HIV-1 Gp120 Research Articles

Antibodies to HIV-1 gp120 in an experimental model of atherosclerosis caused by immunization with native high-density lipoproteins

Antibodies to bacterial and viral antigens are detected in some systematic autoimmune diseases in the absence of infection. Autoimmune MRL mice and patients with mixed connective tissue disease produce antibodies to gp120 HIV-1, despite the fact that they had never been exposed to HIV-1. Conversely, viral infections may be accompanied by pathological autoimmune reactions. Reactivity to viral antigens in autoimmune diseases and autoimmune reactions in viral diseases are caused by the induction of antibodies via idiotype-anti-idiotype interactions between autoclones and lymphocyte clones against foreign antigens or homology of foreign antigens and autoantigens. Cardiovascular diseases caused by atherosclerosis are currently one of the main causes of mortality in HIV infected patients. It is assumed that HIV infection accelerates atherogenesis. The mechanisms of the association between HIV infection and atherosclerosis are not completely clear. Chronic activation of the immune system, disturbance of cytokine regulation caused by HIV infection, induction of autoantibodies against oxidized low-density lipoproteins (LDL) and antibodies to ApoB-D are considered as possible factors of atherogenesis in HIV infection. The aim of this research was to determine whether an atherogenic immune response against native human high-density lipoproteins (nHDL) could induce antibodies to gp120 HIV-1. Studies were conducted on model of autoimmune atherosclerosis caused by immunization with native human HDL in rabbits. Rabbits (n = 6) were one-time intradermally immunized with human nHDL at a dose of 200 μg of protein per animal in incomplete Freund’s adjuvant. Antibodies to gp120 HIV-1 were measured before immunization of rabbits and on the 28th and 42th day after immunization with HDL, and antibodies to HDL were weekly measured within 42 days after immunization. Histological analysis of the aorta was conducted after eight months after immunization. Transient anti-gp120 HIV-1 antibody production was detected in rabbits immunized with native HDL. The appearance of antibodies to gp120 HIV-1 in response to immunization with native HDL which causes atherosclerosis suggests that the immune response to gp120 HIV-1 during HIV infection may be accompanied by the production of atherogenic antibodies to HDL. Consequently, the antibodies to native lipoproteins caused by an immune response against gp120 HIV-1 may be a factor in atherogenesis during HIV-1 infection.

Safety and implementation of a phase 1 randomized GLA-SE-adjuvanted CH505TF gp120 HIV vaccine trial in newborns.

The neonatal immune system is uniquely poised to generate broadly neutralizing antibodies (bnAbs) and thus infants are ideal for evaluating HIV vaccine candidates. We present the design and safety of a novel glucopyranosyl lipid A (GLA)-stable emulsion (SE) adjuvant admixed with a first-in-infant CH505 transmitter-founder (CH505TF) gp120 immunogen designed to induce precursors for bnAbs against HIV. HVTN 135 is a phase I randomized, placebo-controlled trial of CH505TF+GLA-SE or placebo. Healthy infants in South Africa aged ≤5 days, born to mothers living with HIV but HIV nucleic acid negative at birth were randomized to five doses of CH505TF + GLA-SE or placebo at birth and 8, 16, 32, and 54 weeks. 38 infants (median age = 4 days; interquartile range 4, 4.75 days) were enrolled November 2020 to January 2022. Among 28 (10) infants assigned to receive CH505TF + GLA-SE (placebo), most (32/38) completed the 5-dose immunization series and follow-up (35/38). Solicited local and systemic reactions were more frequent in vaccine (8, 28.6% local; 16, 57.1% systemic) vs. placebo recipients (1, 10% local, p = 0.25; 4, 40.0% systemic, p = 0.38). All events were Grade 1 except two Grade 2 events (pain, lethargy). Serious vaccine-related adverse events were not recorded. This study illustrates the feasibility of conducting trials of novel adjuvanted HIV vaccines in HIV-exposed infants receiving standard infant vaccinations. The safety profile of the CH505TF + GLA-SE vaccine was reassuring. ClinicalTrials.gov NCT04607408. National Institute of Allergy and Infectious Diseases (NIAID) at the National Institutes of Health (NIH). This paper summarizes the phase 1 trial design and safety profile of an experimental CH505TF immunogen + GLA-SE HIV vaccine in infants born to mothers living with HIV.

