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Background The high variability in envelope regions of some viruses such as HIV allow the virus to establish infection and to escape subsequent immune surveillance. This variability, as well as increasing incorporation of N-linked glycosylation sites, is fundamental to this evasion. It also creates difficulties for multiple sequence alignment methods (MSA) that provide the first step in their analysis. Existing MSA tools often fail to properly align highly variable HIV envelope sequences requiring extensive manual editing that is impractical with even a moderate number of these variable sequences. Results We developed an automated library building tool NGlyAlign, that organizes similar N-linked glycosylation sites as block constraints and statistically conserved global sites as single site constraints to automatically enforce partial columns in consistency-based MSA methods such as Dialign. This combined method accurately aligns variable HIV-1 envelope sequences. We tested the method on two datasets: a set of 156 founder and chronic gp160 HIV-1 subtype B sequences as well as a set of reference sequences of gp120 in the highly variable region 1. On measures such as entropy scores, sum of pair scores, column score, and similarity heat maps, NGlyAlign+Dialign proved superior against methods such as T-Coffee, ClustalOmega, ClustalW, Praline, HIValign and Muscle. The method is scalable to large sequence sets producing accurate alignments without requiring manual editing. As well as this application to HIV, our method can be used for other highly variable glycoproteins such as hepatitis C virus envelope. Conclusions NGlyAlign is an automated tool for mapping and building glycosylation motif libraries to accurately align highly variable regions in HIV sequences. It can provide the basis for many studies reliant on single robust alignments. NGlyAlign has been developed as an open-source tool and is freely available at https://github.com/UNSW-Mathematical-Biology/NGlyAlign_v1.0 .

Background Precise and cost-efficient human immunodeficiency virus (HIV) incidence and drug resistance surveillances are in high demand for the advancement of the 90-90-90 “treatment for all” target. Methods We developed microdrop HIV sequencing for the HIV incidence and drug resistance assay (HIDA), a single-blood-draw surveillance tool for incidence and drug resistance mutation (DRM) detection. We amplified full-length HIV envelope and pol gene sequences within microdroplets, and this compartmental amplification with long-read high-throughput sequencing enabled us to recover multiple unique sequences. Results We achieved greater precision in determining the stage of infection than current incidence assays, with a 1.2% false recency rate (proportion of misclassified chronic infections) and a 262-day mean duration of recent infection (average time span of recent infection classification) from 83 recently infected and 81 chronically infected individuals. Microdrop HIV sequencing demonstrated an increased capacity to detect minority variants and linked DRMs. By screening all 93 World Health Organization surveillance DRMs, we detected 6 pretreatment drug resistance mutations with 2.6%–13.2% prevalence and cross-linked mutations. Conclusions HIDA with microdrop HIV sequencing may promote global HIV real-time surveillance by serving as a precise and high-throughput cross-sectional survey tool that can be generalized for surveillance of other pathogens.

An effective prophylactic HIV-1 vaccine is essential in order to contain the HIV/AIDS global pandemic. The discovery of different broadly neutralizing antibodies (bnAbs) in the last decades has enabled the characterization of several minimal epitopes on the HIV envelope (Env) spike, including glycan-dependent fragments. Herein, we provide a brief overview of the progress made on the development of synthetic carbohydrate-based epitope mimics for the elicitation of bnAbs directed to certain regions on Env gp120 protein: the outer domain high-mannose cluster and the variable loops V1V2 and V3. We focus on the design, synthesis and biological evaluation of minimal immunogens and discuss key aspects towards the development of a successful protective vaccine against HIV-1.

