A stabilized HIV-1 envelope glycoprotein trimer fused to CD40 ligand targets and activates dendritic cells

<p>Abstract</p> <p>Background</p> <p>One reason why subunit protein and DNA vaccines are often less immunogenic than live-attenuated and whole-inactivated virus vaccines is that they lack the co-stimulatory signals provided by various components of the more complex vaccines. The HIV-1 envelope glycoprotein complex (Env) is no exception to this rule. Other factors that limit the induction of neutralizing antibodies against HIV-1 lie in the structure and instability of Env. We have previously stabilized soluble trimeric mimics of Env by introducing a disulfide bond between gp120 and gp41 and adding a trimer stabilizing mutation in gp41 (SOSIP.R6 gp140).</p> <p>Results</p> <p>We further stabilized the SOSIP.R6 gp140 using a GCN4-based isoleucine zipper motif, creating SOSIP.R6-IZ gp140. In order to target SOSIP.R6-IZ to immune cells, including dendritic cells, while at the same time activating these cells, we fused SOSIP.R6-IZ to the active domain of CD40 ligand (CD40L), which may serve as a '<it>cis</it>-adjuvant'. The Env component of the SOSIP.R6-IZ-CD40L fusion construct bound to CD4 and neutralizing antibodies, while the CD40L moiety interacted with CD40. Furthermore, the chimeric molecule was able to signal efficiently through CD40 and induce maturation of human dendritic cells. Dendritic cells secreted IL-6, IL-10 and IL-12 in response to stimulation by SOSIP.R6-IZ-CD40L and were able to activate naïve T cells.</p> <p>Conclusions</p> <p>Chimeric HIV-1 gp140 - CD40L trimers can target and activate dendritic cells. Targeting and activating immune cells using CD40L and other '<it>cis</it>-adjuvants' may improve subunit protein vaccine immunogenicity for HIV-1 and other infectious diseases.</p

The carbohydrate at asparagine 386 on HIV-1 gp120 is not essential for protein folding and function but is involved in immune evasion

<p>Abstract</p> <p>Background</p> <p>The HIV-1 envelope glycoprotein gp120, which mediates viral attachment to target cells, consists for ~50% of sugar, but the role of the individual sugar chains in various aspects of gp120 folding and function is poorly understood. Here we studied the role of the carbohydrate at position 386. We identified a virus variant that had lost the 386 glycan in an evolution study of a mutant virus lacking the disulfide bond at the base of the V4 domain.</p> <p>Results</p> <p>The 386 carbohydrate was not essential for folding of <it>wt </it>gp120. However, its removal improved folding of a gp120 variant lacking the 385–418 disulfide bond, suggesting that it plays an auxiliary role in protein folding in the presence of this disulfide bond. The 386 carbohydrate was not critical for gp120 binding to dendritic cells (DC) and DC-mediated HIV-1 transmission to T cells. In accordance with previous reports, we found that N386 was involved in binding of the mannose-dependent neutralizing antibody 2G12. Interestingly, in the presence of specific substitutions elsewhere in gp120, removal of N386 did not result in abrogation of 2G12 binding, implying that the contribution of N386 is context dependent. Neutralization by soluble CD4 and the neutralizing CD4 binding site (CD4BS) antibody b12 was significantly enhanced in the absence of the 386 sugar, indicating that this glycan protects the CD4BS against antibodies.</p> <p>Conclusion</p> <p>The carbohydrate at position 386 is not essential for protein folding and function, but is involved in the protection of the CD4BS from antibodies. Removal of this sugar in the context of trimeric Env immunogens may therefore improve the elicitation of neutralizing CD4BS antibodies.</p

Potent Induction of Envelope-Specific Antibody Responses by Virus-Like Particle Immunogens Based on HIV-1 Envelopes from Patients with Early Broadly Neutralizing Responses

