Human Immunodeficiency Virus Envelope Protein Research Articles

Macrophage- and CD4+ T cell-derived SIV differ in glycosylation, infectivity and neutralization sensitivity.

The human immunodeficiency virus (HIV) envelope protein (Env) mediates viral entry into host cells and is the primary target for the humoral immune response. Env is extensively glycosylated, and these glycans shield underlying epitopes from neutralizing antibodies. The glycosylation of Env is influenced by the type of host cell in which the virus is produced. Thus, HIV is distinctly glycosylated by CD4+ T cells, the major target cells, and macrophages. However, the specific differences in glycosylation between viruses produced in these cell types have not been explored at the molecular level. Moreover, it remains unclear whether the production of HIV in CD4+ T cells or macrophages affects the efficiency of viral spread and resistance to neutralization. To address these questions, we employed the simian immunodeficiency virus (SIV) model. Glycan analysis implied higher relative levels of oligomannose-type N-glycans in SIV from CD4+ T cells (T-SIV) compared to SIV from macrophages (M-SIV), and the complex-type N-glycans profiles seem to differ between the two viruses. Notably, M-SIV demonstrated greater infectivity than T-SIV, even when accounting for Env incorporation, suggesting that host cell-dependent factors influence infectivity. Further, M-SIV was more efficiently disseminated by HIV binding cellular lectins. We also evaluated the influence of cell type-dependent differences on SIV's vulnerability to carbohydrate binding agents (CBAs) and neutralizing antibodies. T-SIV demonstrated greater susceptibility to mannose-specific CBAs, possibly due to its elevated expression of oligomannose-type N-glycans. In contrast, M-SIV exhibited higher susceptibility to neutralizing sera in comparison to T-SIV. These findings underscore the importance of host cell-dependent attributes of SIV, such as glycosylation, in shaping both infectivity and the potential effectiveness of intervention strategies.

Impact of Recombinant VSV-HIV Prime, DNA-Boost Vaccine Candidates on Immunogenicity and Viremia on SHIV-Infected Rhesus Macaques.

Currently, no effective vaccine to prevent human immunodeficiency virus (HIV) infection is available, and various platforms are being examined. The vesicular stomatitis virus (VSV) vaccine vehicle can induce robust humoral and cell-mediated immune responses, making it a suitable candidate for the development of an HIV vaccine. Here, we analyze the protective immunological impacts of recombinant VSV vaccine vectors that express chimeric HIV Envelope proteins (Env) in rhesus macaques. To improve the immunogenicity of these VSV-HIV Env vaccine candidates, we generated chimeric Envs containing the transmembrane and cytoplasmic tail of the simian immunodeficiency virus (SIV), which increases surface Env on the particle. Additionally, the Ebola virus glycoprotein was added to the VSV-HIV vaccine particles to divert tropism from CD4 T cells and enhance their replications both in vitro and in vivo. Animals were boosted with DNA constructs that encoded matching antigens. Vaccinated animals developed non-neutralizing antibody responses against both the HIV Env and the Ebola virus glycoprotein (EBOV GP) as well as systemic memory T-cell activation. However, these responses were not associated with observable protection against simian-HIV (SHIV) infection following repeated high-dose intra-rectal SHIV SF162p3 challenges.

Interferon-β deficiency alters brain response to chronic HIV-1 envelope protein exposure in a transgenic model of NeuroHIV