OBJECTIVE The HIV-1-specific antibodies are being considered for prevention and therapy in HIV infection. For effective antibody response, presence of functionally competent memory B cells (MEBs) is important; however, HIV-infection is known to alter the B-cell functionality. Very limited data are available on the HIV-specific memory B-cell population in HIV-infected Indian population. METHODS In this study, the frequencies of HIV-gp140-specific MEBs were measured in individuals with nonprogressive [long-term-nonprogressors (LTNPs), N = 20] and progressive (N = 19) HIV infection using multicolor flow cytometry. The activation and functional status of these MEBs were assessed as frequencies and mean fluorescence intensity (MFI) of the CD38 and CD40 expression, respectively. RESULTS The percentages of gp140 + MEBs were higher in LTNPs than seen in progressors (P = 0.0475) and associated with higher CD4 cell count (P = 0.0312, r = 0.2833). As compared with the progressors, LTNPs also showed higher functional (CD40+) gp140 + MEBs both frequencies (P

Antibody-dependent cellular cytotoxicity (ADCC) has been correlated with reduced risk of HIV-1 infection in several preclinical vaccine trials and the RV144 clinical trial, indicating this is a relevant antibody function to study. Given the diversity of HIV-1, the breadth of vaccine-induced antibody responses is a critical parameter to understand if a universal vaccine is to be realised. Moreover, breadth of ADCC responses can be influenced by different vaccine strategies and regimens, including adjuvants. Therefore, to accurately evaluate ADCC and to compare vaccine regimens, it is important to understand the range of HIV Envelope susceptibility to these responses. These evaluations have been limited because of the complexity of the assay and the lack of a comprehensive panel of viruses for the assessment of these humoral responses. Here, we used twenty-nine HIV-1 infectious molecular clones (IMCs) representing different Envelope subtypes and circulating recombinant forms to characterise susceptibility to ADCC from antibodies in plasma from infected individuals, including thirteen viraemic individuals, ten controllers and six with broadly neutralizing antibody responses. We found in our panel that ADCC susceptibility of the IMCs in our panel did not cluster by subtype, infectivity, level of CD4 downregulation, level of shedding, or neutralization sensitivity. Using partition-around-medoids (PAM) clustering to distinguish smaller groups of IMCs with similar ADCC susceptibility, we identified nested panels of four to eight IMCs that broadly represent the ADCC susceptibility of the entire 29 IMC panel. These panels, together with reagents developed to specifically accommodate circulating viruses at the geographical sites of vaccine trials, will provide a powerful tool to harmonise ADCC data generated across different studies, and detect common themes of ADCC responses elicited by various vaccines. IMPORTANCE Antibody-dependent cellular cytotoxicity (ADCC) responses were found to correlate with reduced risk of infection in the RV144 trial, the only human HIV-1 vaccine to show any efficacy to date. However, reagents to understand the breadth and magnitude of these responses across preclinical and clinical vaccine trials remain underdeveloped. In this study, we characterise HIV-1 infectious molecular clones encoding 29 distinct envelope strains (Env-IMCs) to understand factors which impact virus susceptibility to ADCC and use statistical methods to identify smaller nested panels of four to eight Env-IMCs which accurately represent the full set. These reagents can be used as standardized reagents across studies to fully understand how ADCC may affect efficacy of future vaccine studies, and how studies differed in the breadth of responses developed.

Antibodies that can neutralize diverse HIV-1 strains develop in ~10–20% of HIV-1 infected individuals, and their elicitation is a goal of vaccine design. Such antibodies can also serve as therapeutics for those who have already been infected with the virus. Structural characterizations of broadly reactive antibodies in complex with the HIV-1 spike indicate that there are a limited number of sites of vulnerability on the spike. Analysis of their structures can help reveal commonalities that would be useful in vaccine design and provide insights on combinations of antibodies that can be used to minimize the incidence of viral resistance mutations. In this review, we give an update on recent structures determined of the spike in complex with broadly neutralizing antibodies in the context of all epitopes on the HIV-1 spike identified to date.

