Soluble Gp120 Research Articles

Plasma Human Immunodeficiency Virus 1 Soluble Glycoprotein 120 Association With Correlates of Immune Dysfunction and Inflammation in Antiretroviral Therapy-Treated Individuals With Undetectable Viremia.

Chronic inflammation persists in some people living with human immunodeficiency virus (HIV) during antiretroviral therapy and is associated with premature aging. The glycoprotein 120 (gp120) subunit of HIV-1 envelope sheds and can be detected in plasma, showing immunomodulatory properties even in the absence of detectable viremia. We evaluated whether plasma soluble gp120 (sgp120) and a family of gp120-specific anti-cluster A antibodies, linked to CD4 depletion in vitro, contribute to chronic inflammation, immune dysfunction, and subclinical cardiovascular disease in participants of the Canadian HIV and Aging Cohort Study with undetectable viremia. Cross-sectional assessment of sgp120 and anti-cluster A antibodies was performed in 386 individuals from the cohort. Their association with proinflammatory cytokines and subclinical coronary artery disease was assessed using linear regression models. High levels of sgp120 and anti-cluster A antibodies were inversely correlated with CD4+ T cell count and CD4/CD8 ratio. The presence of sgp120 was associated with increased levels of interleukin 6. In participants with detectable atherosclerotic plaque and detectable sgp120, anti-cluster A antibodies and their combination with sgp120 levels correlated positively with the total volume of atherosclerotic plaques. This study showed that sgp120 may act as a pan toxin causing immune dysfunction and sustained inflammation in a subset of people living with HIV, contributing to the development of premature comorbid conditions.

Temsavir blocks the immunomodulatory activities of HIV-1 soluble gp120

While HIV-1-mediated CD4 downregulation protects infected cells from antibody-dependent cellular cytotoxicity (ADCC), shed gp120 binds to CD4 on uninfected bystander CD4+ Tcells, sensitizing them to ADCC mediated by HIV+ plasma. Soluble gp120-CD4 interaction on multiple immune cells also triggers a cytokine burst. The small molecule temsavir acts as an HIV-1 attachment inhibitor by preventing envelope glycoprotein (Env)-CD4 interaction and alters the overall antigenicity of Env by affecting its processing and glycosylation. Here we show that temsavir also blocks the immunomodulatory activities of shed gp120. Temsavir prevents shed gp120 from interacting with uninfected bystander CD4+ cells, protecting them from ADCC responses and preventing a cytokine burst. Mechanistically, this depends on temsavir's capacity to prevent soluble gp120-CD4 interaction, to reduce gp120 shedding, and to alter gp120 antigenicity. This suggests that the clinical benefits provided by temsavir could extend beyond blocking viral entry.

Self-assembling peptide nanofiber HIV vaccine elicits robust vaccine-induced antibody functions and modulates Fc glycosylation.

To develop vaccines for certain key global pathogens such as HIV, it is crucial to elicit both neutralizing and non-neutralizing Fc-mediated effector antibody functions. Clinical evidence indicates that non-neutralizing antibody functions including antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) contribute to protection against several pathogens. In this study, we demonstrated that conjugation of HIV Envelope (Env) antigen gp120 to a self-assembling nanofiber material named Q11 induced antibodies with higher breadth and functionality when compared to soluble gp120. Immunization with Q11-conjugated gp120 vaccine (gp120-Q11) demonstrated higher tier 1 neutralization, ADCP, and ADCC as compared to soluble gp120. Moreover, Q11 conjugation altered the Fc N-glycosylation profile of antigen-specific antibodies, leading to a phenotype associated with increased ADCC in animals immunized with gp120-Q11. Thus, this nanomaterial vaccine strategy can enhance non-neutralizing antibody functions possibly through modulation of immunoglobulin G Fc N-glycosylation.

MRNA-encoded HIV-1 Env trimer ferritin nanoparticles induce monoclonal antibodies that neutralize heterologous HIV-1 isolates in mice.