Human immunodeficiency virus-1 (HIV-1) infects the central nervous system (CNS) and causes HIV-associated neurocognitive disorders (HAND) in about half of the population living with the virus despite combination anti-retroviral therapy (cART). HIV-1 activates the innate immune system, including the production of type 1 interferons (IFNs) α and β. Transgenic mice expressing HIV-1 envelope glycoprotein gp120 (HIVgp120tg) in the CNS develop memory impairment and share key neuropathological features and differential CNS gene expression with HIV patients, including the induction of IFN-stimulated genes (ISG). Here we show that knocking out IFNβ (IFNβKO) in HIVgp120tg and non-tg control mice impairs recognition and spatial memory, but does not affect anxiety-like behavior, locomotion, or vision. The neuropathology of HIVgp120tg mice is only moderately affected by the KO of IFNβ but in a sex-dependent fashion. Notably, in cerebral cortex of IFNβKO animals presynaptic terminals are reduced in males while neuronal dendrites are reduced in females. The IFNβKO results in the hippocampal CA1 region of both male and female HIVgp120tg mice in an ameliorated loss of neuronal presynaptic terminals but no protection of neuronal dendrites. Only female IFNβ-deficient HIVgp120tg mice display diminished microglial activation in cortex and hippocampus and increased astrocytosis in hippocampus compared to their IFNβ-expressing counterparts. RNA expression for some immune genes and ISGs is also affected in a sex-dependent way. The IFNβKO abrogates or diminishes the induction of MX1, DDX58, IRF7 and IRF9 in HIVgp120tg brains of both sexes. Expression analysis of neurotransmission related genes reveals an influence of IFNβ on multiple components with more pronounced changes in IFNβKO females. In contrast, the effects of IFNβKO on MAPK activities are independent of sex with pronounced reduction of active ERK1/2 but also of active p38 in the HIVgp120tg brain. In summary, our findings show that the absence of IFNβ impairs memory dependent behavior and modulates neuropathology in HIVgp120tg brains, indicating that its absence may facilitate development of HAND. Moreover, our data suggests that endogenous IFNβ plays a vital role in maintaining neuronal homeostasis and memory function.

Impedimetric antimicrobial peptide biosensor for the detection of human immunodeficiency virus envelope protein gp120

This study presents the design and implementation of an antimicrobial peptide-based electrochemical impedance spectroscopy (EIS) based biosensor system. The biosensor consists of a gold coated carbon electrode with MXene and silver nanoparticles (AgNPs) for the label-free detection of the human immunodeficiency virus (HIV) envelope protein gp120. Scanning electron microscopy was used to confirm the presence and distribution of MXene and AgNPs on the biosensor surface. The employment of the antimicrobial peptide on the electrode surface minimized the denaturation of the biorecognition receptor to ensure reliable and stable performance. The biosensor exhibited a linear range of 10-4000 pg mL-1 for gp120 detection, demonstrating good repeatability in real samples. The limit of detection (LOD) and limit of quantification (LOQ) were also calculated as 0.05 pg mL-1 and 0.14 pg mL-1, respectively. This biosensing platform has promising applications in the detection of HIV in clinical and point-of-care settings.

Bispecific antibodies promote natural killer cell-mediated elimination of HIV-1 reservoir cells

The persistence of CD4+ T cells carrying latent human immunodeficiency virus-1 (HIV-1) proviruses is the main barrier to a cure. New therapeutics to enhance HIV-1-specific immune responses and clear infected cells will probably be necessary to achieve reduction of the latent reservoir. In the present study, we report two single-chain diabodies (scDbs) that target the HIV-1 envelope protein (Env) and the human type III Fcγ receptor (CD16). We show that the scDbs promoted robust and HIV-1-specific natural killer (NK) cell activation and NK cell-mediated lysis of infected cells. Cocultures of CD4+ T cells from people with HIV-1 on antiretroviral therapy (ART) with autologous NK cells and the scDbs resulted in marked elimination of reservoir cells that was dependent on latency reversal. Treatment of human interleukin-15 transgenic NSG mice with one of the scDbs after ART initiation enhanced NK cell activity and reduced reservoir size. Thus, HIV-1-specific scDbs merit further evaluation as potential therapeutics for clearance of the latent reservoir.

Accelerated neurodegeneration of basal forebrain cholinergic neurons in HIV-1 gp120 transgenic mice: Critical role of the p75 neurotrophin receptor

Human Immunodeficiency Virus-1 (HIV) infection of the brain induces HIV-associated neurocognitive disorders (HAND). The set of molecular events employed by HIV to drive cognitive impairments in people living with HIV are diverse and remain not completely understood. We have shown that the HIV envelope protein gp120 promotes loss of synapses and decreases performance on cognitive tasks through the p75 neurotrophin receptor (p75NTR). This receptor is abundant on cholinergic neurons of the basal forebrain and contributes to cognitive impairment in various neurological disorders. In this study, we examined cholinergic neurons of gp120 transgenic (gp120tg) mice for signs of degeneration. We observed that the number of choline acetyltransferase-expressing cells is decreased in old (12–14-month-old) gp120tg mice when compared to age matched wild type. In the same animals, we observed an increase in the levels of pro-nerve growth factor, a ligand of p75NTR, as well as a disruption of consolidation of extinction of conditioned fear, a behavior regulated by cholinergic neurons of the basal forebrain. Both biochemical and behavioral outcomes of gp120tg mice were rescued by the deletion of the p75NTR gene, strongly supporting the role that this receptor plays in the neurotoxic effects of gp120. These data indicate that future p75NTR-directed pharmacotherapies could provide an adjunct therapy against synaptic simplification caused by HIV.

