High serum neutralization following BG505 SOSIP.664 envelope trimer immunization was associated with protection against BG505.SHIV challenge in rhesus macaques in a previous study. In an animal that developed high titer, durable neutralization against a glycan hole on envelope gp120, high throughput, longitudinal, antigen-specific B cell receptor sequencing was conducted. This analysis of more than 4,700 antigen-specific B cells revealed marked intra-clonal expansion and divergence from germline, including three abundant clonotypes that produced autologous neutralizing monoclonal antibodies. Monoclonal antibodies from the neutralizing clonotypes and two other expanded non-neutralizing clonotypes targeted epitopes in the same glycan hole, with neutralizers also demonstrating different capacities to obstruct CD4 binding. Cryo-electron microscopy structures of four neutralizing monoclonal antibodies revealed that they bound to glycan hole epitopes using distinct binding modes. One neutralizing antibody displaced a glycan in the loop V5 upon binding and its footprint includes the CD4 binding loop. The findings provide insight into how antibody recognition of a prominent glycan hole could facilitate different mechanisms of neutralization while underscoring how intra-clonal expansion and maturation with repeated BG505 SOSIP.664 immunization drove high serum neutralization.

BACKGROUND:Heavily treatment-experienced (HTE) people with HIV (PWH) have limited antiretroviral therapy (ART) treatment options, putting them at greater risk of unfavorable human immunodeficiency virus 1 (HIV-1) disease progression. Fostemsavir (BMS-663068) efficiently suppressed viremia and increased CD4 count when combined with other antiretrovirals in HTE PWH. Its active metabolite, temsavir (BMS-626529, GSK2616713), binds a conserved pocket within the envelope glycoprotein (Env) CD4 binding site and prevents CD4-induced conformational changes. Temsavir effect on Env conformation profoundly alters its glycosylation and cleavage, thereby modifying its antigenicity and reducing gp120 shedding. Here we evaluated if fostemsavir treatment in PWH modulated the levels of nonneutralizing gp120 CD4-induced (CD4i) antibodies (Abs) associated with CD4 depletion in vitro and in PWH. METHODS:We measured the levels of anti-gp120 CD4i Abs in plasma samples taken before and after fostemsavir treatment in HTE participants from the BRIGHTE trial and the PRESTIGIO registry and compared them to those of a control ART-treated group from the SAILING trial. RESULTS:We observed a significant decline of anti-gp120 CD4i Abs in the 2 fostemsavir-treated HTE groups, but not in the control group. Moreover, this effect was unique to anti-gp120 CD4i Abs, as no significant changes were observed in the levels of antibodies targeting Gag. Functionally, this decline in CD4i Abs was associated with a reduced capacity of plasma to recognize and eliminate uninfected primary CD4+ T cells coated with soluble gp120. CONCLUSIONS:Altogether, fostemsavir may provide additional immune benefits to PWH, beyond blocking viral entry, by reducing anti-gp120 CD4i Abs levels. CLINICAL TRIALS REGISTRATION:NCT04098315, NCT01231516, and NCT02362503.

ABSTRACT The HIV-1 envelope glycoprotein (Env) represents the only viral antigen at the surface of infected cells, making it an ideal target for antibody-based therapies. Most antibodies elicited in people with HIV (PWH) do not recognize Env in its native “closed” conformation but readily bind to Env when it samples the CD4-bound “open” conformation. Downregulation of CD4 at the surface of infected cells by the viral accessory proteins Nef and Vpu prevents the premature opening of Env and has been shown to protect infected cells from antibody-dependent cellular cytotoxicity (ADCC) mediated by PWH plasma. Here, we report that deletion of Nef and Vpu from primary infectious molecular clones renders infected cells vulnerable to antibody-dependent cellular phagocytosis (ADCP) mediated by PWH plasma. This is in part linked to the premature engagement of Env with CD4. In agreement with an “open” Env being vulnerable to ADCP, small CD4-mimetic compounds (CD4mc) sensitize in vitro -infected cells and ex vivo -expanded CD4 T cells to ADCP mediated by autologous monocytes in the presence of PWH plasma. This effect was further improved by increasing cell surface Env through IFN-induced BST-2 upregulation. IMPORTANCE Developing new therapies to eliminate HIV-1-infected cells is essential to decrease the size of the HIV-1 reservoir. Fc-effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), have shown potential in eliminating in vitro- infected cells and ex vivo- expanded infected cells from people with HIV, decreasing the size of the reservoir, and delaying viral rebound in humanized mice. Here, we report that antibody-dependent cellular phagocytosis (ADCP) can also be harnessed to eliminate HIV-1-infected cells. We show that infected cells harboring “open” Env conformations are susceptible to ADCP-mediated killing in the presence of plasma from people with HIV. A better understanding of the contribution of different Fc-effector functions in the elimination of infected cells could help guide the development of new therapeutic approaches toward an HIV-1 cure.

