Distinct conformations of the HIV-1 V3 loop crown are targetable for broad neutralization

The V3 loop of the HIV-1 envelope (Env) protein elicits a vigorous, but largely non-neutralizing antibody response directed to the V3-crown, whereas rare broadly neutralizing antibodies (bnAbs) target the V3-base. Challenging this view, we present V3-crown directed broadly neutralizing Designed Ankyrin Repeat Proteins (bnDs) matching the breadth of V3-base bnAbs. While most bnAbs target prefusion Env, V3-crown bnDs bind open Env conformations triggered by CD4 engagement. BnDs achieve breadth by focusing on highly conserved residues that are accessible in two distinct V3 conformations, one of which resembles CCR5-bound V3. We further show that these V3-crown conformations can, in principle, be attacked by antibodies. Supporting this conclusion, analysis of antibody binding activity in the Swiss 4.5 K HIV-1 cohort (n = 4,281) revealed a co-evolution of V3-crown reactivities and neutralization breadth. Our results indicate a role of V3-crown responses and its conformational preferences in bnAb development to be considered in preventive and therapeutic approaches.

Cooperation Between Systemic IgG1 and Mucosal Dimeric IgA2 Monoclonal Anti-HIV Env Antibodies: Passive Immunization Protects Indian Rhesus Macaques Against Mucosal SHIV Challenges

Understanding the interplay between systemic and mucosal anti-HIV antibodies can provide important insights to develop new prevention strategies. We used passive immunization via systemic and/or mucosal routes to establish cause-and-effect between well-characterized monoclonal antibodies and protection against intrarectal (i.r.) SHIV challenge. In a pilot study, for which we re-used animals previously exposed to SHIV but completely protected from viremia by different classes of anti-HIV neutralizing monoclonal antibodies (mAbs), we made a surprise finding: low-dose intravenous (i.v.) HGN194-IgG1, a human neutralizing mAb against the conserved V3-loop crown, was ineffective when given alone but protected 100% of animals when combined with i.r. applied HGN194-dIgA2 that by itself had only protected 17% of the animals. Here we sought to confirm the unexpected synergy between systemically administered IgG1 and mucosally applied dIgA HGN194 forms using six groups of naïve macaques (n=6/group). Animals received i.v. HGN194-IgG1 alone or combined with i.r.-administered dIgA forms; controls remained untreated. HGN194-IgG1 i.v. doses were given 24 hours before – and all i.r. dIgA doses 30 min before – i.r. exposure to a single high-dose of SHIV-1157ipEL-p. All controls became viremic. Among passively immunized animals, the combination of IgG1+dIgA2 again protected 100% of the animals. In contrast, single-agent i.v. IgG1 protected only one of six animals (17%) – consistent with our pilot data. IgG1 combined with dIgA1 or dIgA1+dIgA2 protected 83% (5/6) of the animals. The dIgA1+dIgA2 combination without the systemically administered dose of IgG1 protected 67% (4/6) of the macaques. We conclude that combining suboptimal antibody defenses at systemic and mucosal levels can yield synergy and completely prevent virus acquisition.

Species-specific valid ternary interactions of HIV-1 Env-gp120, CD4, and CCR5 as revealed by an adaptive single-amino acid substitution at the V3 loop tip

Molecular interactions of the variable envelope gp120 subunit of HIV-1 with two cellular receptors are the first step of viral infection, thereby playing pivotal roles in determining viral infectivity and cell tropism. However, the underlying regulatory mechanisms for interactions under gp120 spontaneous variations largely remain unknown. Here we show an allosteric mechanism in which a single gp120 mutation remotely controls the ternary interactions between gp120 and its receptors for the switch of viral cell tropism. Virological analyses showed that a G310R substitution at the tip of the gp120 V3 loop selectively abolished the viral replication ability in human cells, despite evoking enhancement of viral replication in macaque cells. Molecular dynamics (MD) simulations predicted that the G310R substitution at a site away from the CD4 interaction site selectively impeded the binding ability of gp120 to human CD4. Consistently, virions with the G310R substitution exhibited a reduced binding ability to human lymphocyte cells. Furthermore, the G310R substitution influenced the gp120-CCR5 interaction in a CCR5-type dependent manner as assessed by MD simulations and an infectivity assay using exogenously expressed CCR5s. Interestingly, an I198M mutation in human CCR5 restored the infectivity of the G310R virus in human cells. Finally, MD simulation predicted amino acid interplays that physically connect the V3 loop and gp120 elements for the CD4 and CCR5 interactions. Collectively, these results suggest that the V3 loop tip is a cis-allosteric regulator that remotely controls intra- and inter-molecular interactions of HIV-1 gp120 for balancing ternary interactions with CD4 and CCR5. IMPORTANCE Understanding molecular bases for viral entry into cells leads to the elucidation of one of major viral survival strategies, and thus to the development of new effective antiviral measures. As experimentally shown recently, HIV-1 is highly mutable and adaptable in growth-restrictive cells such as those of macaque origin. HIV-1 initiates its infection by sequential interactions of Env-gp120 with two cell surface receptors, CD4 and CCR5. A recent epoch-making structural study has disclosed that CD4-induced conformation of gp120 is stabilized upon binding of CCR5 to the CD4-gp120 complex, whereas its biological significance remains totally unknown. Here, from a series of mutations found in our extensive studies, we identified a single-amino acid adaptive mutation at the V3 loop tip of Env-gp120 critical for its interaction with both CD4 and CCR5 in a host cell species-specific way. This remarkable finding would certainly provoke and accelerate studies to precisely clarify the HIV-1 entry mechanism.