An update on current HIV treatments

The roll-out of antiretroviral therapies (ART) has greatly assisted in reducing the morbidity and mortality rates caused by HIV. However, ART is incapable of eliminating the latent Human Immunodeficiency Virus (HIV), leading to life-long treatment being undertaken. Current HIV cures can be classified into the sterilizing cure, where the HIV latent reservoir is fully eliminated, or the functional cure, where prolonged suppression of latent HIV is achieved. Broadly Neutralizing Antibody (bNAb) is a type of sterilizing cure which targets an epitope on HIV Envelope (Env) protein. Studies indicated that a combined bNAb regiment is better than bNAb monotherapy due to the ability to avoid HIV escape variants. Another type of sterilizing cure is known as “shock and kill” strategy, comprising a latency-reversing agent which activates latent HIV and eliminates it with a “kill” agent. Alternatively, functional cure such as the “Block and lock” strategy utilizes a latency-promoting agent to “block” viral transcription and prevent it from being activated for a prolonged period. Further research and clinical tests are required to develop an HIV cure.

Refining HIV envelope protein conformational data from different experimental sources using bias-resampling methods and information theory.

Human immunodeficiency virus (HIV) remains a global epidemic, and vaccine development has been challenging. This challenge is due in part to conflicting data regarding which conformational states of the envelope glycoprotein (Env) are effectively targeted by neutralizing antibodies. Env is known to undergo conformational change as part of its function. Identifying the conformations best bound by neutralizing antibodies will improve vaccine design. To this end, we use bias resampling ensemble refinement (BRER) to integrate and compare prior spectroscopic measurements of HIV Env conformation. By using both electron paramagnetic resonance and single-molecule FRET data as restraints in conformational ensemble refinement, we generate models for the sets of HIV Env conformations consistent with each set of measurements. Results show that the “hidden” conformational states identified via single-molecule FRET spectroscopy indeed show differences from reference cryo-EM structures. Further analysis shows that the “hidden” conformational states have different distance distributions for some residue pairs than those measured via DEER. We have developed information-theoretic techniques to identify DEER label sites that would best differentiate conformational states defined by single-molecule FRET. We hope that testing of these proposed DEER experiments will help resolve the discrepancy between observations using different spectroscopic techniques and better elucidate the conformational heterogeneity of HIV Env. Extension to datasets in the presence of bound neutralizing antibodies will help elucidate the key conformations recognized by broadly neutralizing anti-HIV antibodies and, we hope, further assist in vaccine design.

HIV gp120 impairs nucleus accumbens neuroimmune function and dopamine D3 receptor-mediated inhibition of cocaine seeking in male rats

Cocaine Use Disorders (CUDs) are associated with an increased risk of human immunodeficiency virus (HIV) infection. Cocaine and the HIV envelope protein gp120 each induce distinct deficits to mesocorticolimbic circuit function and motivated behavior; however, little is known regarding how they interact to dysregulate these functions or how such interactions impact pharmacotherapeutic efficacy. We have previously shown that the selective, weak partial agonist of the dopamine D3 receptor (D3R), MC-25-41, attenuates cocaine-seeking behavior in male rats. Here, we sought to characterize changes in striatal neuroimmune function in gp120-exposed rats across abstinence from operant access to cocaine (0.75 mg/kg, i.v.) or sucrose (45 mg/pellet), and to examine the impact of gp120 exposure on MC-25-41-reduced cocaine seeking. After establishing a history of cocaine or sucrose self-administration, rats received intracerebroventricular gp120 infusions daily the first 5 days of abstinence and were sacrificed either on day 6 or after 21 days of forced abstinence and a cue-induced cocaine seeking test. We demonstrated that MC-25-41 treatment attenuated cue-induced cocaine seeking among control rats but not gp120-exposed rats. Moreover, postmortem analysis of nucleus accumbens (NAc) core neuroimmune function indicated cocaine abstinence- and gp120-induced impairments, and the expression of several immune factors within the NAc core significantly correlated with cocaine-seeking behavior. We conclude that cocaine abstinence dysregulates striatal neuroimmune function and interacts with gp120 to inhibit the effectiveness of a D3R partial agonist in reducing cocaine seeking. These findings highlight the need to consider comorbidities, such as immune status, when evaluating the efficacy of novel pharmacotherapeutics.