Triple broadly neutralizing antibody (bnAb) combination therapy: a cure for pediatric HIV?

Abstract Background In 2022, UNAIDS estimated out of the 1.7 million children living with HIV, only 57.1% had access to antiretroviral therapy (ART). Though ART has significantly decreased HIV-associated morbidity and mortality, it cannot eliminate the latent viral reservoir. Consequently, most people living with HIV demonstrate viral rebound when ART is interrupted. There is therefore a critical need for alternative therapeutic to achieve long term ART free viral control. Broadly neutralizing antibodies (bnAb) have shown promising results in preclinical and clinical studies, but their potential efficacy is limited by the emergence of escape variants, especially with monotherapy. We previously tested the neutralizing and non-neutralizing functions of different bnAb combinations and identified a triple bnAb combination that showed robust neutralization potency and breadth, as well as non-neutralizing functions. In this project, we investigated the impact of combination bnAb therapy on viral rebound in SHIV infected infant rhesus macaques (RMs). Methods Ten infant rhesus macaques were orally challenged with SHIV.C.CH505 at 4 weeks of age. Oral challenge is a well-established method that is used to mimic breastmilk transmission of HIV, which contributes to almost 50% of pediatric HIV infections every year. A triple bnAb combination of simianized 3BNC117 (CD4 binding site), PGDM1400 (V2 glycan dependent), and PGT151 (interface) was subcutaneously administered twice at 40 mg/kg of each antibody. The initial infusion was administered at ART initiation (8 weeks post-infection) while the second infusion with administered at the time of analytical treatment interruption (ATI) (49 weeks post-infection). RMs were monitored weekly from the beginning of infection until 12 weeks post-ATI. Enzyme-linked Immunosorbent Assay (ELISA) was used to monitor plasma concentrations of each bNab and Env-specific IgG binding responses. Results Env-specific antibody binding levels against HIV envelope proteins gp120 and gp41 peaked in plasma 8 weeks after infection. The antibody levels then slightly decreased during ART and increased again after viral rebound following ATI. BnAb plasma concentrations peaked 1-2 weeks after each bnAb infusion, then slowly declined. After the initial infusion, all RMs had undetectable levels of 3BNC117 and PGT151 by 8 weeks post initial infusion, and only 2/10 RMs had detectable PGDM1400 levels. All animals experienced viral rebound following ATI.). Importantly, the average time to virus rebound (7 weeks, range 3-10 weeks) was significantly longer than a s control group of RMs without any immune-based intervention (~2.5 weeks). By the time of viral rebound, most animals had undetectable plasma levels of 3BNC117, PGDM1400 or PGT151. However, in some animals, rebound occurs when passively administered antibodies were still detectable, suggesting the emergence of bnAb resistant variants. Conclusion Passive immunization with a triple-bnAb combination of 3BNC117, PGT151 and PGDM1400 was associated with delayed viral rebound, but more investigation is needed to understand the potential role of this strategy in the HIV therapeutic portfolio. Future work will investigate the sensitivity of the rebound viruses to the bnAb combination to evaluate the contribution of bnAb escape to viral rebound.

Adaptation of the REINVENT neural network architecture to generate potential HIV-1 entry inhibitors

Objectives. The main purpose of this work is to adapt the architecture of the REINVENT neural network to generate potential inhibitors of the HIV-1 envelope protein gp120 using in the learning process with reinforcement of molecular docking on GPUs.Methods. To modify the initial network model, molecular docking on GPUs implemented in the learning process with reinforcement was used, and an algorithm was developed that allows converting the representations of connections generated by the SMILES network into the PDBQT format necessary for docking. To accelerate the learning of the neural network in the modified version of the REINVENT model, the AutoDock-Vina-GPU-2.1 docking program was used, and to clarify the results of its work, the procedure for revaluing the affinity of compounds to the target using the RFScore-4 evaluation function was used.Results. Using a modified version of the REINVENT model, more than 60,000 compounds were obtained, of which about 52,000 molecules have a binding energy value to the HIV-1 gp120 protein comparable to the value calculated for the HIV-1 inhibitor NBD-14204, used in calculations as a positive control. Of the 52,000 compounds selected, about 34,000 molecules satisfy the restrictions imposed on a potential drug to ensure its bioavailability when taken orally.Conclusion. The results obtained allow us to demonstrate the effectiveness of an adapted neural network by the example of designing new potential inhibitors of the gp120 HIV-1 protein capable of blocking the CD4- binding site of the gp120 virus envelope protein and preventing its penetration into host cells.