p62 promotes HIV-1 gp120 V3 loop-mediated microglial inflammation by promoting noncanonical activation of Nrf2

Combined antiretroviral therapy (cART) has significantly increased the life expectancy of AIDS patients; however, the prevalence of the neurocognitive impairment associated with HIV-1 continues to rise. HIV-1 gp120, an important subunit of the envelope spikes that decorate the surface of virions, is found to activate microglia in central nervous system (CNS) which leads to the cognitive and behavioral dysfunction known as HIV-1 associated neurocognitive disorder(HAND), and the V3 loop is the most important toxic domain of gp120. A study has shown that autophagy plays key role in the activation of microglia, p62 is an important autophagy substrate protein that is elevated in neuroinflammation. In this study, we sought to explore the role of p62 in gp120 V3 loop-mediated microglial activation. Our results demonstrated that exposure of CHME-5 cells to the gp120 V3 loop resulted in elevated inflammatory cytokines, accompanied by autophagy dysfunction and p62 upregulation. Subsequently, we found that the p62-dependent Nrf2 noncanonical signaling pathway was activated and that HO-1, the target protein of Nrf2, was also upregulated. Interestingly, the elevation of inflammatory factors caused by the gp120 V3 loop was significantly alleviated after knocking down p62, Nrf2 and HO-1. Further investigation revealed that in the microglial inflammation induced by the gp120 V3 loop, up-regulated HO-1 promoted the expression of iNOS by interacting with iNOS, while enhanced autophagy by RAPA promoted the degradation of iNOS and alleviated inflammation. These findings provide a new perspective on the relationship between noncanonical Nrf2 activation and autophagy in microglial inflammation and an experimental basis for HAND prevention and treatment.

V3-Loop genotypes do not predict maraviroc susceptibility of CCR5-tropic virus or clinical response through week 48 in HIV-1–infected, treatment-experienced persons receiving optimized background regimens

Viruses from 15 of 35 maraviroc-treated participants with virologic failure and CCR5-tropic (R5) virus in the MOTIVATE studies at Week 24 had reduced maraviroc susceptibility. On-treatment amino acid changes were observed in the viral envelope glycoprotein 120 third variable (V3)–loop stems and tips and differed between viruses. No amino acid change reliably predicted reduced susceptibility, indicating that resistance was genetic context–dependent. Through Week 24, poor adherence was associated with maraviroc-susceptible virologic failure, whereas reduced maraviroc susceptibility was associated with suboptimal background regimen activity, highlighting the importance of overall regimen activity and good adherence. Predictive values of pretreatment V3-loop sequences containing these Week 24 mutations or other variants present at >3% in pretreatment viruses of participants with virologic failure at Week 48 were retrospectively assessed. Week 48 clinical outcomes were evaluated for correlates with pretreatment V3-loop CCR5-tropic sequences from 704 participants (366 responders; 338 virologic failures [83 with R5 virus with maraviroc susceptibility assessment]). Seventy-five amino acid variants with >3% prevalence were identified among 23 V3-loop residues. Previously identified variants associated with resistance in individual isolates were represented, but none were associated reliably with virologic failure alone or in combination. Univariate analysis showed virologic-failure associations with variants 4L, 11R, and 19S ( P < 0.05). However, 11R is a marker for CXCR4 tropism, whereas neither 4L nor 19S was reliably associated with reduced maraviroc susceptibility in R5 failure. These findings from a large study of V3-loop sequences confirm lack of correlation between V3-loop genotype and clinical outcome in participants treated with maraviroc. Clinical trial registration numbers (ClinicalTrials.gov): NCT00098306 and NCT00098722

Highly prevalent Russian HIV-1 V3-loop sequence variants are susceptible to maraviroc