During HIV-1 transmission through T cell virological synapses, the recruitment of the envelope (Env) glycoprotein to the site of cell–cell contact is important for adhesion and for packaging onto nascent virus particles which assemble at the site. Live imaging studies in CD4 T cells have captured the rapid recruitment of the viral structural protein Gag to VSs. We explored the role of endocytic trafficking of Env initiated by a membrane proximal tyrosine motif during HIV transfer into target cells and examined the factors that allow Gag and Env to be transferred together across the synapse. To facilitate tracking of Env in live cells, we adapted an Env tagging method and introduced a biotin acceptor peptide (BAP) into the V4 loop of Env gp120, enabling sensitive fluorescent tracking of V4-biotinylated Env. The BAP-tagged and biotinylated HIVs were replication-competent in cell-free and cell-to-cell infection assays. Live cell fluorescent imaging experiments showed rapid internalized cell surface Env on infected cells. Cell–cell transfer experiments conducted with the Env endocytosis mutant (Y712A) showed increased transfer of Env. Paradoxically, this increase in Env transfer was associated with significantly reduced Gag transfer into target cells, when compared to viral transfer associated with WT Env. This Y712A Env mutant also exhibited an altered Gag/biotin Env fluorescence ratio during transfer that correlated with decreased productive cell-to-cell infection. These results may suggest that the internalization of Env into recycling pools plays an important role in the coordinated transfer of Gag and Env across the VS, which optimizes productive infection in target cells.

BACKGROUND HIV envelope (env) diversity represents a significant challenge for the use of broadly neutralizing antibodies (bNAbs) in HIV treatment and cure studies. Screening for viral sensitivity to bNAbs to select eligible trial participants will be important to improve clinical efficacy; however, no universal approach has been established. METHODS Pre-antiretroviral therapy plasma virus from participants in the Zurich Primary HIV Infection (ZPHI) study was genotyped and phenotyped for sensitivity to the bNAbs elipovimab (EVM, formerly GS-9722) and 3BNC117. The genotyping and phenotyping assessments were performed following the Clinical Laboratory Improvement Amendments of 1988 guidelines as required for entry into clinical trials. The genotypic-based prediction of bNAb sensitivity was based on HIV env amino acid signatures identified from a genotypic-phenotypic correlation algorithm using a subtype B database. RESULTS Genotyping the plasma virus and applying env sensitivity signatures, ZPHI study participants with viral sensitivity to EVM and 3BNC117 were identified. ZPHI study participants with virus sensitive to EVM and 3BNC117 were also identified by phenotyping the plasma virus. Comparison of the genotypic and phenotypic sensitivity assessments showed strong agreement between the 2 methodologies. CONCLUSIONS The genotypic assessment was found to be as predictive as the direct measurement of bNAb sensitivity by phenotyping and may, therefore, be preferred because of more rapid turnaround time and assay simplicity. A significant number of the participants were predicted to have virus sensitive to EVM and 3BNC117 and could, thus, be potential participants for clinical trials involving these bNAbs.

HIV Env trimers elicit antibodies that lead to their own destruction., Rationally designed protein subunit vaccines are being developed for a variety of viruses including influenza, RSV, SARS-CoV-2, and HIV. These vaccines are based on stabilized versions of the primary targets of neutralizing antibodies on the viral surface, namely, viral fusion glycoproteins. While these immunogens display the epitopes of potent neutralizing antibodies, they also present epitopes recognized by non-neutralizing or weakly neutralizing (“off-target”) antibodies. Using our recently developed electron microscopy polyclonal epitope mapping approach, we have uncovered a phenomenon wherein off-target antibodies elicited by HIV trimer subunit vaccines cause the otherwise highly stabilized trimeric proteins to degrade into cognate protomers. Further, we show that these protomers expose an expanded suite of off-target epitopes, normally occluded inside the prefusion conformation of trimer, that subsequently elicit further off-target antibody responses. Our study provides critical insights for further improvement of HIV subunit trimer vaccines for future rounds of the iterative vaccine design process.

PURPOSE OF REVIEW: Close to two million individuals globally become infected with human immunodeficiency virus (HIV-1) each year and just over two thirds will have access to life-prolonging antivirals. However, the rapid development of drug resistance creates challenges, such that generation of more effective therapies is not only warranted but a necessary endeavour. This review discusses a group of HIV-1 entry inhibitors known as CD4 mimics which exploit the highly conserved relationship between the HIV-1 envelope glycoprotein (Env) and the receptor, CD4. RECENT FINDINGS: We review the structure/function guided evolution of these inhibitors, vital mechanistic insights that underpin broad and potent functional antagonism, recent evidence of utility demonstrated in animal and physiologically relevant in vitro models, and current progress towards effective new-generation inhibitors. SUMMARY: This review highlights the promising potential of CD4 mimetics as multifunctional therapeutics.