HIV1 gp120 activates microglia via TLR2-NF-κB signaling to up-regulate inflammatory cytokine expression and induce neuropathic pain

HIV associated neuropathic pain (HANP) is a common complication of AIDS. Intrathecal injection of recombinant HIV-1 gp120 in mice is a well-known model. Previous RNA sequencing revealed spinal TLR2 acts as a differentially expressed gene in HANP mice. The spinal TLR2 is involved in HANP, but its role and underlying mechanism remains unclear. In this study the transcription, expression and distribution characteristics of TLR2 in the spinal cord of HANP male mice have been analyzed by qRT-PCR, Western blotting, and immunofluorescent staining. We found that TLR2 expression was upregulated in the spinal dorsal horn and mainly distributed in microglial cells, and blocking TLR2 relieved pain of HANP mice. Following stimulation by gp120 microglial cells upregulate TLR2 expression and become activated. The activation stimulates their differentiation into the M1 type, increasing IL-1β and TNF-α expression while inhibiting IL-10 expression. Silencing the Tlr2 gene slows down the activation, polarization, and secretion of pro-inflammatory factors in microglial cells induced by gp120, and enhances the expression of anti-inflammatory factors. Further analysis of the impact of gp120 on downstream signaling pathways of TLR2 in microglial cells, including NF-κB, MAPK (p38MAPK, ERK, and JNK) and PI3K/AKT, revealed that TLR2-NF-κB signaling plays a crucial role in the activation and polarization of microglial cells by gp120. Activation of NF-κB signaling aggravates pain in HANP mice, while blocking it lightens pain. This data indicates that gp120, through the TLR2-NF-κB signaling, activates spinal microglial cells, promotes the secretion of inflammatory cytokines, leading to HANP. This provides new targets to develop drugs for HANP.

Comparing Gold-Standard Sanger Sequencing with Two Next-Generation Sequencing Platforms of HIV-1 gp160 Single Genome Amplicons.

Our goal was to assess the accuracy of next generation sequencing (NGS) compared with Sanger. We performed single genome amplification (SGA) of HIV-1 gp160 on extracted tissue DNA from two HIV+ individuals. Amplicons (n = 30) were sequenced with Sanger or reamplified with barcoded primers and pooled before sequencing using Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PB). For each amplicon, a consensus sequence for NGS reads was obtained by (1) mapping reads to the Sanger sequence when available ("reference-based") or (2) mapping reads to a "pseudo-reference" sequence, i.e., a consensus sequence of a subset of NGS reads ("reference-free"). PB reads were clustered based on genetic similarity. A Sanger consensus sequence was obtained for 23/30 amplicons, for which all NGS consensus sequences were identical (n = 9) or nearly identical (n = 14) compared with Sanger. For the nine mismatches between Sanger/NGS, the nucleotide in the NGS sequence matched all other sequences from that patient. Of the 7/30 amplicons without a Sanger sequence, NGS sequences had ≥35 ambiguous calls in five amplicons and 0 ambiguitiesin two amplicons. Analysis of the electropherograms showed failure of a single sequencing primer for the latter two amplicons (consistent with a single template) and overlapping peaks for the other five (consistent with multiple templates). Clustering results closely followed the Sanger/NGS consensus results, where amplicons derived from a single template also had a single cluster and vice versa (with one exception, which could be the result of barcode misidentification). Representative sequences from the clusters contained 2-13 differences compared with Sanger/NGS. In summary, we show that both ONT and PB can produce amplicon consensus sequences with similar or higher accuracy compared with Sanger and, importantly, without the need for a known reference sequence. Clustering could be useful in some circumstances to predict or confirm the presence of multiple starting templates.