Introduction Maraviroc inhibits CCR5-tropic HIV-1 across different subtypes in vitro and has demonstrated efficacy in clinical trials. V3-loop amino acid variants observed in individual maraviroc-resistant viruses have not been found to be predictive of reduced susceptibility. Sequence-database searches have demonstrated that approximately 7.3% of viruses naturally encode these variants, raising concerns regarding potential pre-existing resistance. A study from Russia reported that combinations of these same amino acids are present in the V3 loops of the Russian variant subtype A (IDU-A, now A6) with a much greater prevalence (range: 74.4%–92.3%) depending on the combination. However, these studies and database searches did not include phenotypic evaluation. Methods Sixteen Russian HIV-1 isolates (including sub-subtype A6 viruses) were assessed for V3 loop sequence and phenotypic susceptibility to maraviroc. Results All 12 of the A6 viruses and 2/4 subtype B isolates encoded V3-loop variants that have previously been identified in individual virus isolates with reduced susceptibility to maraviroc. However, despite the prevalence of these V3-loop amino acid variants among the tested viruses, phenotypic sensitivity to maraviroc was observed in all instances. Similarly, reduced susceptibility to maraviroc was not found in virus from participants who experienced virologic failure in a clinical study of maraviroc in Russia (A4001101, [NCT01275625]). Discussion Altogether, these data confirm that the presence of individual or combinations of V3-loop amino acid residues in sub-subtype A6 viruses alone does not predict natural resistance to maraviroc and that V3-loop genotype analysis of R5 virus prior to treatment is not helpful in predicting clinical outcome.

Elucidating the Disparate Inhibitory Mechanisms of Novel 1-Heteroaryl1,3-Propanediamine Derivatives and Maraviroc towards C-C Chemokine Receptor 5: Insights for Structural Modifications in HIV-1 Drug Discovery

Introduction: Blocking Human Immunodeficiency Virus type 1 (HIV-1) entry via C-C chemokine receptor 5 (CCR5) inhibition has remained an essential strategy in HIV drug discovery. This underlies the development of CCR5 blockers, such as Maraviroc, which, however, elicits undesirable side effects despite its potency. Background: Recent lead optimization efforts led to the discovery of novel 1-heteroaryl-1,3-propanediamine derivatives; Compd-21 and -34, which were ~3 times more potent than Maraviroc, with improved pharmacokinetics. However, atomistic molecular interaction mechanism of how slight structural variance between these inhibitors significantly affects their binding profiles have not been elucidated. Method: This study employed explicit lipid bilayer molecular dynamics (MD) simulations, and advance analyses to explore these inhibitory discrepancies. Results: Findings revealed that the thiophene moiety substitution common to Compd-21 and -34 enhanced their CCR5- inhibitory activities due to complementary high-affinity interactions with Trp862.60, Tyr1083.32, Tyr2516.51, Glu2837.39. These cumulatively accounted for their ΔGbind which were higher than Maraviroc. Binding dynamics further revealed that the compounds mediated direct competitive inhibition at CCR5 by blocking the gp120 V3 loop. Furthermore, constituent tropane and triazole moieties in the compounds commonly engaged in interactions with Glu2837.39 and Trp862.60, respectively. Structural analyses also revealed that both Compd-21 and -34 elicited distinct internal dynamic effect on CCR5 relative to Maraviroc. Conclusion: Structural modifications at the thiophene substituent and the addition of new functional groups to the triazole ring may enhance inhibitor competition with gp120 V3-loop. Findings herein highlighted would contribute to future structure-based design of inhibitors of HIV-1 CCR5 with improved potencies.

Common evolutionary features of the envelope glycoprotein of HIV-1 in patients belonging to a transmission chain

The diversity of the HIV-1 envelope glycoproteins (Env) is largely a consequence of the pressure exerted by the adaptive immune response to infection. While it was generally assumed that the neutralizing antibody (NAb) response depended mainly on the infected individual, the concept that virus-related factors could be important in inducing this response has recently emerged. Here, we analyzed the influence of the infecting viral strain in shaping NAb responses in four HIV-1 infected subjects belonging to a transmission chain. We also explored the impact of NAb responses on the functional evolution of the viral quasispecies. The four patients developed a strong autologous neutralizing antibody response that drove viral escape and coincided with a parallel evolution of their infecting quasispecies towards increasing infectious properties, increasing susceptibility to T20 and increasing resistance to both CD4 analogs and V3 loop-directed NAbs. This evolution was associated with identical Env sequence changes at several positions in the V3 loop, the fusion peptide and the HR2 domain of gp41. The common evolutionary pattern of Env in different hosts suggests that the capacity of a given Env to adapt to changing environments may be restricted by functional constraints that limit its evolutionary landscape.

Sensitivity to Monoclonal Antibody 447-52D and an Open Env Trimer Conformation Correlate Poorly with Inhibition of HIV-1 Infectivity by SERINC5

The host protein SERINC5 inhibits the infectivity of HIV-1 virions in an Env-dependent manner and is counteracted by Nef. The conformation of the Env trimer reportedly correlates with sensitivity to SERINC5. Here, we tested the hypothesis that the “open” conformation of the Env trimer revealed by sensitivity to the V3-loop specific antibody 447-52D directly correlates with sensitivity to SERINC5. Of five Envs tested, SF162 was the most sensitive to neutralization by 447-52D, but it was not the most sensitive to SERINC5; instead the Env of LAI was substantially more sensitive to SERINC5 than all the other Envs. Mutational opening of the trimer by substitution of two tyrosines that mediate interaction between the V2 and V3 loops sensitized the Envs of JRFL and LAI to 447-52D as previously reported, but only BaL was sensitized to SERINC5. These data suggest that trimer “openness” is not sufficient for sensitivity to SERINC5.