The success of nucleoside-modified mRNAs in lipid nanoparticles (mRNA-LNP) as COVID-19 vaccines heralded a new era of vaccine development. For HIV-1, multivalent envelope (Env) trimer protein nanoparticles are superior immunogens compared with trimers alone for priming of broadly neutralizing antibody (bnAb) B cell lineages. The successful expression of complex multivalent nanoparticle immunogens with mRNAs has not been demonstrated. Here, we show that mRNAs can encode antigenic Env trimers on ferritin nanoparticles that initiate bnAb precursor B cell expansion and induce serum autologous tier 2 neutralizing activity in bnAb precursor VH + VL knock-in mice. Next-generation sequencing demonstrates acquisition of critical mutations, and monoclonal antibodies that neutralize heterologous HIV-1 isolates are isolated. Thus, mRNA-LNP can encode complex immunogens and may be of use in design of germline-targeting and sequential boosting immunogens for HIV-1 vaccine development.

The Genesis and Future Prospects of Small Molecule HIV-1 Attachment Inhibitors.

Gp120 is a critical viral proteins required for HIV-1 entry and infection. It facilitates HIV-1 binding to target cells, human-to-human transmission, relocation of virus from mucosa to lymph nodes, cell-cell infection and syncytium formation, and the bystander effect that kills uninfected CD4+ T-cells and other human cells. Molecules that bind to gp120 can inhibit its function by stabilizing conformations of the protein, leading to the inability to infect cells, and resulting in non-permissive. Small molecule-mediated stabilization of certain conformations of gp120 may also enhance recognition of HIV-1 infected cells by neutralizing antibodies and make the virus more susceptible to effector functions such as ADCC, which could potentially be part of future cure regimens. Additionally, HIV attachment inhibitors can complex with free gp120 and potentially repress both cytopathic effects from membrane-bound or soluble gp120. Fostemsavir (RukobiaTM), a phosphate prodrug of an HIV-1 attachment inhibitor that was recently approved for use in highly treatment experienced (HTE) patients with multidrug resistant HIV-1 is a first-in-class drug with a favorable safety profile that provides an additional treatment option for treatment in this population of patients with a high medical need.

TAMI-50. PATHWAYS ACTIVATION IN GL261 MICE GLIOMA CELLS BY HIV-1 GLYCOPROTEIN GP120

Abstract Patients infected with human immunodeficiency virus type 1 (HIV-1) are more prone to developing cancers, including glioblastomas (GBMs). The median survival for GBM patients with HIV is significantly shorter than for HIV-negative GBM patients, even though they receive the same treatments. This difference indicates that HIV infection is associated with more aggressive tumor behavior and with treatment resistance. Earlier we demonstrated that gp120, a main glycoprotein in the HIV shell, stimulates glycolysis and protein synthesis in glioma cells. The purpose of this study was to evaluate the underlying gp120 dependent signaling mechanism in glioma cell. Mouse glioma cells GL-261 were continuously cultured for 7 days in medium with and without soluble gp120 Bal III (100ng/ml) and collected for Western blot and Cell cycle assays. Western blot analysis presented an increase in phosphorylation of Proline-rich tyrosine kinase (Pyk2(Y402)), p38(YT100/Y182) and p70s6(T421/S424), the key proteins of the Pyk2 pathway, along with the increased levels of Akt(S473) and Glycogen Synthase Kinase 3b (GSK3b (S9)) phosphorylation. Flow cytometry analysis of Cell Cycle revealed an increase of G2/M phase in cells cultured in gp120 Bal III when compared to control cells. Furthermore, GL-261 cells with knock-out of Pyk2 via CRISPR Cas 9 gene editing showed no significant change in cell cycle regulation when cultured with gp120 Bal III.Overall, these results demonstrate that gp120 triggers activation of Pyk2/MAPK and Akt/GSK3b pathways and alter cell cycle regulation in GBM. This research was made possible by NIH grant number 1SC1GM122691.