Maternal antiretroviral therapy (ART) has effectively reduced but not eliminated the burden of mother-to-child transmission of HIV across the globe, as an estimated 160,000 children were newly infected with HIV in 2018. Thus, additional preventive strategies beyond ART will be required to close the remaining gap and end the pediatric HIV epidemic. A maternal active immunization strategy that synergizes with maternal ART could further reduce infant HIV infections. In this study, we found that two historic HIV Env vaccines did not enhance the ability of HIV-infected pregnant women to neutralize autologous viruses. Therefore, next-generation maternal HIV vaccine candidates must employ alternate approaches to achieve potent neutralizing antibody and perhaps nonneutralizing antibody responses to effectively impede vertical virus transmission. Moreover, these approaches must reflect the broad diversity of HIV strains and widespread availability of ART worldwide., A maternal vaccine capable of boosting neutralizing antibody (NAb) responses directed against circulating viruses in HIV-infected pregnant women could effectively decrease mother-to-child transmission of HIV. However, it is not known if an HIV envelope (Env) vaccine administered to infected pregnant women could enhance autologous virus neutralization and thereby reduce this risk of vertical HIV transmission. Here, we assessed autologous virus NAb responses in maternal plasma samples obtained from AIDS Vaccine Evaluation Group (AVEG) protocols 104 and 102, representing historical phase I safety and immunogenicity trials of recombinant HIV Env subunit vaccines administered to HIV-infected pregnant women (ClinicalTrials registration no. NCT00001041). Maternal HIV Env-specific plasma binding and neutralizing antibody responses were characterized before and after vaccination in 15 AVEG 104 (n = 10 vaccine recipients, n = 5 placebo recipients) and 2 AVEG 102 (n = 1 vaccine recipient, n = 1 placebo recipient) participants. Single-genome amplification (SGA) was used to obtain HIV env gene sequences of autologous maternal viruses for pseudovirus production and neutralization sensitivity testing in pre- and postvaccination plasma of HIV-infected pregnant vaccine recipients (n = 6 gp120, n = 1 gp160) and placebo recipients (n = 3). We detected an increase in Env subunit MN gp120-specific IgG binding in the group of vaccine recipients between the first immunization visit and the last visit at delivery (P = 0.027, 2-sided Wilcoxon test). While no difference was observed in the levels of autologous virus neutralization potency between groups, in both groups maternal plasma collected at delivery more effectively neutralized autologous viruses from early pregnancy than late pregnancy. Immunization strategies capable of further enhancing these autologous virus NAb responses in pregnant women will be important to block vertical transmission of HIV. IMPORTANCE Maternal antiretroviral therapy (ART) has effectively reduced but not eliminated the burden of mother-to-child transmission of HIV across the globe, as an estimated 160,000 children were newly infected with HIV in 2018. Thus, additional preventive strategies beyond ART will be required to close the remaining gap and end the pediatric HIV epidemic. A maternal active immunization strategy that synergizes with maternal ART could further reduce infant HIV infections. In this study, we found that two historic HIV Env vaccines did not enhance the ability of HIV-infected pregnant women to neutralize autologous viruses. Therefore, next-generation maternal HIV vaccine candidates must employ alternate approaches to achieve potent neutralizing antibody and perhaps nonneutralizing antibody responses to effectively impede vertical virus transmission. Moreover, these approaches must reflect the broad diversity of HIV strains and widespread availability of ART worldwide.

