) glycoprotein of HIV is the only intact viral protein expressed on the surface of both virions and infected cells. Env is the target of neutralizing antibodies (Abs) and has been the subject of intense study in efforts to produce HIV vaccines. Therapeutic anti-Env Abs can also exert antiviral effects via Fc-mediated effector mechanisms or as cytotoxic immunoconjugates, such as immunotoxins (ITs). In the course of screening monoclonal antibodies (MAbs) for their ability to deliver cytotoxic agents to infected or Env-transfected cells, we noted disparities in their functional activities. Different MAbs showed diverse functions that did not correlate with each other. For example, MAbs against the external loop region of gp41 made the most effective ITs against infected cells but did not neutralize virus and bound only moderately to the same cells that they killed so effectively when they were used in ITs. There were also differences in IT-mediated killing among transfected and infected cell lines that were unrelated to the binding of the MAb to the target cells. Our studies of a well-characterized antigen demonstrate that MAbs against different epitopes have different functional activities and that the binding of one MAb can influence the interaction of other MAbs that bind elsewhere on the antigen. These results have implications for the use of MAbs and ITs to kill HIV-infected cells and eradicate persistent reservoirs of HIV infection., Importance: There is increased interest in using antibodies to treat and cure HIV infection. Antibodies can neutralize free virus and kill cells already carrying the virus. The virus envelope (Env) is the only HIV protein expressed on the surfaces of virions and infected cells. In this study, we examined a panel of human anti-Env antibodies for their ability to deliver cell-killing toxins to HIV-infected cells and to perform other antiviral functions. The ability of an antibody to make an effective immunotoxin could not be predicted from its other functional characteristics, such as its neutralizing activity. Anti-HIV immunotoxins could be used to eliminate virus reservoirs that persist despite effective antiretroviral therapy., (Copyright © 2017 American Society for Microbiology.)

The envelope (Env) glycoprotein of HIV is expressed on the surface of productively infected cells and can be used as a target for cytotoxic immunoconjugates (ICs), in which cell-killing moieties, including toxins, drugs, or radionuclides, are chemically or genetically linked to monoclonal antibodies (MAbs) or other targeting ligands. Such ICs could be used to eliminate persistent reservoirs of HIV infection. We have found that MAbs which bind to the external loop of gp41, e.g., MAb 7B2, make highly effective ICs, particularly when used in combination with soluble CD4. We evaluated the toxicity, immunogenicity, and efficacy of the ICs targeted with 7B2 in mice and in simian-human immunodeficiency virus-infected macaques. In the macaques, we tested immunotoxins (ITs), consisting of protein toxins bound to the targeting agent. ITs were well tolerated and initially efficacious but were ultimately limited by their immunogenicity. In an effort to decrease immunogenicity, we tested different toxic moieties, including recombinant toxins, cytotoxic drugs, and tubulin inhibitors. ICs containing deglycosylated ricin A chain prepared from ricin toxin extracted from castor beans were the most effective in killing HIV-infected cells. Having identified immunogenicity as a major concern, we show that conjugation of IT to polyethylene glycol limits immunogenicity. These studies demonstrate that cytotoxic ICs can target virus-infected cells in vivo but also highlight potential problems to be addressed., Importance: It is not yet possible to cure HIV infection. Even after years of fully effective antiviral therapy, a persistent reservoir of virus-infected cells remains. Here we propose that a targeted conjugate consisting of an anti-HIV antibody bound to a toxic moiety could function to kill the HIV-infected cells that constitute this reservoir. We tested this approach in HIV-infected cells grown in the lab and in animal infections. Our studies demonstrated that these immunoconjugates are effective both in vitro and in test animals. In particular, ITs constructed with the deglycosylated A chain prepared from native ricin were the most effective in killing cells, but their utility was blunted because they provoked immune reactions that interfered with the therapeutic effects. We then demonstrated that coating of the ITs with polyethylene glycol minimized the immunogenicity, as has been demonstrated with other protein therapies., (Copyright © 2017 American Society for Microbiology.)