BACKGROUND:Despite effective antiretroviral therapy (ART), neurocognitive impairment (NCI) persists in 30-50% of virally suppressed people with HIV (VS PWH). The mechanisms underlying this persisting NCI remain incompletely understood. Soluble gp120 (sgp120) has been detected in the plasma of PWH on ART and may contribute to inflammation and CD4 + T-cell depletion in the presence of non-neutralizing anti-cluster A antibodies (Abs). gp120 can induce neurotoxicity, leading to neuronal damage both in vitro and in transgenic mice. OBJECTIVE:To determine whether sgp120 and anti-cluster A Abs are detectable in the cerebrospinal fluid (CSF) of VS PWH and to evaluate whether their presence is associated with domain-specific neurocognitive performance. METHODS:We analyzed paired plasma and CSF samples from 146 VS PWH and 37 unsuppressed PWH. Neurocognitive performance was assessed using a comprehensive neuropsychological testing battery across multiple domains, from which demographically adjusted T -scores for each test were derived. Levels of sgp120 and anti-cluster A Abs were measured to evaluate their association with neurocognitive performance in PWH. RESULTS:Sgp120 and anti-cluster A Abs were detected in the CSF of 11 and 26% of VS PWH, respectively. Detection of CSF sgp120 was associated with lower neurocognitive motor-domain T -scores (Mann-Whitney U , P  = 0.039; Cliff's δ  = -0.33). This association was confined to sgp120 in the CSF and was not observed for sgp120 in plasma or for anti-HIV antibodies in the CSF. CONCLUSION:These findings suggest that central nervous system (CNS) exposure to sgp120 may contribute to poorer motor neurocognitive performance in VS PWH. Strategies targeting CSF sgp120-related biomarkers warrant further investigation as potential approaches to mitigate NCI in VS PWH.

HIV-1 envelope glycoproteins (Env) from primary HIV-1 isolates typically adopt a pretriggered "closed" conformation that resists to CD4-induced (CD4i) non-neutralizing antibodies (nnAbs) mediating antibody-dependent cellular cytotoxicity (ADCC). CD4-mimetic compounds (CD4mcs) "open-up" Env allowing binding of CD4i nnAbs, thereby sensitizing HIV-1-infected cells to ADCC. Two families of CD4i nnAbs, the anti-cluster A and anti-coreceptor binding site (CoRBS) Abs, are required to mediate ADCC in combination with the indane CD4mc BNM-III-170. Recently, new indoline CD4mcs with improved potency and breadth have been described. Here, we show that the lead indoline CD4mc, CJF-III-288, sensitizes HIV-1-infected cells to ADCC mediated by anti-CoRBS Abs alone, contributing to improved ADCC activity. When administrated along with the anti-CoRBS 17b, CJF-III-288 delayed viral rebound after ART interruption in HIV-1-infected humanized mice, demonstrating potential for eliciting ADCC in vivo. Structural and conformational analyses reveal that CJF-III-288, in combination with this anti-CoRBS Abs, potently stabilizes an asymmetric "open" State-3 Env conformation. This Env conformation orients the anti-CoRBS Ab to improve ADCC activity and therapeutic potential.