Characterization of clinical envelopes with lack of sensitivity to the HIV-1 inhibitors temsavir and ibalizumab

Previous data suggest a lack of cross-resistance between the gp120-directed attachment inhibitor temsavir (active moiety of fostemsavir) and the CD4-directed post-attachment inhibitor ibalizumab. Recently, analysis of HIV-1 envelopes with reduced sensitivity to both inhibitors was undertaken to determine whether they shared genotypic correlates of resistance. Sequences from 2 envelopes with reduced susceptibility to both agents were mapped onto a temsavir-bound gp120 structure. Residues within 5.0 Å of the temsavir binding site were evaluated using reverse genetics. Broader applicability and contextual determinants of key substitutions were further assessed using envelopes from participants in the phase 3 BRIGHTE study. Temsavir sensitivity was measured by half-maximal inhibitory concentration (IC50) and ibalizumab sensitivity by IC50 and maximum percent inhibition (MPI). One envelope required substitutions of E113D and T434M for full restoration of temsavir susceptibility. Neither substitution nor their combination affected ibalizumab sensitivity. However, in the second envelope, an E202 substitution (HXB2, T202) was sufficient for observed loss of susceptibility to both inhibitors. One BRIGHTE participant with no ibalizumab exposure had an emergent K202E substitution at protocol-defined virologic failure, with reduced sensitivity to both inhibitors. Introducing T202E into previously susceptible clinical isolates reduced temsavir potency by ≥ 40-fold and ibalizumab MPI from >99% to ∼80%. Interestingly, introduction of the gp120 V5 region from a highly ibalizumab-susceptible envelope mitigated the E202 effect on ibalizumab but not temsavir. A rare HIV-1 gp120 E202 mutation reduced temsavir susceptibility, and depending on sequence context, could result in reduced susceptibility to ibalizumab.

A computational approach to evaluate caffeoylquinic acids and flavonoids in Pluchea indica Less. leaves as potential anti-HIV agents

Context: The attachment of human immunodeficiency virus type 1 glycoprotein 120 (HIV-1 gp120) to the CD4 receptor of human immune cells is the beginning of HIV-1 infection. Stimulation of reactive oxygen species (ROS) production through upregulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-2 (NOX-2) and -4 (NOX-4), and cytochrome P450 2E1 (CYP2E1) of the virus can be a potential target for anti-HIV agents. Aims: To evaluate the inhibitory effects of caffeoylquinic acids (CQAs) and flavonoids of Pluchea indica leaves against the binding of HIV-1 gp120 with CD4 receptor and their antioxidant activities via interactions with NOX-2, NOX-4, and CYP2E1 through in silico study. Methods: Ten CQAs and nine flavonoids of P. indica were docked to the 3TGS (gp120 HIV-1), 2CDU (NOX-2), 3A1F (NOX-4), and 3T3Z (CYP2E1) receptors using the AutoDockTools 1.5.7. Physicochemical and pharmacokinetics properties were predicted using the pkCSM online tool, while toxicity was predicted using the ProTox-II webserver. Results: Mostly, all of the CQAs and flavonoids were able to bind to all receptors. 3,4-Di-O-caffeoylquinic acid has the lowest binding energy (-8.79 kcal/mol) against 3TGS (gp120). 5-O-Caffeoylquinic acid and apigenin have great potential as antioxidants due to their good binding with NOX-2 and CYP2E1. However, CQAs might have ADME problems. Most test compounds did not cause hepatotoxicity, carcinogenicity, or mutagenicity. All test compounds have no cytotoxic potential. However, all CQAs have the potential to be immunotoxins. Conclusions: The findings indicated that 3,4-di-O-caffeylquinic acid could be a potential inhibitor of HIV-1 gp120-CD4 binding, while 5-O-caffeoylquinic acid and apigenin demonstrated strong antioxidant activities via NOX-2 and CYP2E1 inhibition. However, in-depth studies, including experimental in vitro and in vivo studies, are required to validate the anti-HIV activity of the compounds further.