The induction by vaccination of broadly neutralizing antibodies (bNAbs) capable of neutralizing various HIV-1 viral strains is challenging, but understanding how a subset of HIV-infected individuals develops bNAbs may guide immunization strategies. Here, we describe the isolation and characterization of the bNAb ACS202 from an elite neutralizer that recognizes a new, trimer-specific and cleavage-dependent epitope at the gp120-gp41 interface of the envelope glycoprotein (Env), involving the glycan N88 and the gp41 fusion peptide. In addition, an Env trimer, AMC011 SOSIP.v4.2, based on early virus isolates from the same elite neutralizer, was constructed, and its structure by cryo-electron microscopy at 6.2 Å resolution reveals a closed, pre-fusion conformation similar to that of the BG505 SOSIP.664 trimer. The availability of a native-like Env trimer and a bNAb from the same elite neutralizer provides the opportunity to design vaccination strategies aimed at generating similar bNAbs against a key functional site on HIV-1.

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.

While the search for an efficacious HIV-1 vaccine remains elusive, emergence of a new generation of virus-neutralizing monoclonal antibodies (mAbs) has re-ignited the field of passive immunization for HIV-1 prevention. However, the plasticity of HIV-1 demands additional improvements to these mAbs to better ensure their clinical utility. Here, we report engineered bispecific antibodies that are the most potent and broad HIV-neutralizing antibodies to date. One bispecific antibody, 10E8V2.0/iMab, neutralized 118 HIV-1 pseudotyped viruses tested with a mean 50% inhibitory concentration (IC50) of 0.002 μg/mL. 10E8V2.0/iMab also potently neutralized 99% of viruses in a second panel of 200 HIV-1 isolates belonging to clade C, the dominant subtype accounting for ∼50% of new infections worldwide. Importantly, 10E8V2.0/iMab reduced virus load substantially in HIV-1-infected humanized mice and also provided complete protection when administered prior to virus challenge. These bispecific antibodies hold promise as novel prophylactic and/or therapeutic agents in the fight against HIV-1., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Prime-boost regimens comprising ALVAC-HIV (prime) and human immunodeficiency virus type 1 (HIV) Env (boost) induce HIV-specific neutralizing antibody and cell-mediated immune responses, but the impact of boost schedule and adjuvant requires further definition., Methods: A phase 1 trial was conducted. In part A (open label), 19 volunteers received oligomeric glycoprotein 160 from HIV strains MN and LAI-2 (ogp160 MN/LAI-2) with dose escalation (25, 50, 100 μg) and either polyphosphazene (pP) or alum adjuvant. In part B, 72 volunteers received either placebo (n=12) or recombinant canarypox virus expressing HIV antigens (ALVAC-HIV [vCP205]) with different doses and schedules of ogp160 MN/LAI-2 in pP or alum (n = 60)., Results: The vaccines were safe and well tolerated, with no vaccine-related serious adverse events. Anti-gp70 V1V2 antibody responses were detected in 17 of 19 part A volunteers (89%) and 10%-100% of part B volunteers. Use of a peripheral blood mononuclear cell-based assay revealed that US-1 primary isolate neutralization was induced in 2 of 19 recipients of ogp160 protein alone (10.5%) and 5 of 49 prime-boost volunteers (10.2%). Among ogp160 recipients, those who received pP were more likely than those who received alum to have serum that neutralized tier 2 viruses (12% vs 0%; P = .015)., Conclusions: Administration of ogp160 with pP induces primary isolate tier 2 neutralizing antibody responses in a small percentage of volunteers, demonstrating proof of concept and underscoring the importance of further optimization of prime-boost strategies for HIV infection prevention., Clinical Trials Registration: NCT00004579., (Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

The partial success of RV144 human clinical trial demonstrated that ALVAC prime/envelope protein boost vaccine regimen may represent a promising strategy for the development of an effective HIV-1 vaccine. Our earlier study demonstrated that a trimeric HIV-1 envelope gp145 from an Indian clade C isolate elicited cross clade neutralizing antibodies primarily towards Tier 1 isolates. In the present study, we examined the immunogenicity of DNA prime/envelope protein boost vaccine in rabbits using gp160 DNA of the Indian clade C isolate with various cytoplasmic tail truncations and trimeric gp145 protein. Cytoplasmic tail mutants of gp160 exposed epitopes that reacted strongly with a number of broadly neutralizing human monoclonal antibodies against