mRNA vaccines have demonstrated considerable efficacy and safety against SARS-CoV-2, limiting the pandemic burden worldwide. The emergence of new variants of concern and the decline in neutralizing activity observed several weeks post-vaccination reinforced the call for repeated mRNA vaccination. We and others have shown that vaccine efficacy does not exclusively rely on antibody neutralizing activites; Fc-effector functions play an important role as well. However, it is well known that long-term exposure and repeated antigen stimulation elicit the IgG4 subclass of antibodies, which are inefficient at mediating Fc-effector functions. In this regard, recent studies highlighted concerns about IgG4 induction by mRNA vaccines. Here, we explored the impact of repeated mRNA vaccination on IgG4 induction and its impact on Fc-effector functions. We observed anti-Spike IgG4 elicitation after three doses of mRNA vaccine; the antibody levels further increased with additional doses. Vaccine-elicited IgG4 preferentially bound the ancestral D614G Spike. We also observed that Breakthrough Infection (BTI) after several doses of vaccine strongly increased IgG1 levels but had no impact on IgG4 levels, thereby improving Fc-effector functions. Finally, we observed that elderly donors vaccinated with Moderna mRNA vaccines elicited higher IgG4 levels and presented lower Fc-effector functions than donors vaccinated with the Pfizer mRNA vaccine. Altogether, our results highlight the importance of monitoring the IgG subclasses elicited by vaccination.

The affinity of Fc γ receptor IIIa (FcγRIIIa) binding to immunoglobulin G1 (IgG1) correlates with patient responses for antibody-based therapeutics. Among multiple factors affecting affinity, a mechanism defining how the composition of the FcγRIIIa N162 glycan regulates affinity remains undefined. Here, we evaluate the binding modes of two competitive FcγRIIIa ligands. IgG1 Fc binding is sensitive to N162 glycan composition, unlike the antigen-binding fragment (Fab) of the FcγRIII-specific antibody 3G8. Both ligands bound to overlapping surfaces, utilizing different angles of attack such that the IgG1 Fc but not 3G8 Fab limited the space available to the FcγRIII N162 N-glycan. FcγRIII binding to IgG1 Fc generated a 2.1 kcal/mol penalty from a loss of N162 glycan conformational entropy, greater than the 0.3 kcal/mol penalty for 3G8 and consistent with binding measurements. Thus, the conformational entropy of the FcγRIIIa N162-glycan is the predominant force modulating differential binding affinity compared to 3G8 Fab binding for endogenous FcγRIIIa glycoforms.

IntroductionAntibodies play a critical role in immunity in part by mediating clearance of pathogens and infected cells by antibody-dependent cellular phagocytosis (ADCP) through engagement of Fc gamma receptors (FcγRs) on innate immune cells. Among these, FcγRIIa (CD32a) is a key activating receptor expressed on macrophages, dendritic cells, and other antigen-presenting cells. Its affinity for IgG and ability to mediate ADCP is influenced by allelic polymorphisms. In humans, a single amino acid polymorphism at position 131, where histidine (H) is substituted with arginine (R), leads to decreased IgG1 and IgG2 subclass binding affinity and, consequently, lower efficiency of phagocytic responses. Rhesus macaques (Macaca mulatta), which are widely used as nonhuman primate models, exhibit a similar polymorphism at position 131 of FcγRIIa, but with arginine replaced by proline (P). Here, we investigated structure-function relationships associated with the FcγRIIa polymorphism at position 131 in both species, specifically with respect to IgG1 and IgG2.MethodsWe determined the structures of complexes formed by each variant with IgG1 Fc and those formed by the higher affinity variant with IgG2 Fc for both species by x-ray crystallography and linked these structures to affinity and activity using SPR and an ADCP assay. We also determined the structure of human inhibitory FcγRIIb (CD32b) in complex with IgG1 Fc by x-ray crystallography. ResultsThrough analysis of these structures, our studies reveal that FcγRIIa engagement is minimally influenced by Fc glycan composition, distinguishing it from FcγRIIIa whose affinity is strongly influenced by glycan-composition. Comparative structures of human and macaque FcγRIIa variants demonstrate species- and allele-specific differences in Fc binding, but our functional assays showed only minimal allele-specific effects in humans. In contrast, allele-specific effects in macaques were highly significant; the macaque P131 variant showing uniformly reduced IgG affinity. ConclusionThese insights highlight fundamental interspecies and allelic distinctions that are critical for interpreting FcγRIIa-mediated effector functions in macaque models and for optimizing translational antibody and vaccine design.