HIV Env-specific nonneutralizing Abs (NnAbs) able to mediate ADCC have been implicated in protection from HIV infection. However, Env-specific NnAbs have the capacity to support ADCC of both HIV-infected and HIV-uninfected bystander cells, potentially leading to misinterpretations when the assay used to measure ADCC does not distinguish between the two target cell types present in HIV cultures. Using a novel ADCC assay, which simultaneously quantifies the killing activity of Env-specific Abs on both infected and uninfected bystander cells, we observed that only a minority of Env-specific Abs in HIV+ plasma mediated ADCC of genuinely HIV-infected cells displaying Env in its native closed conformation. This assay can be used for the development of vaccine strategies aimed at eliciting Env-specific Ab responses capable of controlling HIV infection., Measuring Envelope (Env)-specific antibody (Ab)-dependent cellular cytotoxicity (ADCC)-competent Abs in HIV+ plasma is challenging because Env displays distinctive epitopes when present in a native closed trimeric conformation on infected cells or in a CD4-bound conformation on uninfected bystander cells. We developed an ADCC model which distinguishes Env-specific ADCC-competent Abs based on their capacity to eliminate infected, bystander, or Env rgp120-coated cells as a surrogate for shed gp120 on bystander cells. A panel of monoclonal Abs (MAbs), used to opsonize these target cells, showed that infected cells were preferentially recognized/eliminated by MAbs to CD4 binding site, V3 loop, and viral spike epitopes whereas bystander/coated cells were preferentially recognized/eliminated by Abs to CD4-induced (CD4i) epitopes. In HIV-positive (HIV+) plasma, Env-specific Abs recognized and supported ADCC of infected cells, though a majority were directed toward CD4i epitopes on bystander cells. For ADCC activity to be effective in HIV control, ADCC-competent Abs need to target genuinely infected cells.

Anti-HIV broadly neutralizing antibodies (bnAbs) have revealed vaccine targets on the virus’s envelope (Env) protein and are themselves promising immunotherapeutics. The efficacy of bnAb-based therapies and vaccines depends in part on how readily the virus can escape neutralization. While structural studies can define contacts between bnAbs and Env, only functional studies can define mutations that confer escape. Here we mapped how all possible single amino-acid mutations in Env affect neutralization of HIV by nine bnAbs targeting five epitopes. For most bnAbs, mutations at only a small fraction of structurally defined contact sites mediated escape, and most escape occurred at sites that are near but do not directly contact the antibody. The Env mutations selected by two pooled bnAbs were similar to those expected from the combination of the bnAbs’ independent action. Overall, our mutation-level antigenic atlas provides a comprehensive dataset for understanding viral immune escape and refining therapies and vaccines.

HIV-infected macrophages contribute to persistence of HIV reservoirs in people living with HIV receiving antiretroviral therapy. A potential strategy to eliminate reservoirs is the use of antibody-dependent cellular cytotoxicity (ADCC) against infected cells expressing the HIV envelope (Env) protein on their surface. Designing ADCC strategies requires knowledge of exposed Env epitopes on the cell surface and identifying antibodies capable of opsonising infected cells, yet little is known regarding the ability of HIV-infected macrophages to be targeted with such strategies. Using a panel of neutralising and poorly-neutralising anti-Env antibodies we compared Env epitopes expressed on infected monocyte-derived macrophages (MDM) and autologous T cells. Our results reveal potential differences in epitope expression on macrophage- and T cell-expressed Env. Notably, HIVBaL-infected macrophages were more susceptible to opsonisation by NIH45-46 (median = 40.4%) compared to infected T cells (13.6%; p = 0.002), which were more susceptible to opsonisation by 17b and 447.52D (88.6% and 45.6% respectively) compared to MDM (30% and 6.7%, p = 0.002 and 0.004 respectively). Furthermore, neutralising antibodies 10E8 and PGT145 were relatively ineffective at opsonising Env expressed on the surface of infected T cells or macrophages, indicating that the context in which Env is presented on infected cells may differ to that of cell-free virions.

An HIV-1 vaccine is needed to curtail the HIV epidemic. Only one (RV144) out of the 6 HIV-1 vaccine efficacy trials performed showed efficacy. A potential mechanism of protection is the induction of functional antibodies to V1V2 region of HIV envelope. The 2 main current approaches to the generation of protective immunity are through broadly neutralizing antibodies (bnAb) and induction of functional antibodies (non-neutralizing Abs with other potential anti-viral functions). Passive immunization using bnAb has advanced into phase II clinical trials. The induction of bnAb using mimics of the natural Env trimer or B-cell lineage vaccine design is still in pre-clinical phase. An attempt at optimization of protective functional antibodies will be assessed next with the efficacy trial (HVTN702) about to start. With on-going optimization of prime/boost strategies, the development of mosaic immunogens, replication competent vectors, and emergence of new strategies designed to induce bnAb, the prospects for a preventive HIV vaccine have never been more promising.