Highly mutated monoclonal antibody 3F2 targets a conformational and strain-restricted epitope in human immunodeficiency virus gp41 with significant antibody-dependent cell cytotoxicity.

Identifying epitope targets by studying the native antibody (Ab) response can identify potential novel vaccine constructs. Studies suggest that long-term non-progressor (LTNP) subjects have inherent immune mechanisms that help to control viremia and disease progression. To explore a role for antibodies (Abs) in LTNP progression, our lab has previously characterized a number of highly mutated Abs that target conformational epitopes of the human immunodeficiency virus (HIV) envelope protein from a single LTNP subject (10076). One Ab clone, 10076-Q3-2C6, had significant cross-clade Ab-dependent cell cytotoxicity. To assess if other LTNP subjects produced similar Abs, we expressed another highly mutated Ab from another subject; subject 10002, clone 10002-Q1-3F2 (variable heavy chain, 63.2% amino acid sequence identity to predicted germline). After expression with its native light chain, the recombinant Ab 3F2 bound to the trimeric envelope protein of HIV (trimer), as well as to the ectodomain of gp41. 3F2 binding to gp41 peptide libraries was consistent with non-linear epitope binding and showed possible overlap with the epitope of 2C6. Ab competition assays suggested that 3F2 may bind near the immunodominant epitope 1 loop region (ID1) of gp41. 2C6 blocked the binding of ID1-loop-binding Abs and 3F2 to the trimer, but 3F2 failed to block 2C6 binding. Together, these results suggest that 3F2 binds to a non-linear conformational epitope primarily localized between the epitope of 2C6 and the ID1. Since they are targeted by functional Abs, a more complete understanding of these ID1 and near-ID1 epitopes may be exploited in future immunization strategies.

Modeling and simulation of fully-glycosylated full-length HIV envelope protein embedded in a viral membrane

According to World Health Organization (WHO), human immunodeficiency virus (HIV) continues to be a major global health issue. Although the current antiretroviral therapy can suppress viral load and improve life quality, there is no cure for HIV infection. The HIV envelope (Env) spike trimer, stabilized in prefusion-closed conformation, is a promising antigenic target to trigger the host immune system to produce broadly neutralizing antibodies. Numerous structures of soluble Env spike trimer mimics have been solved by X-ray crystallography and cryo-electron microscopy (cryo-EM), but nearly all of them have missing residues and the transmembrane and membrane-proximal external regions are truncated to prevent aggregation.

Refining HIV envelope protein conformational data from different experimental sources using bias-resampling methods

Human immunodeficiency virus (HIV) continues to cause a global epidemic, and vaccine development has been challenging. This is in part because there exist conflicting data regarding which conformational states of the envelope glycoprotein (Env) are effectively targeted by neutralizing antibodies. Env is known to undergo conformational change as part of its function, but identifying the conformations best bound by neutralizing antibodies will improve vaccine design. Here, we use bias resampling ensemble refinement (BRER) to integrate and compare prior spectroscopic measurements of HIV Env conformation.

Neuroinflammation in HIV-Related Neuropathic Pain.

In the management of human immunodeficiency virus (HIV) infection around the world, chronic complications are becoming a new problem along with the prolonged life expectancy. Chronic pain is widespread in HIV infected patients and even affects those with a low viral load undergoing long-term treatment with antiviral drugs, negatively influencing the adherence to disease management and quality of life. A large proportion of chronic pain is neuropathic pain, which defined as chronic pain caused by nervous system lesions or diseases, presenting a series of nervous system symptoms including both positive and negative signs. Injury caused by HIV protein, central and peripheral sensitization, and side effects of antiretroviral therapy lead to neuroinflammation, which is regarded as a maladaptive mechanism originally serving to promote regeneration and healing, constituting the main mechanism of HIV-related neuropathic pain. Gp120, as HIV envelope protein, has been found to be the major toxin that induces neuropathic pain. Particularly, the microglia, releasing numerous pro-inflammatory substances (such as TNFα, IL-1β, and IL-6), not only sensitize the neurons but also are the center part of the crosstalk bridging the astrocytes and oligodendrocytes together forming the central sensitization during HIV infection, which is not discussed detailly in recent reviews. In the meantime, some NRTIs and PIs exacerbate the neuroinflammation response. In this review, we highlight the importance of clarifying the mechanism of HIV-related neuropathic pain, and discuss about the limitation of the related studies as future research directions.