The viral reservoir in long-lived memory CD4+ cells, established in the early stages of HIV infection, represents the main obstacle to an HIV cure. Some strategies being developed to target the reservoir rely on rendering HIV-1 envelope glycoproteins (Env) visible to the immune system. Small molecule CD4-mimetics (CD4mcs) expose vulnerable Env epitopes, which can be targeted by non-neutralizing antibodies (nnAbs) that are abundant in the plasma of people living with HIV (PLWH) and can mediate antibody-dependent cellular cytotoxicity (ADCC). Administration of CD4mcs in combination with plasma from PLWH or nnAbs efficiently reduces the size of the HIV-1 reservoir and postpones viral rebound upon antiretroviral therapy (ART) interruption in humanized mice. However, it remains unclear when these nnAbs are elicited after HIV infection. In this study, we collected longitudinal plasma samples before acquisition, at diagnosis, and at multiple time points up to 33 weeks after the estimated date of detectable infection (EDDI) and before ART treatment initiation. We found that plasma samples collected as early as 3 to 10 weeks after EDDI neutralized viral particles and mediated ADCC in the presence of the CJF-III-288 CD4mc. Recognition of HIV-1-infected cells and ADCC progressively increased over time, reaching a plateau by 19-25 weeks after EDDI. ADCC activity increased concomitantly with the elicitation of anti-gp120 and anti-gp41 CD4-induced (CD4i) Abs and improved over time with the appearance of anti-coreceptor binding site antibodies. Our results show that CD4i nnAbs, able to eliminate HIV-1-infected cells in the presence of CD4mc, are elicited within a few weeks after HIV acquisition. IMPORTANCE:A viral reservoir is established at the early stages of HIV-1 infection. This reservoir persists during antiretroviral therapy (ART) treatment, and viral rebound is observed after ART interruption. New strategies are needed to reduce the size of the viral reservoir and prevent virus rebound. Several families of non-neutralizing antibodies, which are abundant in plasma from people living with HIV-1, neutralize viral particles and mediate the elimination of HIV-1-infected cells through antibody-dependent cellular cytotoxicity (ADCC) when combined with CD4-mimetic compounds. The presence of non-neutralizing antibodies in plasma during early-stage HIV-1 infection would support the use of CD4-mimetic compounds as an early intervention to decrease the size of the latent HIV-1 reservoir by eliminating infected cells.

ABSTRACT The HIV-1 envelope glycoprotein (Env) is expressed at the surface of infected cells and, as such, can be targeted by non-neutralizing antibodies (nnAbs) that mediate antibody-dependent cellular cytotoxicity (ADCC). Previous single-molecule Förster resonance energy transfer (smFRET) studies demonstrated that Envs from clinical isolates predominantly adopt a “closed” conformation (State 1), which is resistant to nnAbs. After interacting with the cellular receptor CD4, the conformational equilibrium of Env shifts toward States 2 and 3, exposing the coreceptor-binding site (CoRBS) and permitting targeting by CD4-induced (CD4i) antibodies. We showed that the binding of anti-CoRBS Abs enables the engagement of other nnAbs that target the cluster A epitopes on Env. Anti-cluster A nnAbs stabilize an asymmetric Env conformation, State 2A, and have potent ADCC activity. CRF01_AE strains were suggested to be intrinsically susceptible to ADCC mediated by nnAbs. This may be due to the presence of a histidine at position 375, known to shift Env toward more “open” conformations. In this work, through adaptation of an established smFRET imaging approach, we report that native, unliganded CRF01_AE HIV-1 Envs frequently sample the State 2A conformation. This is in striking contrast with Envs from clades A and B, for example HIV-1JR-FL, which do not transition to State 2A in the absence of ligands. These findings inform on the conformational dynamics of CRF01_AE Env, which are relevant for structure-based design of both synthetic inhibitors of receptor binding and enhancers of ADCC as therapeutic alternatives.IMPORTANCEA concerning increase in infections with HIV-1 from CRF01_AE has occurred globally and regionally in recent years, especially in Southeast Asia. Despite the advances made in understanding HIV-1 envelope glycoprotein (Env) conformational dynamics, the knowledge about Env from CRF01_AE HIV-1 is limited. Here, we demonstrate that the unliganded CRF01_AE Env readily samples an “open” conformation (State 2A), which is susceptible to antibody-dependent cellular cytotoxicity (ADCC). This is in contrast with the subtypes previously studied from HIV-1 group M that rely on anti-cluster A antibodies to adopt State 2A. These findings are relevant for the structure-based design of novel synthetic inhibitors of CD4 binding and enhancers of ADCC for the elimination of infected cells.