The HIV envelope protein contains five hypervariable domains (V1–V5) that are fundamental for cell entry. We contrasted modifications in the variable domains derived from a panel of 24 tissues from 7 subjects with no measurable plasma viral load (NPVL) to variable domains from 76 tissues from 15 subjects who had a detectable plasma viral load (PVL) at death. NPVL subject's V1 and V2 domains were usually highly length variable, whereas length variation in PVL sequences was more conserved. Longer V1s contained more charged residues, whereas longer V2s were more glycosylated. Structural analysis demonstrated V1/V2 charge, and N-site additions/subtractions were localized to the CD4 binding pocket. Diversified envelopes in tissues during therapy may represent a mechanism for HIV persistence in tissues, as binding pocket complexity is associated with HIV that may escape neutralization, whereas shorter envelopes are associated with increased infectivity. Further analysis of tissue-derived envelope sequences may enable better understanding of potential immunological approaches targeting the persistent HIV reservoir.

VRC01 broadly neutralizing antibodies (bnAbs) target the CD4-binding site (CD4BS) of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (Env). Unlike mature antibodies, corresponding VRC01 germline precursors poorly bind to Env. Immunogen design has mostly relied on glycan removal from trimeric Env constructs and has had limited success in eliciting mature VRC01 bnAbs. To better understand elicitation of such bnAbs, we characterized the inferred germline precursor of VRC01 in complex with a modified trimeric 426c Env by cryo-electron microscopy and a 426c gp120 core by X-ray crystallography, biolayer interferometry, immunoprecipitation, and glycoproteomics. Our results show VRC01 germline antibodies interacted with a wild-type 426c core lacking variable loops 1–3 in the presence and absence of a glycan at position Asn276, with the latter form binding with higher affinity than the former. Interactions in the presence of an Asn276 oligosaccharide could be enhanced upon carbohydrate shortening, which should be considered for immunogen design.

Neutralizing antibodies to the V2 apex antigenic region of the HIV-1 envelope (Env) trimer are among the most prevalent cross-reactive antibodies elicited by natural infection. Two recently described V2-specific antibodies, PGDM1400 and CAP256-VRC26.25, have demonstrated exquisite potency and neutralization breadth against HIV-1. However, little data exist on the protective efficacy of V2-specific neutralizing antibodies. We created a novel SHIV-325c viral stock that included a clade C HIV-1 envelope and was susceptible to neutralization by both of these antibodies. Rhesus macaques received a single infusion of either antibody at three different concentrations (2, 0.4, and 0.08 mg/kg) before challenge with SHIV-325c. PGDM1400 was fully protective at the 0.4 mg/kg dose, whereas CAP256-VRC26.25-LS was fully protective even at the 0.08 mg/kg dose, which correlated with its greater in vitro neutralization potency against the challenge virus. Serum antibody concentrations required for protection were

Viral load quantification is a critical need for HIV management worldwide. However, the diagnostic technologies currently available are too limited by their size and expense to reach many remote and resource-limited populations. Toward the development of techniques which can be leveraged for point-of-care assays, we have investigated affinity capture of whole viruses using magnetic microparticles functionalized with antibodies or proteins targeting components of the HIV envelope. Results show the best performance from T-20, a small peptide employed in antiretroviral pharmacotherapy which targets an HIV envelope protein. This demonstration introduces an interesting alternative to antibodies for future affinity-capture applications in HIV diagnostics.

HIV incidence is a primary metric for epidemic surveillance and prevention efficacy assessment. HIV incidence assay performance is evaluated via false recency rate (FRR) and mean duration of recent infection (MDRI). We conducted a meta-analysis of 438 incident and 305 chronic specimens’ HIV envelope genes from a diverse global cohort. The genome similarity index (GSI) accurately characterized infection stage across diverse host and viral factors. All except one chronic specimen had GSIs below 0.67, yielding a FRR of 0.33 [0-0.98] %. We modeled the incidence assay biomarker dynamics with a logistic link function assuming individual variabilities in a Beta distribution. The GSI probability density function peaked close to 1 in early infection and 0 around two years post infection, yielding MDRI of 420 [361, 467] days. We tested the assay by newly sequencing 744 envelope genes from 59 specimens of 21 subjects who followed from HIV negative status. Both standardized residuals and Anderson-Darling tests showed that the test dataset was statistically consistent with the model biomarker dynamics. This is the first reported incidence assay meeting the optimal FRR and MDRI performance standards. Signatures of HIV gene diversification can allow precise cross-sectional surveillance with a desirable temporal range of incidence detection.