Longitudinal studies in HIV-1 infected individuals have indicated that 2-3 years of infection are required to develop broadly neutralizing antibodies. However, we have previously identified individuals with broadly neutralizing activity (bNA) in early HIV-1 infection, indicating that a vaccine may be capable of bNA induction after short periods of antigen exposure. Here, we describe 5 HIV-1 envelope sequences from individuals who have developed bNA within the first 100 days of infection (early neutralizers) and selected two of them to design immunogens based on HIV-1-Gag virus like particles (VLPs). These VLPs were homogeneous and incorporated the corresponding envelopes (7 to 9μg of gp120 in 1010 VLPs). Both envelopes bound to well-characterized bNAbs, including trimer-specific antibodies (PGT145, VRC01 and 35022). For immunogenicity testing, we immunized rabbits with the Env-VLPs or with the corresponding stabilized soluble Envelope trimers. A short immunization protocol (105 days) was used to recapitulate the early nAb induction observed after HIV-1 infection in these two individuals. All VLP and trimeric Envelope immunogens induced a comparably strong anti-gp120 response, despite having immunized rabbits with 30 times less gp120 in the case of the Env-VLPs. In addition, animals immunized with VLP-formulated Envs induced antibodies that cross-recognized the corresponding soluble stabilized trimer and vice versa, even though no neutralizing activity was observed. Nevertheless, our data may provide a new platform of immunogens, based on HIV-1 envelopes from patients with early broadly neutralizing responses, with the potential to generate protective immune responses using vaccination protocols similar to those used in classical preventive vaccines. Importance: It is generally accepted that an effective HIV-1 vaccine should be able to induce broad-spectrum neutralizing antibodies. Since most of these antibodies require long periods of somatic maturation in vivo, several groups are developing immunogens, based on the HIV envelope protein, that require complex and lengthy immunization protocols that would be difficult to implement to the general population. Here, we show that rabbits immunized with new envelopes (VLP-formulated) from two individuals who demonstrated broadly neutralizing activity very early after infection, induced specific HIV-1 antibodies after a short immunization protocol. This evidence provides the basis for generating protective immune responses with classic vaccination protocols with vaccine prototypes based on HIV envelope sequences from individuals who have developed early broadly neutralizing responses.This project received funding from the European Union's Horizon 2020 research and innovation program under grant agreement no. 681137 to I.B., N.G., A.O., C.B., J.A., R.W.S., and E.Y. It was also partially supported by the Spanish AIDS Research Network (RIS), funded by the Instituto de Salud Carlos III and co-funded by the European Regional Development Fund (ERDF) “A way to build Europe” (projects RD16CIII/0002/0001, RD16CIII/0002/0005, and RD16CIII/0025/0041), Plan Estatal de I1D1I 2013-2016 to N.G., A.M.M., J.A., V.S.M., E.Y., M.P., A.O., and C.B.; by IDIBAPS to J.M.M. (80:20 Research grant); by the Fondation Dormeur, Vaduz to C.B.; by the Ministerio de Economía, Industria y Competitividad to N.G., V.S.M., and E.Y. (PI17CIII/00049); by the Ministerio de Ciencia e Innovación to N.G., V.S.M., and E.Y. (PI20CIII/00039); by the Consejo Nacional de Innovación, Ciencia y Tecnología to C.B.P.; and by the HHS/ National Institutes of Health (NIH) to C.B. (P01-AI131568).S

Bispecific antibodies combine breadth, potency, and avidity of parental antibodies to neutralize sarbecoviruses

SARS-CoV-2 variants evade current monoclonal antibody therapies. Bispecific antibodies (bsAbs) combine the specificities of two distinct antibodies taking advantage of the avidity and synergy provided by targeting different epitopes. Here we used controlled Fab-arm exchange to produce bsAbs that neutralize SARS-CoV and SARS-CoV-2 variants, including Omicron and its subvariants, by combining potent SARS-CoV-2-specific neutralizing antibodies with broader antibodies that also neutralize SARS-CoV. We demonstrated that the parental antibodies rely on avidity for neutralization using bsAbs containing one irrelevant Fab arm. Using mass photometry to measure the formation of antibody:spike complexes, we determined that bsAbs increase binding stoichiometry compared to corresponding cocktails, without a loss of binding affinity. The heterogeneous binding pattern of bsAbs to spike, observed by negative-stain electron microscopy and mass photometry provided evidence for both intra- and inter-spike crosslinking. This study highlights the utility of cross-neutralizing antibodies for designing bivalent agents to combat circulating and future SARS-like coronaviruses