HIV-1. Overall, envelope specific titers were found to be similar in all rabbit groups with higher pseudovirus neutralization in protein only immunized rabbits. The complete linear epitope mapping of rabbit immune sera revealed strong binding to C1, C2, V3, C3 and C4 domains of gp145. Importantly, reactivity of gp41 ecto-domain peptides was observed in DNA prime/protein boost sera but not in the sera of rabbits immunized with protein alone. Moreover, membrane anchored but not soluble envelope encoding DNA immunization elicited antibodies against linear epitopes on the conserved gp41 ecto-domain. Together, these results suggest that priming with DNA encoding cytoplasmic domains of Env alters the quality of antibodies elicited following protein boost and hence may be utilized to generate protective immunity by HIV-1 vaccine., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Human immunodeficiency virus type 1 (HIV-1) acquisition occurs predominantly through mucosal transmission. We hypothesized that greater mucosal immune responses and protective efficacy against mucosal HIV-1 infection may be achieved by prime-boost immunization at mucosal sites. We used a macaque model to determine the safety, immunogenicity, and protective efficacy of orally delivered, replication-competent but attenuated recombinant vaccinia viruses expressing full-length HIV-1 SF162 envelope (Env) or simian immunodeficiency virus (SIV) Gag-Pol proteins. We examined the dose and route that are suitable for oral immunization with recombinant vaccinia viruses. We showed that sublingual inoculation of two vaccinia virus-naive pigtailed macaques with 5 × 10(8) PFU of recombinant vaccinia viruses was safe. However, sublingual inoculation with a higher dose or tonsillar inoculation resulted in secondary oral lesions, indicating the need to optimize the dose and route for oral immunization with replication-competent vaccinia virus vectors. Oral priming alone elicited antibody responses to vaccinia virus and to the SF162 Env protein. Intramuscular immunization with the SF162 gp120 protein at either 20 or 21 weeks postpriming resulted in a significant boost in antibody responses in both systemic and mucosal compartments. Furthermore, we showed that immune responses induced by recombinant vaccinia virus priming and intramuscular protein boosting provided protection against intrarectal challenge with the simian-human immunodeficiency virus SHIV-SF162-P4., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The human immunodeficiency virus (HIV) is the causative agent of acquired immune deficiency syndrome (AIDS) and is thus responsible for significant morbidity and mortality worldwide. Despite considerable effort, preparation of an effective vaccine for AIDS has been elusive and it has become clear that a fundamental understanding of the relevant antigenic targets on HIV is essential. The Env trimer spike is the only viral antigen present on the surface of the viral particle and it is the target of all broadly neutralizing antibodies isolated to date. Thus, a soluble, homogeneous, and well-defined preparation of Env trimers is an important first step toward biochemical and structural characterization of the antigenic spike. Phospholipid bilayer nanodiscs represent a relatively new technology that can serve as a platform for the assembly of membrane proteins into a native membrane-like environment. Here we describe the preparation and characterization of unprocessed full-length, natively glycoslyated gp160 Env proteins incorporated into nanodiscs (gp160-ND). The particles are soluble and well defined in the absence of detergent, and possess a morphology anticipated of Env incorporated into a lipid ND. Importantly, the gp160-NDs retain CD4 and Env antibody binding characteristics expected of a functional trimer spike and their incorporation into a lipid membrane allows interrogation of epitopes associated with the membrane-proximal ectodomain region of gp41. These studies provide the groundwork for the use of gp160-ND in more detailed biochemical and structural studies that may set the stage for their use in vaccine development., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Newcastle disease virus (NDV) expressing HIV-1 BaL gp160 was evaluated either alone or with monomeric BaL gp120 and BaL SOSIP gp140 protein in a prime-boost combination in guinea pigs to enhance envelope (Env)-specific humoral and mucosal immune responses. We showed that a regimen consisting of an NDV prime followed by a protein boost elicited stronger serum and mucosal Th-1-biased IgG responses and neutralizing antibody responses than NDV-only immunizations. Additionally, these responses were higher after