Alpaca-derived nanobody targeting the hydrophobic pocket of the HIV-1 gp41 NHR broadly neutralizes HIV-1 by blocking six-helix bundle formation

The highly conserved hydrophobic pocket region of HIV-1 gp41 NHR triple-stranded coiled coil is crucial for the binding of CHR to NHR to form a six-helix bundle (6-HB). This pocket is only exposed instantaneously during fusion, making it an ideal target for antibody drug design. However, IgG molecule is too big to enter the pocket during the fusion process. Therefore, to overcome the steric hindrance and kinetic obstacles caused by the formation of gp41 pre-hairpin fusion intermediate, we obtained nanobodies (Nbs) targeting NHR by immunizing alpaca with an NHR-trimer mimic. Specifically, we identified a Nb, Nb-172, that exhibited potent and broadly neutralizing activity against HIV-1 pseudoviruses, HIV-1 primary isolates, and T20-resistant HIV-1 strains. In addition, the combinatorial use of mD1.22 and Nb-172 exhibited synergism in inhibiting HIV-1 infection inactivating cell-free virions. Nb-172 can competitively bind to the hydrophobic pocket of gp41 NHR to inhibit 6-HB formation. These findings suggest that Nb-172 merits further investigation as a potential therapeutic for HIV-1 infection.

The sensitivity of HIV-1 gp120 polymorphs to inhibition by temsavir correlates to temsavir binding on-rate

Temsavir binds directly to the HIV-1 envelope glycoprotein gp120 and selectively inhibits interactions between HIV-1 and CD4 receptors. Previous studies identified gp120 amino acid positions where substitutions are associated with reduced susceptibility to temsavir. The mechanism by which temsavir susceptibility is altered in these envelope glycoproteins was evaluated. Pseudoviruses encoding gp120 substitutions alone (S375H/I/M/N, M426L, M434I, M475I) or in combination (S375H + M475I) were engineered on a wild-type JRFL background. Temsavir-gp120 and CD4-gp120 binding kinetics and ability of temsavir to block CD4-gp120 binding were evaluated using the purified polymorphic gp120 proteins and a Creoptix® WAVE Delta grating-coupled interferometry system. Fold-change in half-maximal inhibitory concentration (IC50) in JRFL-based pseudoviruses containing the aforementioned polymorphisms relative to that of wild-type ranged from 4-fold to 29,726-fold, while temsavir binding affinity for the polymorphic gp120 proteins varied from 0.7-fold to 73.7-fold relative to wild-type gp120. Strong correlations between temsavir IC50 and temsavir binding affinity (r = 0.7332; P = 0.0246) as well as temsavir binding on-rate (r = −0.8940; P = 0.0011) were observed. Binding affinity of gp120 proteins for CD4 varied between 0.4-fold and 3.1-fold compared with wild-type gp120; no correlations between temsavir IC50 and CD4 binding kinetic parameters were observed. For all polymorphic gp120 proteins, temsavir was able to fully block CD4 binding; 3 polymorphs required higher temsavir concentrations. Loss of susceptibility to temsavir observed for gp120 polymorphisms strongly correlated with reductions in temsavir binding on-rate. Nonetheless, temsavir retained the ability to fully block CD4-gp120 engagement given sufficiently high concentrations.

Scyllatoxin-based peptide design for E. coli expression and HIV gp120 binding

Targeting the hydrophobic Phe43 pocket of HIV's envelope glycoprotein gp120 is a critical strategy for antiviral interventions due to its role in interacting with the host cell's CD4. Previous inhibitors, including small molecules and CD4 mimetic peptides based on scyllatoxin, have demonstrated significant binding and neutralization capabilities but were often chemically synthesized or contained non-canonical amino acids. Microbial expression using natural amino acids offers advantages such as cost-effectiveness, scalability, and efficient production of fusion proteins. In this study, we enhanced the previous scyllatoxin-based synthetic peptide by substituting natural amino acids and successfully expressed it in E. coli. The peptide was optimized by mutating the C-terminal amidated valine to valine and glutamine, and by reducing the disulfide bonds from three to two. Circular dichroism confirmed proper secondary structure formation, and fluorescence polarization analysis revealed specific, concentration-dependent binding to HIV gp120, supported by molecular dynamics simulations. These findings indicate the potential for scalable microbial production of effective antiviral peptides, with significant applications in pharmaceutical development for HIV treatment.