While antiretroviral therapy efficiently suppresses viral replication, inflammation and immune dysfunction persist in some people living with HIV-1 (PLWH). Soluble gp120 (sgp120) has been detected in PLWH plasma and its presence is linked to immune dysfunction. It was reported that sgp120 binding to CD4 on uninfected bystander CD4 + T cells sensitizes them to antibody-dependent cellular-cytotoxicity (ADCC) mediated by non-neutralizing antibodies present in PLWH plasma. Using three independent PLWH cohorts, we observed that non-neutralizing anti-cluster A antibodies are negatively associated with CD4 + T cell counts. Anti-CD4BS antibodies blocked the coating of uninfected bystander cells by sgp120, thereby preventing their elimination by ADCC. Supporting a protective role of anti-CD4BS antibodies, PLWH having these antibodies didn't show a negative association between CD4 T cell counts and anti-cluster A. Our results reveal that anti-cluster A antibodies are associated with immune dysfunction in PLWH and anti-CD4BS antibodies might have a beneficial impact in these individuals.

One characteristic of the HIV-1 CRF01_AE strain is that it contains a bulkier histidine residue at position 375 (H375) in its envelope glycoproteins (Env). This residue is part of the Phe43 cavity, where residue 43 of CD4 engages with gp120. It has been shown that H375 contributes to resistance against small molecule inhibitors targeting gp120. Residue 375 co-evolved with a few residues of the gp120 inner domain layers, and together they modulate the susceptibility of Env to small molecule gp120 inhibitors. Since residue 629 within the HR2 region of gp41 has also been proposed to have co-evolved with residue 375, we explored its role in the susceptibility of HIV-1 Env to two classes of small molecule gp120 inhibitors: the conformational blocker temsavir and the CD4-mimetic (CD4mc) BNM-III-170. Reversion of CRF01_AE isoleucine to a major clade methionine at position 629 had a significant but opposite impact on the susceptibility of the virus to temsavir and BNM-III-170. Mechanistically, this is associated with the capacity of residue 629 to modulate Env stability, as attested by its impact on cold inactivation. Overall, our results show how a single residue of HR2 contributes to the overall Env trimer stability and its susceptibility to gp120-targeted small molecule inhibitors.IMPORTANCECRF01_AE envelope glycoproteins (Env) have a well-conserved histidine at position 375. This residue is key in modulating the susceptibility of HIV-1 to small molecule Env inhibitors. Here, we report that a residue of the gp41 HR2 region affects Env trimer stability and its susceptibility to gp120-directed small molecule inhibitors. This work adds to our understanding of HIV-1 Env resistance to small molecule inhibitors.