the gp120 than after the SOSIP gp140 protein boost., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: HIV-1 is typically CCR5 using (R5) and T cell tropic (T-tropic), targeting memory CD4(+) T cells throughout acute and chronic infections. However, viruses can expand into alternative cells types. Macrophage-tropic (M-tropic) HIV-1 variants have evolved to infect macrophages, which have only low levels of surface CD4. Most M-tropic variants have been isolated from the central nervous system during late-stage chronic infection. We used the HIV-1 env genes of well-defined, subject-matched M-tropic and T-tropic viruses to characterize the phenotypic features of the M-tropic Env protein. We found that, compared to T-tropic viruses, M-tropic viruses infect monocyte-derived macrophages (MDMs) on average 28-fold more efficiently, use low-density CD4 more efficiently, have increased sensitivity to soluble CD4 (sCD4), and show trends toward sensitivity to some CD4 binding site antibodies but no difference in sensitivity to antibodies targeting the CD4-bound conformation. M-tropic viruses also displayed a trend toward resistance to neutralization by monoclonal antibodies targeting the V1/V2 region of Env, suggesting subtle changes in Env protein conformation. The paired M- and T-tropic viruses did not differ in autologous serum neutralization, temperature sensitivity, entry kinetics, intrinsic infectivity, or Env protein incorporation. We also examined viruses with modestly increased CD4 usage. These variants have significant sensitivity to sCD4 and may represent evolutionary intermediates. CD4 usage is strongly correlated with infectivity of MDMs over a wide range of CD4 entry phenotypes. These data suggest that emergence of M-tropic HIV-1 includes multiple steps in which a phenotype of increased sensitivity to sCD4 and enhanced CD4 usage accompany subtle changes in Env conformation., Importance: HIV-1 typically replicates in CD4(+) T cells. However, HIV-1 can evolve to infect macrophages, especially within the brain. Understanding how CCR5-using macrophage-tropic viruses evolve and differ from CCR5-using T cell-tropic viruses may provide insights into viral evolution and pathogenesis within the central nervous system. We characterized the HIV-1 env viral entry gene from subject-matched macrophage-tropic and T cell-tropic viruses to identify entry features of macrophage-tropic viruses. We observed several differences between T cell-tropic and macrophage-tropic Env proteins, including functional differences with host CD4 receptor engagement and possible changes in the CD4 binding site and V1/V2 region. We also identified viruses with phenotypes between that of "true" macrophage-tropic and T cell-tropic viruses, which may represent evolutionary intermediates in a multistep process to macrophage tropism., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: The structure of HIV-1 envelope glycoprotein (Env) is flexible and heterogeneous on whole virions. Although functional Env complexes are thought to require trimerization of cleaved gp41/gp120 heterodimers, variable processing can result in the potential incorporation of non-functional uncleaved proteins (gp160), non-trimeric arrangements of gp41/gp120 heterodimers, and gp120 depleted gp41 stumps. The potential distribution of functional and non-functional Env forms across replication-competent viral populations may have important implications for neutralizing and non-neutralizing antibody functions. This study applied an immuno-bead viral capture assay (VCA) to interrogate the potential distribution (heterologous vs homologous) of functional and non-functional forms of virion associated Env., Results: The VCA revealed a significant association between depletion of infectious virions and virion Env incorporation, but not between infectivity and p24-gag. Three distinct subpopulations of virions were identified within pools of genetically homogenous viral particles. Critically, a significant subpopulation of infectious virions were exclusively captured by neutralizing antibodies (nAbs) indicative of a homologous distribution of functional trimeric Env forms. A second infectious subpopulation bound both neutralizing and non-neutralizing antibodies (nnAbs) representative of a heterologous distribution of Env forms, while a third non-infectious subpopulation was predominantly bound by nnAbs recognizing gp41 stumps., Conclusions: The observation that a distinct and significant subpopulation of infectious virions is exclusively captured by neutralizing antibodies has important implications for understanding antibody binding and neutralization, as well as other antibody effector functions.