Safety and Immunogenicity of a DNA Vaccine With Subtype C gp120 Protein Adjuvanted With MF59 or AS01B: A Phase 1/2a HIV-1 Vaccine Trial.

An effective vaccine is required to end the HIV pandemic. We evaluated the safety and immunogenicity of a DNA (DNA-HIV-PT123) vaccine with low- or high-dose bivalent (TV1.C and 1086.C glycoprotein 120) subtype C envelope protein combinations, adjuvanted with MF59 or AS01B. HIV Vaccine Trials Network (HVTN)108 was a randomized, placebo-controlled, double-blind, phase 1/2a trial conducted in the United States and South Africa. HIV-negative adults were randomly assigned to 1 of 7 intervention arms or placebo to assess DNA prime with DNA/protein/adjuvant boosts, DNA/protein/adjuvant co-administration, and low-dose protein/adjuvant regimens. HVTN111 trial participants who received an identical regimen were also included. Outcomes included safety and immunogenicity 2 weeks and 6 months after final vaccination. From June 2016 to July 2018, 400 participants were enrolled (N = 334 HVTN108, N = 66 HVTN111); 370 received vaccine and 30 received placebo. There were 48 grade 3 and 3 grade 4 reactogenicity events among 39/400 (9.8%) participants, and 32 mild/moderate-related adverse events in 23/400 (5.8%) participants. All intervention groups demonstrated high IgG response rates (>89%) and high magnitudes to HIV-1 Env gp120 and gp140 proteins; response rates for AS01B-adjuvanted groups approached 100%. V1V2 IgG magnitude, Fc-mediated functions, IgG3 Env response rates, and CD4+ T-cell response magnitudes and rates were higher in the AS01B-adjuvanted groups. The AS01B-adjuvanted low-dose protein elicited greater IgG responses than the higher protein dose. The vaccine regimens were generally well tolerated. Co-administration of DNA with AS01B-adjuvanted bivalent Env gp120 elicited the strongest humoral responses; AS01B-adjuvanted regimens elicited stronger CD4+ T-cell responses, justifying further evaluation.ClinicalTrials.gov registration: NCT02915016, registered 26 September 2016.

Discovery of a Double-Stapled Short Peptide as a Long-Acting HIV-1 Inactivator with Potential for Oral Bioavailability.

Different from most antiretroviral drugs that act as passive defenders to inhibit HIV-1 replication inside the host cell, virus inactivators can attack and inactivate HIV-1 virions without relying on their replication cycle. Herein, we describe the discovery of a hydrocarbon double-stapled helix peptide, termed D26. D26 is based on the HIV-1 gp41 protein lentiviral lytic peptide-3 motif (LLP3) sequence, which can efficiently inhibit HIV-1 infection and inactivate cell-free HIV-1 virions. It was noted that D26 was highly resistant to proteolytic degradation and exhibited a remarkably extended in vivo elimination half-life. Additionally, relative to its linear, nonstapled version, D26 exhibited much higher exposure in sanctuary sites for HIV-1. Amazingly, this lead compound also demonstrated detectable oral absorption. Thus, it can be concluded that D26 is a promising candidate for further development as a long-acting, orally applicable HIV-1 inactivator for the treatment of HIV-1 infection.