The treatment or cure of HIV infection by cell and gene therapy has been a goal for decades. Recent advances in both gene editing and chimeric antigen receptor (CAR) technology have created new therapeutic possibilities for a variety of diseases. Broadly neutralizing monoclonal antibodies (bNAbs) with specificity for the HIV envelope glycoprotein provide a promising means of targeting HIV-infected cells. Here we show that primary human T cells engineered to express anti-HIV CARs based on bNAbs (HIVCAR) show specific activation and killing of HIV-infected versus uninfected cells in the absence of HIV replication. We also show that homology-directed recombination of the HIVCAR gene expression cassette into the CCR5 locus enhances suppression of replicating virus compared with HIVCAR expression alone. This work demonstrates that HIV immunotherapy utilizing potent bNAb-based single-chain variable fragments fused to second-generation CAR signaling domains, delivered directly into the CCR5 locus of T cells by homology-directed gene editing, is feasible and effective. This strategy has the potential to target HIV-infected cells in HIV-infected individuals, which might help in the effort to cure HIV.

Carbohydrate-binding agents (CBAs) are potential HIV microbicidal agents with a high genetic barrier to resistance. We wanted to evaluate whether two mannose-specific CBAs, recognizing multiple and often distinct glycan structures on the HIV envelope gp120, can interact synergistically against HIV-1, HIV-2, and HIV-1 strains that were selected for resistance against particular CBAs [i.e., 2G12 mAb and microvirin (MVN)]. Paired CBA/CBA combinations mainly showed synergistic activity against both wild-type HIV-1 and HIV-2 but also 2G12 mAb- and MVN-resistant HIV-1 strains as based on the median effect principle with combination indices (CIs) ranging between 0.29 and 0.97. Upon combination, an increase in antiviral potency of griffithsin (GRFT) up to ∼12-fold (against HIV-1), ∼8-fold (against HIV-2), and ∼6-fold (against CBA-resistant HIV-1) was observed. In contrast, HHA/GNA combinations showed additive activity against wild-type HIV-1 and HIV-2 strains, but remarkable synergy with HHA and GNA was observed against 2G12 mAb- and MVN-resistant HIV-1 strains (CI, 0.64 and 0.49, respectively). Overall, combinations of GRFT and other CBAs showed synergistic activity against HIV-1, HIV-2, and even against certain CBA-resistant HIV-1 strains. The CBAs tested appear to have distinct binding patterns on the gp120 envelope and therefore do not necessarily compete with each other's glycan binding sites on gp120. As a result, there might be no steric hindrance between two different CBAs in their competition for glycan binding (except for the HHA/GNA combination). These data are encouraging for the use of paired CBA combinations in topical microbicide applications (e.g., creams, gels, or intravaginal rings) to prevent HIV transmission.

The dependency between the primary structure of HIV envelope glycoproteins (ENV) and the neutralization data for given antibodies is very complicated and depends on a large number of factors, such as the binding affinity of a given antibody for a given ENV protein, and the intrinsic infection kinetics of the viral strain. This paper presents a first approach to learning these dependencies using an artificial feedforward neural network which is trained to learn from experimental data. The results presented here demonstrate that the trained neural network is able to generalize on new viral strains and to predict reliable values of neutralizing activities of given antibodies against HIV-1.