Histone deacetylase 6 inhibition rescues axonal transport impairments and prevents the neurotoxicity of HIV-1 envelope protein gp120

Despite successful antiretroviral drug therapy, a subset of human immunodeficiency virus-1 (HIV)-positive individuals still display synaptodendritic simplifications and functional cognitive impairments referred to as HIV-associated neurocognitive disorders (HANDs). The neurological damage observed in HAND subjects can be experimentally reproduced by the HIV envelope protein gp120. However, the complete mechanism of gp120-mediated neurotoxicity is not entirely understood. Gp120 binds to neuronal microtubules and decreases the level of tubulin acetylation, suggesting that it may impair axonal transport. In this study, we utilized molecular and pharmacological approaches, in addition to microscopy, to examine the relationship between gp120-mediated tubulin deacetylation, axonal transport, and neuronal loss. Using primary rat cortical neurons, we show that gp120 decreases acetylation of tubulin and increases histone deacetylase 6 (HDAC6), a cytoplasmic enzyme that regulates tubulin deacetylation. We also demonstrate that the selective HDAC6 inhibitors tubacin and ACY-1215, which prevented gp120-mediated deacetylation of tubulin, inhibited the ability of gp120 to promote neurite shortening and cell death. We further observed by co-immunoprecipitation and confirmed with mass spectroscopy that exposure of neurons to gp120 decreases the association between tubulin and motor proteins, a well-established consequence of tubulin deacetylation. To assess the physiological consequences of this effect, we examined the axonal transport of brain-derived neurotrophic factor (BDNF). We report that gp120 decreases the velocity of BDNF transport, which was restored to baseline levels when neurons were exposed to HDAC6 inhibitors. Overall, our data suggest that gp120-mediated tubulin deacetylation causes impairment of axonal transport through alterations to the microtubule cytoskeleton.

Go go gadget glycoprotein!: HSV-1 draws on its sizeable glycoprotein tool kit to customize its diverse entry routes.

All viruses must enter cells to replicate [1]. Entry is thus the first hurdle a virus must overcome for a successful infection. Given the astonishing diversity of viruses that infect mammals alone—hundreds of thousands of different viruses according to some estimates [2]—it is unsurprising that their entry routes into cells are just as diverse. A major structural distinction that dictates the entry mode is the presence or absence of a lipid bilayer surrounding the viral nucleocapsid, which defines a virus as enveloped or nonenveloped, respectively. Nonenveloped viruses enter cells by endocytosis and subsequently penetrate the endosomal membrane by a variety of mechanisms including pore formation and endosomal fragmentation due to disruptive changes in membrane curvature [3], whereas all enveloped viruses must fuse their envelope with a host cell membrane: either the plasma membrane or the membrane of the endosomal vesicle following cellular uptake [4]. Regardless of the entry route, all viruses initially attach to the surface of the host cell by binding a cellular receptor. After attachment, enveloped viruses must employ fusogens—specialized viral surface glycoproteins that mediate the merger of the viral and host membranes by bringing them together as they undergo large, energetically favorable conformational changes. To do this, a spring-loaded fusogen must be triggered once the virus arrives at the right cell and/or the right intracellular compartment (such as an endosome, for example), either by binding a receptor (or a coreceptor) or by sensing the acidic pH of the endosome [4]. In many enveloped viruses, the receptor-binding and the fusogenic functions are mediated by different domains of a single glycoprotein. For example, the human immunodeficiency virus (HIV) envelope protein, Env, the sole glycoprotein encoded by HIV, binds the cellular glycoprotein cluster of differentiation 4 (CD4) and a coreceptor, C-X-C chemokine receptor 4 (CXCR4) or C-C chemokine receptor 5 (CCR5), on the surface of CD4+ T cells and also serves as the fusogen [5]. The influenza virus glycoprotein hemagglutinin binds an attachment receptor, sialic acid, and undergoes low-pH-triggered fusogenic conformational changes upon endocytosis [6]. In some cases, for example, in paramyxoviruses, the receptor-binding and the fusogenic functions are mediated by separate glycoproteins, and the fusogen receives the triggering signal from the receptor-binding viral protein [7]. Most enveloped viruses thus contain multiple copies of only one or two glycoproteins, which mediate viral attachment and entry into target cells [5–15]. Yet, entry of herpesviruses—large enveloped viruses that infect a wide variety of cells—is more complex, as it requires multiple viral glycoproteins (typically, at least three) and diverse host receptors [16]. Moreover, the coordinated activity of these multiple viral glycoproteins permits entry into different cell types by different routes. Whereas in some herpesviruses, such as human cytomegalovirus (HCMV) or Epstein–Barr virus (EBV), the use of particular entry routes correlates with the involvement of specific viral glycoprotein complexes [17, 18], in other herpesviruses, notably, herpes simplex virus type 1 (HSV-1), the picture is less clear [19]. Nonetheless, the entry mechanisms of all herpesviruses into a given cell, and particularly, the selection of the entry route, are complex and incompletely understood. The HSV-1 replication cycle in humans necessitates the infection of different cell types, chiefly, epithelial and neuronal cells. Although it is known that HSV-1 enters these cells by different mechanisms—endocytosis (epithelial cells) and fusion at the plasma membrane (neurons) [20, 21]—knowledge regarding HSV-1 glycoprotein involvement in the entry route–selection process is minimal. This raises the following question: How does HSV-1 select a particular route to enter different cell types? Although the answer remains elusive, this Pearl will summarize the current understanding of HSV-1 entry strategies and the players involved.