HIV envelope antigen valency on peptide nanofibers modulates antibody magnitude and binding breadth

A major challenge in developing an effective vaccine against HIV-1 is the genetic diversity of its viral envelope. Because of the broad range of sequences exhibited by HIV-1 strains, protective antibodies must be able to bind and neutralize a widely mutated viral envelope protein. No vaccine has yet been designed which induces broadly neutralizing or protective immune responses against HIV in humans. Nanomaterial-based vaccines have shown the ability to generate antibody and cellular immune responses of increased breadth and neutralization potency. Thus, we have developed supramolecular nanofiber-based immunogens bearing the HIV gp120 envelope glycoprotein. These immunogens generated antibody responses that had increased magnitude and binding breadth compared to soluble gp120. By varying gp120 density on nanofibers, we determined that increased antigen valency was associated with increased antibody magnitude and germinal center responses. This study presents a proof-of-concept for a nanofiber vaccine platform generating broad, high binding antibody responses against the HIV-1 envelope glycoprotein.

Exploration of a Sequential Gp140-Gp145 Immunization Regimen with Heterologous Envs to Induce a Protective Cross-Reactive HIV Neutralizing Antibody Response In Non-human Primates.

Raising a heterologous tier 2 neutralizing antibody (nAb) response remains a daunting task for HIV vaccine development. In this study, we explored the utility of diverse HIV-1 envelope (Env) immunogens in a sequential immunization scheme as a solution to this task. This exploration stemmed from the rationale that gp145, a membrane-bound truncation form of HIV Env, may facilitate the focusing of induced antibody response on neutralizing epitopes when sequentially combined with the soluble gp140 form as immunogens in a prime-boost mode. We first showed that gp140 DNA prime-gp145 Tiantan vaccinia (TV) boost likely represents a general format for inducing potent nAb response in mice. However, when examined in rhesus macaque, this modality showed little effectiveness. To improve the efficacy, we extended the original modality by adding a strong protein boost, namely native-like SOSIP.664 trimer displayed on ferritin-based nanoparticle (NP), which was generated by a newly developed click approach. The resulting three-immunization regimen succeeded in eliciting tier-2 nAb response with substantial breadth when implemented in rhesus macaque over a short 8-week schedule. Importantly, the elicited nAb response was able to effectively contain viremia upon a heterologous SHIV challenge. Collectively, our studies highlighted that diversification of Env immunogens, in both types and formulations, under the framework of a sequential immunization scheme might open new opportunity toward HIV vaccine development.Electronic supplementary materialThe online version of this article (10.1007/s12250-021-00361-3) contains supplementary material, which is available to authorized users.

Exploring Viral Interference Using Peptides: Molecular Determinants of HIV-1 Inhibition by a Peptide Derived from Human Pegivirus-1 Envelope Protein E2.

Co‐infection with the human pegivirus 1 (HPgV‐1) often has a beneficial effect on disease progression in HIV‐1‐infected individuals. Several HPgV‐1 proteins and peptides, including a 20‐mer peptide (P6‐2) derived from the N‐terminal region of the HPgV‐1 surface protein E2, have been associated with this phenomenon, which is referred to as viral interference. We identified the cysteine residues, the hydrophobic core tetrapeptide, as well as the C‐terminal negative charge as key factors for the HIV‐1 inhibitory activity of P6‐2. Analysis of mutations in P6‐2‐resistant HIV‐1 indicated a binding site for the peptide in the HIV‐1 envelope glycoprotein gp120. In fact, P6‐2 was shown to bind to soluble gp120, as well as to a peptide presenting the gp120 V3 loop. Furthermore, the HIV‐1 inhibitory activity of P6‐2 could be revoked by the V3 loop peptide, thus indicating a molecular mechanism that involves interaction of P6‐2 with the gp120 V3 loop.

Co-expression of human calreticulin significantly improves the production of HIV gp140 and other viral glycoproteins in plants.