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission(2,10,11). This cell to cell transfer between CD4(+) T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell (8). In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions(1,4,5,6,7,13). VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4(+) T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3ζ, ZAP70, LAT, SLP-76, Itk, and PLCγ(15). In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

International audience; Broadly neutralizing antibodies (bnAbs) directed to the V2 apex of the HIV envelope (Env) trimer isolated from individual HIV-infected donors potently neutralize diverse HIV strains, but strategies for designing immunogens to elicit bnAbs have not been identified. Here, we compared four prototypes (PG9, CH01, PGT145, and CAP256.VRC26.09) of V2 apex bnAbs and showed that all recognized a core epitope of basic V2 residues and the glycan-N160. Two prototype bnAbs were derived from VH-germlines that were 99% identical and used a common germline D-gene encoded YYD-motif to interact with the V2-epitope. We identified isolates that were neutralized by inferred germline (iGL) versions of three of the prototype bnAbs. Soluble Env derived from one of these isolates was shown to form a well-ordered Env trimer that could serve as an immunogen to initiate a V2-apex bnAb response. These studies illustrate a strategy to transition from panels of bnAbs to vaccine candidates.

The origin of broadly neutralizing HIV-specific antibodies and their relation to HIV evolution are not well defined. Here we examined virus evolution and neutralizing antibody escape in a subtype A infected individual with a broad, cross subtype, antibody response. The majority of envelope variants isolated over the first approximately 5 years after infection were poorly neutralized by contemporaneous plasma that neutralized variants from earlier in infection, consistent with a dynamic process of escape. The majority of variants could be neutralized by later plasma, suggesting these evolving variants may have contributed to the elicitation of new antibody responses. However, some variants from later in infection were recognized by plasma from earlier in infection, including one notably neutralization-sensitive variant that was sensitive due to a proline at position 199 in V2. These studies suggest a complex pattern of virus evolution in this individual with a broad NAb response, including persistence of neutralization-sensitive viruses.

A major difference between binding and fusion assays commonly used to study the human immunodeficiency virus (HIV) envelope is the use of monomeric envelope for the former assay and oligomeric envelope for the latter. Due to discrepancies in their readouts for some mutants, envelope regions involved in CCR5 coreceptor usage were systematically studied to determine whether the discordance is due to inherent differences between the two assays or whether it genuinely reflects functional differences at each entry step. By adding the binding inhibitor TAK-779 to delay coreceptor binding kinetics in the fusion assay, the readouts were found comparable between the assays for the mutants analysed in this study. Our finding indicates that monomeric binding reflects oligomeric envelope–CCR5 interaction, thus discordant results between binding and fusion assays do not necessarily indicate differences in coreceptor usage by oligomeric envelope and monomeric gp120.

HIV entry into cells is mediated through sequential interactions between HIV envelope proteins (Env) and two cellular molecules: CD4 and a co-receptor, typically either CCR5 or CXCR4. Co-receptor preference has been associated with other viral traits; specifically, CXCR4-tropic viruses have been associated with increased host cell pathogenicity and more rapid progression of disease. Recently, much attention has been focused on the development of CCR5 and CXCR4 antagonists as antiviral agents and several are set to enter phase III trials in 2004 and 2005. The development of assays to assess the co-receptor tropism of HIV populations is critical for the optimal design and performance of clinical trials to evaluate these agents. In addition, the use of these agents in a clinical setting is likely to benefit from a reliable methodology for tropism determination prior to the selection of an optimal antiviral therapy or to evaluate continued efficacy of a regimen.Tropism assays that use recombinant viruses and pseudotyped HIV are becoming more commonly employed and comparative data with standard assays have begun to be accumulated. These assays are being used to expand on earlier studies of the epidemiology and natural history of HIV tropism. In addition, tropism assays have facilitated the study of co-receptor inhibitors in vitro and in phase I and II trials.The development of rapid, reliable tropism assays has been useful in advancing the development of novel antiviral agents. Defining the role of these assays in routine clinical practice will be the next important step.

Development of a safe and preventive HIV-1 vaccine is a high priority. Recent advances in HIV vaccine development include an improved understanding of HIV envelope structure, development of techniques that enable a detailed analysis of vaccine-induced immune responses in humans, expansion of the pipeline of promising candidate vaccines, and completion of the first vaccine efficacy trials. A common feature of several preventive vaccine strategies in early clinical trials is their ability to attenuate clinical disease rather than completely prevent HIV infection in nonhuman primates. One or more candidate vaccines will likely advance into efficacy trials within the next few years, while efforts to identify new designs that induce broadly neutralizing antibodies continue with incremental success.