Structure and Interactions of HIV-1 gp41 CHR-NHR Reverse Hairpin Constructs Reveal Molecular Determinants of Antiviral Activity

Engineered reverse hairpin constructs containing a partial C-heptad repeat (CHR) sequence followed by a short loop and full-length N-heptad repeat (NHR) were previously shown to form trimers in solution and to be nanomolar inhibitors of HIV-1 Env mediated fusion. Their target is the in situ gp41 fusion intermediate, and they have similar potency to other previously reported NHR trimers. However, their design implies that the NHR is partially covered by CHR, which would be expected to limit potency. An exposed hydrophobic pocket in the folded structure may be sufficient to confer the observed potency, or they may exist in a partially unfolded state exposing full length NHR. Here we examined their structure by crystallography, CD and fluorescence, establishing that the proteins are folded hairpins both in crystal form and in solution. We examined unfolding in the milieu of the fusion reaction by conducting experiments in the presence of a membrane mimetic solvent and by engineering a disulfide bond into the structure to prevent partial unfolding. We further examined the role of the hydrophobic pocket, using a hairpin-small molecule adduct that occluded the pocket, as confirmed by X-ray footprinting. The results demonstrated that the NHR region nominally covered by CHR in the engineered constructs and the hydrophobic pocket region that is exposed by design were both essential for nanomolar potency and that interaction with membrane is likely to play a role in promoting the required inhibitor structure. The design concepts can be applied to other Class 1 viral fusion proteins.

Stabilized trimeric peptide immunogens of the complete HIV-1 gp41 N-heptad repeat and their use as HIV-1 vaccine candidates

Efforts to develop an HIV-1 vaccine include those focusing on conserved structural elements as the target of broadly neutralizing monoclonal antibodies. MAb D5 binds to a highly conserved hydrophobic pocket on the gp41 N-heptad repeat (NHR) coiled coil and neutralizes through prevention of viral fusion and entry. Assessment of 17-mer and 36-mer NHR peptides presenting the D5 epitope in rodent immunogenicity studies showed that the longer peptide elicited higher titers of neutralizing antibodies, suggesting that neutralizing epitopes outside of the D5 pocket may exist. Although the magnitude and breadth of neutralization elicited by NHR-targeting antigens are lower than that observed for antibodies directed to other epitopes on the envelope glycoprotein complex, it has been shown that NHR-directed antibodies are potentiated in TZM-bl cells containing the FcγRI receptor. Herein, we report the design and evaluation of covalently stabilized trimeric 51-mer peptides encompassing the complete gp41 NHR. We demonstrate that these peptide trimers function as effective antiviral entry inhibitors and retain the ability to present the D5 epitope. We further demonstrate in rodent and nonhuman primate immunization studies that our 51-mer constructs elicit a broader repertoire of neutralizing antibody and improved cross-clade neutralization of primary HIV-1 isolates relative to 17-mer and 36-mer NHR peptides in A3R5 and FcγR1-enhanced TZM-bl assays. These results demonstrate that sensitive neutralization assays can be used for structural enhancement of moderately potent neutralizing epitopes. Finally, we present expanded trimeric peptide designs which include unique low-molecular-weight scaffolds that provide versatility in our immunogen presentation strategy.

Phospho-Chitooligosaccharides below 1 kDa Inhibit HIV-1 Entry In Vitro.

Despite present antiviral agents that can effectively work against HIV-1 replication, side effects and drug resistance have pushed researchers toward novel approaches. In this context, there is a continued focus on discovering new and more effective antiviral compounds, particularly those that have a natural origin. Polysaccharides are known for their numerous bioactivities, including inhibiting HIV-1 infection and replication. In the present study, phosphorylated chitosan oligosaccharides (PCOSs) were evaluated for their anti-HIV-1 potential in vitro. Treatment with PCOSs effectively protected cells from HIV-1-induced lytic effects and suppressed the production of HIV-1 p24 protein. In addition, results show that PCOSs lost their protective effect upon post-infection treatment. According to the results of ELISA, PCOSs notably disrupted the binding of HIV-1 gp120 protein to T cell surface receptor CD4, which is required for HIV-1 entry. Overall, the results point out that PCOSs might prevent HIV-1 infection at the entry stage, possibly via blocking the viral entry through disruption of virus-cell fusion. Nevertheless, the current results only present the potential of PCOSs, and further studies to elucidate its action mechanism in detail are needed to employ phosphorylation of COSs as a method to develop novel antiviral agents.

Nanocapsules Comprised of Purified Protein: Construction and Applications in Vaccine Research.