A major goal for HIV-1 vaccine development is the production of an immunogen to mimic native, functional HIV-1 envelope trimeric spikes (Env) on the virion surface. We lack a reliable description of a native, functional trimer, however, because of inherent instability and heterogeneity in most preparations. We describe here two conformationally homogeneous Envs derived from difficult-to-neutralize primary isolates. All their non-neutralizing epitopes are fully concealed and independent of their proteolytic processing. Most broadly neutralizing antibodies (bnAbs) recognize these native trimers. Truncation of their cytoplasmic tail has little effect on membrane fusion, but it diminishes binding to trimer-specific bnAbs while exposing non-neutralizing epitopes. These results yield a more accurate antigenic picture than hitherto possible of a genuinely untriggered and functional HIV-1 Env; they can guide effective vaccine development., (Copyright © 2015, American Association for the Advancement of Science.)

Several HIV envelope-targeting (Env-targeting) antibodies with broad and potent neutralizing activity have been identified and shown to have unusual features. Of these, the PG9 antibody has a long heavy chain complementarity determining region 3 (HCDR3) and possesses unique structural elements that interact with protein and glycan features of the HIV Env glycoprotein. Here, we used the Rosetta software suite to design variants of the PG9 antibody HCDR3 loop with the goal of identifying variants with increased potency and breadth of neutralization for diverse HIV strains. One variant, designated PG9&#95;N100(F)Y, possessed increased potency and was able to neutralize a diverse set of PG9-resistant HIV strains, including those lacking the Env N160 glycan, which is critical for PG9 binding. An atomic resolution structure of the PG9&#95;N100(F)Y fragment antigen binding (Fab) confirmed that the mutated residue retains the paratope surface when compared with WT PG9. Differential scanning calorimetry experiments revealed that the mutation caused a modest increase in thermodynamic stability of the Fab, a feature predicted by the computational model. Our findings suggest that thermodynamic stabilization of the long HCDR3 in its active conformation is responsible for the increased potency of PG9&#95;N100(F)Y, and strategies aimed at stabilizing this region in other HIV antibodies could become an important approach to in silico optimization of antibodies.

To construct an HSV-1 vector vaccine carrying HIV-1 antigens, HIV-1 gp160, gag, protease and the expression elements were chained together, and then inserted into the internal inverted repeat sequence region of HSV-1 by bacterial artificial chromosome technology. Firstly, HIV-1 gp160 (including type B and C), gag and protease genes were cloned into pcDNA3 in series to generate the pcDNA/gBgp and pcDNA/gCgp, then the recombinant plasmids were transfected into 293FT cells, and HIV-1 antigen was detected from transfected cells by Western blotting. Then the expression cassettes from pcDNA/gBgp and pcDNA/gCgp, comprising HIV-1 antigen genes and expression elements, were cloned into pKO5/BN to generate the shuttle plasmids pKO5/BN/gBgp and pKO5/BN/gCgp. The shuttle plasmids were electroporated into E. coli cells that harbor an HSV-BAC, the recombinant bacteria were screened, and the recombinant DNA was extracted and transfected into Vero cells. The recombinant virus was purified through picking plaques, the virus' DNAs were identified by Southern blotting; HIV-1 antigen was detected from the recombinant HSV-1 infected cells by Western blotting, and the virus' replication competent was analyzed. As the results, gp160 and gag proteins were detected from 293FT cells transfected with pcDNA/gBgp and pcDNA/gCgp by Western blotting. The recombinant bacteria were generated from the E. coli electroporated with pKO5/BN/gBgp or pKO5/BN/gCgp. The recombinant HSV was purified from the Vero cells transfected with the recombinant DNA, the unique DNA fragment was detected from the genome of recombination HSV by Southern blotting; gp120 and gp41 were detected from the infected cells by Western blotting, and the recombinant HSV retained replication competent in mammalian cells. The results indicate that the recombinant HSV carrying HIV-1 gp160, gag and protease genes was generated, the virus retains replication competent in mammalian cells, and could be used as a replicated viral vector vaccine.