Co-display of diverse spike proteins on nanoparticles broadens sarbecovirus neutralizing antibody responses

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants poses continuous challenges in combating the virus. Here, we describe vaccination strategies to broaden SARS-CoV-2 and sarbecovirus immunity by combining spike proteins based on different viruses or viral strains displayed on two-component protein nanoparticles. First, we combined spike proteins based on ancestral and Beta SARS-CoV-2 strains to broaden SARS-CoV-2 immune responses. Inclusion of Beta spike improved neutralizing antibody responses against SARS-CoV-2 Beta, Gamma, and Omicron BA.1 and BA.4/5. A third vaccination with ancestral SARS-CoV-2 spike also improved cross-neutralizing antibody responses against SARS-CoV-2 variants, in particular against the Omicron sublineages. Second, we combined SARS-CoV and SARS-CoV-2 spike proteins to broaden sarbecovirus immune responses. Adding SARS-CoV spike to a SARS-CoV-2 spike vaccine improved neutralizing responses against SARS-CoV and SARS-like bat sarbecoviruses SHC014 and WIV1. These results should inform the development of broadly active SARS-CoV-2 and pan-sarbecovirus vaccines and highlight the versatility of two-component nanoparticles for displaying diverse antigens

Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection

Brouwer et al. present preclinical evidence in support of a COVID-19 vaccine candidate, designed as a self-assembling two-component protein nanoparticle displaying multiple copies of the SARS-CoV-2 spike protein, which induces strong neutralizing antibody responses and protects from high-dose SARS-CoV-2 challenge.The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is continuing to disrupt personal lives, global healthcare systems, and economies. Hence, there is an urgent need for a vaccine that prevents viral infection, transmission, and disease. Here, we present a two-component protein-based nanoparticle vaccine that displays multiple copies of the SARS-CoV-2 spike protein. Immunization studies show that this vaccine induces potent neutralizing antibody responses in mice, rabbits, and cynomolgus macaques. The vaccine-induced immunity protects macaques against a high-dose challenge, resulting in strongly reduced viral infection and replication i

Stabilized HIV-1 Envelope Glycoprotein Trimers Lacking the V1V2 Domain, Obtained by Virus Evolution*

The envelope glycoproteins (Env) are the focus of HIV-1 vaccine development strategies based on the induction of humoral immunity, but the mechanisms the virus has evolved to limit the induction and binding of neutralizing antibodies (NAbs) constitute substantial obstacles. Conserved neutralization epitopes are shielded by variable regions and carbohydrates, so one strategy to increase their exposure and, it is hoped, their immunogenicity is to delete the overlying variable loops. However, deleting the variable regions from Env trimers can be problematic, because hydrophobic patches that are normally solvent-inaccessible now become exposed, causing protein misfolding or aggregation, for example. Here, we describe the construction and characterization of recombinant gp140 trimers lacking variable domains 1 and 2 (ΔV1V2). The design of the trimers was guided by HIV-1 evolution studies that identified compensatory changes in V1V2-deleted but functional Env proteins (Bontjer, I., Land, A., Eggink, D., Verkade, E., Tuin, K., Baldwin, C., Pollakis, G., Paxton, W. A., Braakman, I., Berkhout, B., and Sanders, R. W. (2009) J. Virol. 83, 368–383). We now show that specific compensatory changes improved the function of ΔV1V2 Env proteins and hence HIV-1 replication. The changes acted by reducing the exposure of a hydrophobic surface either by replacing a hydrophobic residue with a hydrophilic one or by covering the surface with a glycan. The compensatory changes allowed the efficient expression of well folded, soluble gp140 trimers derived from various HIV-1 isolates. The evolved ΔV1V2 Env viruses were extremely sensitive to NAbs, indicating that neutralization epitopes are well exposed, which was confirmed by studies of NAb binding to the soluble ΔV1V2 gp140 trimers. These evolved ΔV1V2 trimers could be useful reagents for immunogenicity and structural studies