SummaryPlant molecular farming (PMF) is rapidly gaining traction as a viable alternative to the currently accepted paradigm of producing biologics. While the platform is potentially cheaper and more scalable than conventional manufacturing systems, expression yields and appropriate post‐translational modifications along the plant secretory pathway remain a challenge for certain proteins. Viral fusion glycoproteins in particular are often expressed at low yields in plants and, in some cases, may not be appropriately processed. Recently, however, transiently or stably engineering the host plant has shown promise as a strategy for producing heterologous proteins with more complex maturation requirements. In this study we investigated the co‐expression of a suite of human chaperones to improve the production of a human immunodeficiency virus (HIV) type 1 soluble gp140 vaccine candidate in Nicotiana benthamiana plants. The co‐expression of calreticulin (CRT) resulted in a dramatic increase in Env expression and ameliorated the endoplasmic reticulum (ER) stress response ‐ as evidenced by lower transcript abundance of representative stress‐responsive genes. The co‐expression of CRT similarly improved accumulation of glycoproteins from Epstein‐Barr virus (EBV), Rift Valley fever virus (RVFV) and chikungunya virus (CHIKV), suggesting that the endogenous chaperone machinery may impose a bottleneck for their production. We subsequently successfully combined the co‐expression of human CRT with the transient expression of human furin, to enable the production of an appropriately cleaved HIV gp140 antigen. These transient plant host engineering strategies are a promising approach for the production of high yields of appropriately processed and cleaved viral glycoproteins.

Production and Immunogenicity of Soluble Plant-Produced HIV-1 Subtype C Envelope gp140 Immunogens.

The development of effective vaccines is urgently needed to curb the spread of human immunodeficiency virus type 1 (HIV-1). A major focal point of current HIV vaccine research is the production of soluble envelope (Env) glycoproteins which reproduce the structure of the native gp160 trimer. These antigens are produced in mammalian cells, which requires a sophisticated infrastructure for manufacture that is mostly absent in developing countries. The production of recombinant proteins in plants is an attractive alternative for the potentially cheap and scalable production of vaccine antigens, especially for developing countries. In this study, we developed a transient expression system in Nicotiana benthamiana for the production of soluble HIV Env gp140 antigens based on two rationally selected virus isolates (CAP256 SU and Du151). The scalability of the platform was demonstrated and both affinity and size exclusion chromatography (SEC) were explored for recovery of the recombinant antigens. Rabbits immunized with lectin affinity-purified antigens developed high titres of binding antibodies, including against the V1V2 loop region, and neutralizing antibodies against Tier 1 viruses. The removal of aggregated Env species by gel filtration resulted in the elicitation of superior binding and neutralizing antibodies. Furthermore, a heterologous prime-boost regimen employing a recombinant modified vaccinia Ankara (rMVA) vaccine, followed by boosts with the SEC-purified protein, significantly improved the immunogenicity. To our knowledge, this is the first study to assess the immunogenicity of a near-full length plant-derived Env vaccine immunogen.

SOSIP Changes Affect Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Conformation and CD4 Engagement.

The entry of human immunodeficiency virus into host cells is mediated by the envelope glycoprotein (Env) trimeric spike, which consists of three exterior gp120 subunits and three transmembrane gp41 subunits. The trimeric Env undergoes extensive conformational rearrangement upon interaction with the CD4 receptor, transitioning from the unliganded, "closed" State 1 to more-open downstream State 2 and State 3 conformations. Changes in "restraining" amino acid residues, such as leucine 193 and isoleucine 423, destabilize State 1 Env, which then assumes entry-competent, downstream conformations. The introduction of an artificial disulfide bond linking the gp120 and gp41 subunits (SOS) in combination with the I559P (IP) change has allowed structural characterization of soluble gp140 (sgp140) trimers. The conformation of these SOSIP-stabilized sgp140 trimers has been suggested to represent the closed native State 1 conformation. Here we compare the impact on the membrane Env conformation of the SOSIP changes with that of the well-characterized changes (L193R and I423A) that shift Env to downstream States 2 and 3. The results presented here suggest that the SOSIP changes stabilize Env in a conformation that differs from State 1 but also from the downstream Env conformations stabilized by L193R or I423A.IMPORTANCE The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer is triggered by receptor binding to mediate the entry of the virus into cells. Most structural studies of Env trimers have utilized truncated soluble gp140 Envs stabilized with the I559P and SOS changes. Here we present evidence indicating that these stabilizing changes have a profound impact on the conformation of Env, moving Env away from the native pretriggered Env conformation. Our studies underscore the need to acquire structural information on the pretriggered Env conformation, which is recognized by most broadly reactive neutralizing antibodies.

Comparison of Uncleaved and Mature Human Immunodeficiency Virus Membrane Envelope Glycoprotein Trimers.