Method to identify efficiently cleaved, membrane-bound, functional HIV-1 (Human Immunodeficiency Virus-1) envelopes

An ideal vaccine against HIV-1 will specifically elicit bNAbs (broadly neutralizing antibodies) which can cross-neutralize a wide spectrum of circulating viral strains belonging to different clades. The current paradigm for developing such a vaccine is to generate HIV-1 envelope (Env)-based immunogens which can specifically elicit bNAbs. For this purpose, it is necessary to identify Envs, belonging to different clades, suitable for immunogen design. Efficient cleavage of the HIV-1 Env precursor gp160 polypeptide into its constituent subunits determines its ability to selectively bind to bNAbs and poorly to non-NAbs (non-neutralizing antibodies), properties desirable in Env-based immunogens. Thus, efficiently cleaved HIV-1 Envs with desirable antigenic properties can be good candidates for developing immunogens. Here we describe in detail a six step method we have used in our laboratory to identify such efficiently cleaved Envs. Some of these protocols are optimizations of previously reported assays such as FACS-based cell surface antibody binding assay, pseudovirus neutralization assay and gp120 shedding assay. Other protocols like biotinylation-neutravidin-agarose pull-down assay and plasma membrane protein immunoprecipitation assay have been developed by taking inputs from reagent/kit manufacturer’s protocols and previous studies. These protocols will help the field in identifying more such Envs which can be used for immunogen development.•Six step process to identify efficiently cleaved, membrane-bound, functional HIV-1 Envs with high degree of repeatability.•Method applicable for characterizing any HIV-1 envelope protein.•New method of immunoprecipitation of plasma membrane fraction to validate efficiently cleaved HIV-1 envelopes.

641. Development of Structural Epitope Targeting During B-cell Ontogeny by Exploration of Relatives of Gp41 Structural Epitope Binding Antibody 6F5