Developing a vaccine that overcomes the diversity of HIV-1 is likely to require a strategy that directs antibody (Ab) responses toward conserved regions of the viral Envelope (Env). However, the generation of neutralizing Abs (NAbs) targeting these regions through vaccination has proven to be difficult. One conserved region of particular interest is the membrane proximal external region (MPER) of Env located within the gp41 ectodomain. In order to direct the immune response to this region, the MPER and gp41 ectodomain were expressed separately as N-terminal fusions to the E2 protein of Geobacillus stearothermophilus. The E2 protein acts as a scaffold by self-assembling into 60-mer particles, displaying up to 60 copies of the fused target on the surface. Rabbits were immunized with E2 particles displaying MPER and/or the gp41 ectodomain in conjunction with DNA encoding full-length gp160. Only vaccines including E2 particles displaying MPER elicited MPER-specific Ab responses. NAbs were elicited after two immunizations that largely targeted the V3 loop. To overcome V3 immunodominance in the DNA component, E2 particles displaying MPER were used in conjunction with gp160 DNA lacking hypervariable regions V2, V3, or combined V1V2V3. All rabbits had HIV binding Ab responses and NAbs following the second vaccination. Using HIV-2/HIV-1 MPER chimeric viruses as targets, NAbs were detected in 12/16 rabbits after three immunizations. Low levels of NAbs specific for Tier 1 and 2 viruses were observed in all groups. This study provides evidence that co-immunizing E2 particles displaying MPER and gp160 DNA can focus Ab responses toward conserved regions of Env.

We sought to test whether vaccine-induced immune responses could protect rhesus macaques (RMs) against upfront heterologous challenges with an R5 simian-human immunodeficiency virus, SHIV-2873Nip. This SHIV strain exhibits many properties of transmitted HIV-1, such as tier 2 phenotype (relatively difficult to neutralize), exclusive CCR5 tropism, and gradual disease progression in infected RMs. Since no human AIDS vaccine recipient is likely to encounter an HIV-1 strain that exactly matches the immunogens, we immunized the RMs with recombinant Env proteins heterologous to the challenge virus. For induction of immune responses against Gag, Tat, and Nef, we explored a strategy of immunization with overlapping synthetic peptides (OSP). The immune responses against Gag and Tat were finally boosted with recombinant proteins. The vaccinees and a group of ten control animals were given five low-dose intrarectal (i.r.) challenges with SHIV-2873Nip. All controls and seven out of eight vaccinees became systemically infected; there was no significant difference in viremia levels of vaccinees vs. controls. Prevention of viremia was observed in one vaccinee which showed strong boosting of virus-specific cellular immunity during virus exposures. The protected animal showed no challenge virus-specific neutralizing antibodies in the TZM-bl or A3R5 cell-based assays and had low-level ADCC activity after the virus exposures. Microarray data strongly supported a role for cellular immunity in the protected animal. Our study represents a case of protection against heterologous tier 2 SHIV-C by vaccine-induced, virus-specific cellular immune responses., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

To protect against human immunodeficiency virus (HIV-1) infection, broadly neutralizing antibodies (bnAbs) must be active at the portals of viral entry in the gastrointestinal or cervicovaginal tracts. The localization and persistence of antibodies at these sites is influenced by the neonatal Fc receptor (FcRn), whose role in protecting against infection in vivo has not been defined. Here, we show that a bnAb with enhanced FcRn binding has increased gut mucosal tissue localization, which improves protection against lentiviral infection in non-human primates. A bnAb directed to the CD4-binding site of the HIV-1 envelope (Env) protein (denoted VRC01) was modified