The mature envelope glycoprotein (Env) spike on the surfaces of human immunodeficiency virus type 1 (HIV-1)-infected cells and virions is derived from proteolytic cleavage of a trimeric gp160 glycoprotein precursor. In these studies, we compared the conformations of cleaved and uncleaved membrane Envs with truncated cytoplasmic tails to those of stabilized soluble gp140 SOSIP.664 Env trimers. Deletion of the gp41 cytoplasmic tail did not significantly affect the sensitivity of viruses with the HIV-1AD8 Env to inhibition by antibodies or a CD4-mimetic compound. After glutaraldehyde fixation and purification from membranes, a cleaved Env exhibited a hydrodynamic radius of ∼10 nm and an antibody-binding profile largely consistent with that expected based on virus neutralization sensitivity. The purified cleaved Env trimers exhibited a hollow architecture with a central void near the trimer axis. Uncleaved Env, cross-linked and purified in parallel, exhibited a hydrodynamic radius similar to that of the cleaved Env. However, the uncleaved Env was recognized by poorly neutralizing antibodies and appeared by negative-stain electron microscopy to sample multiple conformations. Compared with membrane Envs, stabilized soluble gp140 SOSIP.664 Env trimers appear to be more compact, as reflected in their smaller hydrodynamic radii and negative-stain electron microscopy structures. The antigenic features of the soluble gp140 SOSIP.664 Env trimers differed from those of the cleaved membrane Env, particularly in gp120 V3 and some CD4-binding-site epitopes. Thus, proteolytic maturation allows the membrane-anchored Env to achieve a conformation that retains functional metastability but masks epitopes for poorly neutralizing antibodies.IMPORTANCE The entry of human immunodeficiency virus type 1 (HIV-1) into host cells is mediated by the envelope glycoprotein (Env) spike on the surface of the virus. Host antibodies elicited during natural HIV-1 infection or by vaccination can potentially recognize the Env spike and block HIV-1 infection. However, the changing shape of the HIV-1 Env spike protects the virus from antibody binding. Understanding the shapes of natural and man-made preparations of HIV-1 Envs will assist the development of effective vaccines against the virus. Here, we evaluate the effects of several Env modifications commonly used to produce Env preparations for vaccine studies and the determination of structure. We found that the cleavage of the HIV-1 Env precursor helps Env to assume its natural shape, which resists the binding of many commonly elicited antibodies. Stabilized soluble Envs exhibit more compact shapes but expose some Env elements differently than the natural Env.

P-C4 Associating HIV-1 Env trimer structures with functional Env conformational states by smFRET analysis

The HIV-1 envelope glycoprotein (Env) trimer mainly exists in a closed conformation (state 1), which is driven by CD4 binding through an intermediate conformation (state 2) to the open CD4-bound conformation (state 3). These functional Env states can be visualized by single-molecule Fluorescence Resonance Energy Transfer (smFRET). A breakthrough in the structural characterization of the HIV-1 Env trimer has been the generation of recombinant cleaved soluble gp140 SOSIP.664 trimers. Parallel cryoelectron microscopy studies have been performed with the mature HIV-1JR-FL Env in complex with the PGT151 neutralizing antibody. Both approaches resulted in similar structures. It is currently generally assumed that these structures represent the ground state of HIV-1 Env (state 1). Here we apply smFRET to probe the conformational state of HIV-1 Env in these constructs and antibody complexes. Fluorophores were introduced at the identical positions in the HIV-1 Env proteins used for structural studies and the native Env on the surface of virions, and the resulting smFRET values compared. Surprisingly, smFRET data reveal that both the soluble gp140 SOSIP.664 and PGT151-HIV-1JR-FL Env structures correspond to the state 2 gp120 conformation observed on the virus. Our data suggest that the all-important structure of state 1 of HIV-1 Env, which is the target of the majority of broadly neutralizing antibodies, remains unknown. Determining the structure of this additional conformation observed on native virions should allow the design of second generation immunogens that specifically present the state 1 conformation of HIV-1 Env.

Transgenic mice expressing HIV-1 envelope protein gp120 in the brain as an animal model in neuroAIDS research.