HIV-1 anchor inhibitors and membrane fusion inhibitors target distinct but overlapping steps in virus entry

HIV-1 entry into cells is mediated by the envelope glycoprotein (Env) and represents an attractive target for therapeutic intervention. Two drugs that inhibit HIV entry are approved for clinical use: the membrane fusion-inhibitor T20 (Fuzeon, enfuvirtide) and the C-C chemokine receptor type 5 (CCR5) blocker maraviroc (Selzentry). Another class of entry inhibitors supposedly target the fusion peptide (FP) and are termed anchor inhibitors. These include the VIRIP peptide and VIRIP derivatives such as VIR165, VIR353, and VIR576. Here, we investigated the mechanism of inhibition by VIR165. We show that substitutions within the FP modulate sensitivity to VIR165, consistent with the FP being the drug target. Our results also revealed that VIR165 acts during an intermediate post-CD4 – binding entry step that is overlapping but not identical to the step inhibited by fusion inhibitors such as T20. We found that some but not all resistance mutations to heptad repeat 2 (HR2)-targeting fusion inhibitors can provide cross-resistance to VIR165. In contrast, resistance mutations in the HR1-binding site for the fusion inhibitors did not cause cross-resistance to VIR165. However, Env with mutations located outside this binding site and thought to affect fusion kinetics, exhibited decreased sensitivity to VIR165. Although we found a strong correlation between Env stability and resistance to HR2-based fusion inhibitors, such correlation was not observed for Env stability and VIR165 resistance. We conclude that VIRIP analogs target the FP during an intermediate, post-CD4 – binding entry step that overlaps with but is distinct from the step(s) inhibited by HR2-based fusion inhibitors

The Phylogeography of Orangutan Foamy Viruses Supports the Theory of Ancient Repopulation of Sumatra

Phylogenetic analysis of foamy virus sequences obtained from Bornean and Sumatran orangutans showed a distinct clustering pattern. One subcluster was represented by both Bornean and Sumatran orangutan simian foamy viruses (SFV). Combined analysis of host mitochondrial DNA and SFV phylogeny provided evidence for the hypothesis of the repopulation of Sumatra by orangutans from Borneo

HIV-1 Escape from a Peptidic Anchor Inhibitor through Stabilization of the Envelope Glycoprotein Spike

The trimeric HIV-1 envelope glycoprotein spike (Env) mediates viral entry into cells by using a spring-loaded mechanism that allows for the controlled insertion of the Env fusion peptide into the target membrane, followed by membrane fusion. Env is the focus of vaccine research aimed at inducing protective immunity by antibodies as well as efforts to develop drugs that inhibit the viral entry process. The molecular factors contributing to Env stability and decay need to be understood better in order to optimally design vaccines and therapeutics. We generated viruses with resistance to VIR165, a peptidic inhibitor that binds the fusion peptide of the gp41 subunit and prevents its insertion into the target membrane. Interestingly, a number of escape viruses acquired substitutions in the C1 domain of the gp120 subunit (A60E, E64K, and H66R) that rendered these viruses dependent on the inhibitor. These viruses could infect target cells only when VIR165 was present after CD4 binding. Furthermore, the VIR165-dependent viruses were resistant to soluble CD4-induced Env destabilization and decay. These data suggest that VIR165-dependent Env proteins are kinetically trapped in the unliganded state and require the drug to negotiate CD4-induced conformational changes. These studies provide mechanistic insight into the action of the gp41 fusion peptide and its inhibitors and provide new ways to stabilize Env trimer vaccines. Because of the rapid development of HIV-1 drug resistance, new drug targets need to be explored continuously. The fusion peptide of the envelope glycoprotein can be targeted by anchor inhibitors. Here we describe virus escape from the anchor inhibitor VIR165. Interestingly, some escape viruses became dependent on the inhibitor for cell entry. We show that the identified escape mutations stabilize the ground state of the envelope glycoprotein and should thus be useful in the design of stabilized envelope-based HIV vaccine