BackgroundIn previous studies, our lab has characterized a number of highly mutated antibodies against structural epitopes of the human immunodeficiency virus (HIV) envelope protein. These antibodies were first isolated from long-term nonprogressors (LTNPs). We have previous mapped 6F5 to a novel structural epitope that encompasses areas in both heptad repeats of GP41, mapping to amino acids of 557, 654 and 657 of reference sequence HXB2. In these studies, three other antibodies that were <90% homologous to 6F5 also resolved amino acid 657. On sequence analysis, 6F5 and its relatives had the same gene usage and general structure. These similarities and the similar epitope mapping implied these were once distantly related to a single B-cell lineage. As fusion of the viral membrane to the target cell depends on these heptad repeat regions associating and forming a six-helix postfusion bundle, antibodies that can interfere in this may be highly useful.MethodsSee results.ResultsBecause 6F5 maps to 557 and 654/657 which are widely separated on the primary sequence, we explored if there was differential binding to the postfusion six-helix-bundle form. Two peptides (N36 and C34) each containing one of the heptad repeats can form the post-fusion six-helix-bundle in vitro. On sandwich ELISA testing, 6F11 and 7B6 did not bind any form. Interestingly, 4E4 specifically captured both peptides alone, but not the six-helix-bundle and 6F5 only bound the six-helix-bundle but not the other peptide.A small number of samples were obtained to assess the prevalence of these responses in LTNPs. Antibodies that compete 6F11 are much more prevalent in LTNPs than normal progressors (75% vs. 20%). Functionally, we found that despite being mapped to a similar portion of Gp41 (657), only 6F5 is shown to have significant ADCC activity, however relative 6F11 does not.ConclusionIf targeting these epitopes correlates with the LTNP state, then these sites may be highly significant as targets of therapeutics or in vaccine strategies. Further studies on a larger cohort of LTNPs are ongoing. Additionally, deep sequencing of antibody sequences are being done to explore the development of structural epitope targeting by this family of antibodies.Disclosures All authors: No reported disclosures.

Differential T follicular helper cell responses induced by candidate HIV vaccine strategies utilizing ALVAC versus Ad5 vectors

Abstract The development of a safe and effective vaccine against Human Immunodeficiency Virus (HIV) remains a major priority in global health research. Several candidate HIV vaccines have undergone late-stage clinical trials in recent years, several of which utilized non-replicating viral vectors based on canarypox (ALVAC) or human adenovirus (Ad5.) Thus far, the only trial to show moderate (31.2%) efficacy in preventing HIV infection is the RV144 trial, which utilized an ALVAC prime/protein boost strategy. Subsequent analyses showed that protection from HIV infection correlated with the production of IgG3 antibodies directed against the highly conserved V1V2 loop of the HIV envelope (Env) protein, and was abrogated by the production of Env-specific serum IgA antibodies. Although the ineffective Ad5-based vaccines also induced strong Env-specific antibody responses, they failed to induce protective IgG3 and/or V1V2-specific antibodies. To better understand the mechanisms underlying the differential antibody profiles generated by these two vectors, we measured the frequency and functional phenotype of follicular T helper-like cells found in the peripheral blood of vaccine recipients (pTfh). We found that subjects who received an ALVAC prime/protein boost vaccine regimen had higher levels of Env-specific pTfh cells than subjects who received a DNA prime/Ad5 boost regimen, and that these pTfh cells have enhanced ability to induce B cell activation and class switching compared to those found in Ad5-based vaccine recipients. We hypothesize that the higher frequency and functionality of these ALVAC-induced pTfh cells contributes to the uniquely protective antibody response generated by ALVAC prime/protein boost vaccination.

Fitness landscape of the human immunodeficiency virus envelope protein that is targeted by antibodies

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.

The viral protein gp120 decreases the acetylation of neuronal tubulin: potential mechanism of neurotoxicity.

The human immunodeficiency virus (HIV) envelope protein gp120 promotes axonal damage and neurite pruning, similar to that observed in HIV-positive subjects with neurocognitive disorders. Thus, gp120 has been used to examine molecular and cellular pathways underlying HIV-mediated neuronal dysfunction. Gp120 binds to tubulin beta III, a component of neuronal microtubules. Microtubule function, which modulates the homeostasis of neurons, is regulated by polymerization and post-translational modifications. Based on these considerations, we tested the hypothesis that gp120 induces dynamic instability of neuronal microtubules. We first observed that gp120 prevents the normal polymerization of tubulin invitro. We then tested whether gp120 alters the post-translational modifications in tubulin by examining the ability of gp120 to change the levels of acetylated tubulin in primary rat neuronal cultures. Gp120 elicited a time-dependent decrease in tubulin acetylation thatwas reversed by Helix-A peptide, a compound that competitively displaces the binding of gp120 to neuronal microtubules. To determine whether post-translational modifications in tubulin also occur invivo, we measured acetylated tubulin in the cerebral cortex of HIV transgenic rats (HIV-tg). We observed a decrease in tubulin acetylation in 5- and 9-month-old HIV-tg rats when compared to age-matched wild type. Neither changes in microglia morphology nor alterations in mRNA levels for interleukin-1β and tumor necrosis factor α were detected in 5-month-old animals. Our findings propose neuronal microtubule instability as a novel mechanism of HIV neurotoxicity, without evidence of enhanced inflammation.