HIV-1 infection causes injury to the central nervous system (CNS) and is often associated with neurocognitive disorders. One model for brain damage seen in AIDS patients is the transgenic (tg) mouse expressing a soluble envelope protein gp120 of HIV-1 LAV in the brain in astrocytes under the control of the promoter of glial fibrillary acidic protein. These GFAP-gp120tg mice manifest several key neuropathological features observed in AIDS brains, such as decreased synaptic and dendritic density, increased numbers of activated microglia, and pronounced astrocytosis. Several recent studies show that brains of GFAP-gp120tg mice and neurocognitively impaired HIV patients share also a significant number of differentially regulated genes, activation of innate immunity and other cellular signaling pathways, disturbed neurogenesis, and learning deficits. These findings support the continued relevance of the GFAP-gp120tg mouse as a useful model to investigate neurodegenerative mechanisms and develop therapeutic strategies to mitigate the consequences associated with HIV infection of the CNS, neuroAIDS, and HAND.

Structure and topology around the cleavage site regulate post-translational cleavage of the HIV-1 gp160 signal peptide.

Like all other secretory proteins, the HIV-1 envelope glycoprotein gp160 is targeted to the endoplasmic reticulum (ER) by its signal peptide during synthesis. Proper gp160 folding in the ER requires core glycosylation, disulfide-bond formation and proline isomerization. Signal-peptide cleavage occurs only late after gp160 chain termination and is dependent on folding of the soluble subunit gp120 to a near-native conformation. We here detail the mechanism by which co-translational signal-peptide cleavage is prevented. Conserved residues from the signal peptide and residues downstream of the canonical cleavage site form an extended alpha-helix in the ER membrane, which covers the cleavage site, thus preventing cleavage. A point mutation in the signal peptide breaks the alpha helix allowing co-translational cleavage. We demonstrate that postponed cleavage of gp160 enhances functional folding of the molecule. The change to early cleavage results in decreased viral fitness compared to wild-type HIV.

A heterologous prime-boosting strategy with replicating Vaccinia virus vectors and plant-produced HIV-1 Gag/dgp41 virus-like particles

Showing modest efficacy, the RV144 HIV-1 vaccine clinical trial utilized a non-replicating canarypox viral vector and a soluble gp120 protein boost. Here we built upon the RV144 strategy by developing a novel combination of a replicating, but highly-attenuated Vaccinia virus vector, NYVAC-KC, and plant-produced HIV-1 virus-like particles (VLPs). Both components contained the full-length Gag and a membrane anchored truncated gp41 presenting the membrane proximal external region with its conserved broadly neutralizing epitopes in the pre-fusion conformation. We tested different prime/boost combinations of these components in mice and showed that the group primed with NYVAC-KC and boosted with both the viral vectors and plant-produced VLPs have the most robust Gag-specific CD8 T cell responses, at 12.7% of CD8 T cells expressing IFN-γ in response to stimulation with five Gag epitopes. The same immunization group elicited the best systemic and mucosal antibody responses to Gag and dgp41 with a bias towards IgG1.

Antigenic and immunosuppressive properties of a trimeric recombinant transmembrane envelope protein gp41 of HIV-1.

The transmembrane envelope (TM) protein gp41 of the human immunodeficiency virus—1 (HIV-1) plays an important role during virus infection inducing the fusion of the viral and cellular membranes. In addition, there are indications that the TM protein plays a role in the immunopathogenesis leading to the acquired immunodeficiency syndrome (AIDS). Inactivated virus particles and recombinant gp41 have been reported to inhibit lymphocyte proliferation, as well as to alter cytokine release and gene expression. The same was shown for a peptide corresponding to a highly conserved domain of all retroviral TM proteins, the immunosuppressive domain. Due to its propensity to aggregate and to be expressed at low levels, studies comprising authentic gp41 produced in eukaryotic cells are extremely rare. Here we describe the production of a secreted, soluble recombinant gp41 in 293 cells. The antigen was purified to homogeneity and characterised thoroughly by various biochemical and immunological methods. It was shown that the protein was glycosylated and assembled into trimers. Binding studies by ELISA and surface plasmon resonance using conformation-specific monoclonal antibodies implied a six-helix bundle conformation. The low binding of broadly neutralising antibodies (bnAb) directed against the membrane proximal external region (MPER) suggested that this gp41 is probably not suited as vaccine to induce such bnAb. Purified gp41 bound to monocytes and to a lesser extent to lymphocytes and triggered the production of specific cytokines when added to normal peripheral blood mononuclear cells. In addition, gp41 expressed on target cells inhibited the antigen-specific response of murine CD8+ T cells by drastically impairing their IFNγ production. To our knowledge, this is the first comprehensive analysis of a gp41 produced in eukaryotic cells including its immunosuppressive properties. Our data provide another line of evidence that gp41 might be directly involved in HIV-1 immunopathogenesis through modulation of the cytokine release and active inhibition of immune responses.