by site-directed mutagenesis to increase its binding affinity for FcRn. This enhanced FcRn-binding mutant bnAb, denoted VRC01-LS, displayed increased transcytosis across human FcRn-expressing cellular monolayers in vitro while retaining FcγRIIIa binding and function, including antibody-dependent cell-mediated cytotoxicity (ADCC) activity, at levels similar to VRC01 (the wild type). VRC01-LS had a threefold longer serum half-life than VRC01 in non-human primates and persisted in the rectal mucosa even when it was no longer detectable in the serum. Notably, VRC01-LS mediated protection superior to that afforded by VRC01 against intrarectal infection with simian-human immunodeficiency virus (SHIV). These findings suggest that modification of FcRn binding provides a mechanism not only to increase serum half-life but also to enhance mucosal localization that confers immune protection. Mutations that enhance FcRn function could therefore increase the potency and durability of passive immunization strategies to prevent HIV-1 infection.

Latently HIV-1-infected cells are recognized as the last barrier toward viral eradication and cure. To purge these cells, we combined a provirus stimulant with a blocker of human CD59, a key member of the regulators of complement activation, to trigger Ab-dependent complement-mediated lysis. Provirus stimulants including prostratin and histone deacetylase inhibitors such as romidepsin and suberoylanilide hydroxamic acid activated proviruses in the latently HIV-1-infected T cell line ACH-2 as virion production and viral protein expression on the cell surface were induced. Romidepsin was the most attractive provirus stimulant as it effectively activated proviruses at nanomolar concentrations that can be achieved clinically. Antiretroviral drugs including two protease inhibitors (atazanavir and darunavir) and an RT inhibitor (emtricitabine) did not affect the activity of provirus stimulants in the activation of proviruses. However, saquinavir (a protease inhibitor) markedly suppressed virus production, although it did not affect the percentage of cells expressing viral Env on the cell surface. Provirus-activated ACH-2 cells expressed HIV-1 Env that colocalized with CD59 in lipid rafts on the cell surface, facilitating direct interaction between them. Blockage of CD59 rendered provirus-activated ACH-2 cells and primary human CD4(+) T cells that were latently infected with HIV-1 sensitive to Ab-dependent complement-mediated lysis by anti-HIV-1 polyclonal Abs or plasma from HIV-1-infected patients. Therefore, a combination of provirus stimulants with regulators of complement activation blockers represents a novel approach to eliminate HIV-1., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Antibodies capable of neutralizing HIV-1 often target variable regions 1 and 2 (V1V2) of the HIV-1 envelope, but the mechanism of their elicitation has been unclear. Here we define the developmental pathway by which such antibodies are generated and acquire the requisite molecular characteristics for neutralization. Twelve somatically related neutralizing antibodies (CAP256-VRC26.01-12) were isolated from donor CAP256 (from the Centre for the AIDS Programme of Research in South Africa (CAPRISA)); each antibody contained the protruding tyrosine-sulphated, anionic antigen-binding loop (complementarity-determining region (CDR) H3) characteristic of this category of antibodies. Their unmutated ancestor emerged between weeks 30-38 post-infection with a 35-residue CDR H3, and neutralized the virus that superinfected this individual 15 weeks after initial infection. Improved neutralization breadth and potency occurred by week 59 with modest affinity maturation, and was preceded by extensive diversification of the virus population. HIV-1 V1V2-directed neutralizing antibodies can thus develop relatively rapidly through initial selection of B cells with a long CDR H3, and limited subsequent somatic hypermutation. These data provide important insights relevant to HIV-1 vaccine development.