Nanoparticles show great promise as a platform for developing vaccines for the prevention of infectious disease. We have been investigating a method whereby nanocapsules can be formulated from protein, such that the final capsules contain only the cross-linked protein itself. Such nanocapsules are made using a silica templating system and can be customised in terms of size and porosity. Here we compare the construction and characteristics of nanocapsules from four different proteins: one a model protein (ovalbumin) and three from infectious disease pathogens, namely the influenza virus, Helicobacter pylori and HIV. Two of the nanocapsules were assessed further. We confirm that nanocapsules constructed from the urease A subunit of H. pylori can reduce subsequent infection in a vaccinated mouse model. Further, we show that capsules constructed from the HIV gp120 protein can be taken up by dendritic cells in tissue culture and can be recognised by antibodies raised against the virus. These results point to the utility of this method in constructing protein-only nanocapsules from proteins of varying sizes and isoelectric points.

CoPoP liposomes displaying stabilized clade C HIV-1 Env elicit tier 2 multiclade neutralization in rabbits

One of the strategies towards an effective HIV-1 vaccine is to elicit broadly neutralizing antibody responses that target the high HIV-1 Env diversity. Here, we present an HIV-1 vaccine candidate that consists of cobalt porphyrin-phospholipid (CoPoP) liposomes decorated with repaired and stabilized clade C HIV-1 Env trimers in a prefusion conformation. These particles exhibit high HIV-1 Env trimer decoration, serum stability and bind broadly neutralizing antibodies. Three sequential immunizations of female rabbits with CoPoP liposomes displaying a different clade C HIV-1 gp140 trimer at each dosing generate high HIV-1 Env-specific antibody responses. Additionally, serum neutralization is detectable against 18 of 20 multiclade tier 2 HIV-1 strains. Furthermore, the peak antibody titers induced by CoPoP liposomes can be recalled by subsequent heterologous immunization with Ad26-encoded membrane-bound stabilized Env antigens. Hence, a CoPoP liposome-based HIV-1 vaccine that can generate cross-clade neutralizing antibody immunity could potentially be a component of an efficacious HIV-1 vaccine.

Structural Basis of HIV-1 gp41 N-trimer for Designing Bifunctional Peptide-based Fusion Inhibitors.

Pathogenic viruses that cause large-scale global or regional outbreaks almost always contain class I fusion proteins. Although the viruses differ in morphology, they all require fusion protein-mediated virus-host cell membranes during the early stages of host cell invasion. The CHR region and NHR region of fusion proteins can form the 6-HB structure to drive the fusion pore formation between viruses and host cells through metastable interactions. Here, we obtained bifunctional N-peptides with inhibitory activities against two viruses, HIV-1 and MERS-CoV, based on the sequences in the HIV-1 NHR region by constructing N-trimer conformation interacting with the CHR region. This study demonstrates that N-peptides with the coiled triple helix structure obtained from the NHR region in 6-HB are able to target the CHR region and exhibit inhibitory activity against a variety of viruses. Moreover, this strategy can be used to investigate antivirals against unknown viruses for future outbreaks.

Using a Drug Repurposing Strategy to Virtually Screen Potential HIV-1 Entry Inhibitors That Block the NHR Domain of the Viral Envelope Protein gp41

Using a drug repurposing strategy, virtual screening of potential inhibitors of the NHR domain of the HIV-1 gp41 protein, a conserved region critical for the virus-cell membrane fusion and viral infectivity, was carried out. The used computational approach included: (1) molecular docking of this functionally significant region of the HIV-1 envelope with compounds from a library of bioactive molecules containing clinically approved drugs, experimental drugs, and investigational drug candidates; (2) assessing the binding affinity of these compounds to the therapeutic target; (3) molecular dynamics simulations of ligand/NHR-gp41 complexes; (4) calculations of the binding free energy followed by the analysis of molecular dynamics trajectories and selection of compounds promising to test for anti-HIV-1 activity. As a result, six compounds that exhibited the high binding affinity to the NHR domain of the HIV-1 gp41 protein and showed acceptable pharmacological properties were identified. The predicted compounds are assumed to form a promising basis for the development of new, effective and safe broad-spectrum antiviral agents able to inhibit the HIV-1 entry into the host cell.