Differential Antibody Responses to Conserved HIV-1 Neutralizing Epitopes in the Context of Multivalent Scaffolds and Native-Like gp140 Trimers.

ABSTRACTBroadly neutralizing antibodies (bNAbs) have provided valuable insights into the humoral immune response to HIV-1. While rationally designed epitope scaffolds and well-folded gp140 trimers have been proposed as vaccine antigens, a comparative understanding of their antibody responses has not yet been established. In this study, we probed antibody responses to the N332 supersite and the membrane-proximal external region (MPER) in the context of heterologous protein scaffolds and native-like gp140 trimers. Ferritin nanoparticles and fragment crystallizable (Fc) regions were utilized as multivalent carriers to display scaffold antigens with grafted N332 and MPER epitopes, respectively. Trimeric scaffolds were also identified to stabilize the MPER-containing BG505 gp140.681 trimer in a native-like conformation. Following structural and antigenic evaluation, a subset of scaffold and trimer antigens was selected for immunization in BALB/c mice. Serum binding revealed distinct patterns of antibody responses to these two bNAb targets presented in different structural contexts. For example, the N332 nanoparticles elicited glycan epitope-specific antibody responses that could also recognize the native trimer, while a scaffolded BG505 gp140.681 trimer generated a stronger and more rapid antibody response to the trimer apex than its parent gp140.664 trimer. Furthermore, next-generation sequencing (NGS) of mouse splenic B cells revealed expansion of antibody lineages with long heavy-chain complementarity-determining region 3 (HCDR3) loops upon activation by MPER scaffolds, in contrast to the steady repertoires primed by N332 nanoparticles and a soluble gp140.664 trimer. These findings will facilitate the future development of a coherent vaccination strategy that combines both epitope-focused and trimer-based approaches.

Computational Evaluation of HIV-1 gp120 Conformations of Soluble Trimeric gp140 Structures as Targets for de Novo Docking of First- and Second-Generation Small-Molecule CD4 Mimics.

Small-molecule CD4 mimics (SMCM's) bind to the gp120 subunit of the HIV-1 envelope glycoprotein (Env) and have been optimized to block cell infection in vitro. The lack of the V1/2 and V3 loops and the presence of the β2/3 and β20/21 strands (bridging sheet) in the available structures of the monomeric gp120 core may limit its applicability as a target for further synthetic optimization of SMCM potency and/or breadth. Here, we employ a combination of binding-site search, docking, estimation of protein-ligand interaction energy, all-atom molecular dynamics, and ELISA-based CD4-binding competition assays to create, characterize, and rationalize models of first- and second-generation of SMCM's bound to the distinct, trimeric BG505 SOSIP.664 structures 4NCO and 4TVP containing V1/2 and V3 loops with no bridging sheet. We demonstrate that the in silico neutralization of the highly conserved D368 is necessary to obtain the correct orientation of SMCM in their binding site when docking against the monomeric gp120 core. The computational results correlate with IC50's measured in CD4 binding competition ELISA and with KD's measured on gp120 core monomer. This supports the hypothesis that the 4NCO trimeric structure represents a viable target for further SMCM's optimization with advantages over both the 4TVP trimer and gp120 core monomer. Finally, the docking protocol has been optimized to screen compounds that can clearly interact with the highly conserved residue D368, increasing the likelihood of future optimizations to arrive at SMCM's with a broader spectrum of activity.