ABSTRACT Anti-HIV-1 antibodies capable of mediating ADCC are elicited by the majority of people with HIV-1 and preferentially target the “open,” CD4-bound conformation of HIV-1 envelope glycoproteins (Env). However, due to the “closed” conformation sampled by unliganded HIV-1-Envs, these antibodies are ineffective at eliminating infected cells. BNM-III-170 is a small-molecule CD4-mimetic compound that binds the Phe43 cavity of the gp120 subunit of Env, forcing Env to “open up,” thus exposing epitopes targeted by CD4-induced (CD4i), ADCC-mediating antibodies. Here, we assessed the safety, pharmacokinetics, and biological activity of BNM-III-170 in uninfected and SHIV-AD8-EO-infected rhesus macaques (RMs). In uninfected RMs, single subcutaneous administrations of 3–36 mg/kg BNM-III-170 were well-tolerated, with serum half-lives ranging from 3 to 6 h. In SHIV-infected RMs, four different regimens were evaluated: 2 × 36 mg/kg daily, 1 × 24 mg/kg, 3 × 36 mg/kg every 7 days, and 3 × 36 mg/kg every 3 days. While toxicity was observed with daily doses, all other regimens demonstrated reasonable safety profiles. No changes in plasma viral loads were observed in SHIV-infected RMs following any of the evaluated BNM-III-170 dosing regimens. However, plasma collected following BNM-III-170 administration was shown to have increased binding to infected cells and to sensitize SHIV AD8-EO virions to neutralization by otherwise non-neutralizing antibodies. In addition, the plasma of treated animals mediated ADCC in the presence of BNM-III-170. These results establish a well-tolerated BNM-III-170 dosing regimen in SHIV-infected RMs and serve as proof of concept for its biological activity in promoting the targeting of infected cells by CD4i ADCC-mediating antibodies. Thus, they inform future studies evaluating CD4mc treatment in ART-treated animals. IMPORTANCE A therapeutic regimen able to eradicate or functionally cure HIV-1 remains elusive and may require a “shock-and-kill” approach to reactivate and then purge the latent HIV-1 reservoir. The small-molecule CD4-mimetic compound BNM-III-170 has previously been shown to (i) sensitize HIV-1-infected cells to ADCC mediated by plasma from people with HIV-1 (PWH) in vitro and (ii) significantly delay the time to viral rebound following ART interruption when combined with anti-CoRBS + anti-cluster A Abs or plasma from PWH in humanized mice. To evaluate the use of BNM-III-170 as part of a kill approach, we characterized the safety, pharmacokinetics, and biological activity of BNM-III-170 in uninfected and SHIV-infected RMs. Our study identifies a tolerable BNM-III-170 dosing regimen in SHIV-infected RMs and provides insights into its antiviral activities; as such, it informs future studies evaluating the efficacy of BNM-III-170 in reducing the viral reservoir.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remains a threat to human health, particularly among immunocompromised and elderly individuals, given their heightened vulnerability to coronavirus disease 2019 (COVID-19)-associated morbidity and mortality. Recently, omicron subvariants such as KP.3.1.1 and XEC have emerged with an enhanced ability to evade humoral immunity. The development of new strategies against these variants of concern remains an intense area of research. The small molecule VE607 is an entry inhibitor that targets the Spike glycoprotein and delays virus spread in vivo. To improve the potency of this new class of SARS-CoV-2 entry inhibitors, we generated and characterized VE607 analogs and identified candidates with enhanced activity against variants, including KP.3.1.1 and XEC. Promising analogs exhibited higher inhibitory potency than the original compound and stabilized the receptor-binding domain in its "up" conformation. Among these, DY-III-281 also reduced viral burden and delayed death in SARS-CoV-2-challenged K18-hACE2 transgenic mice. Furthermore, combining DY-III-281 with a non-neutralizing antibody engineered for Fc-enhanced functions exhibited an additive effect in reducing SARS-CoV-2-induced disease burden in mice. Our findings support the continued development of small-molecule entry inhibitors, alone or in combination with antibody-based therapies, as a promising strategy to counteract emerging SARS-CoV-2 variants. IMPORTANCE:Mutations in the Spike glycoprotein drive viral evolution and confer resistance to current vaccines and some therapeutic interventions against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we report new analogs of the SARS-CoV-2 small-molecule entry inhibitor VE607. These analogs exhibited improved potency against emerging SARS-CoV-2 variants, including KP.3.1.1 and XEC. One analog, DY-III-281, delayed viral replication in SARS-CoV-2WA1-challenged K18-hACE2 transgenic mice, suggesting that small-molecule compounds targeting viral entry might be useful in fighting evolving SARS-CoV-2 variants.

Anti-HIV envelope broadly neutralizing antibodies (bnAbs) are alternatives to conventional antiretrovirals with the potential to prevent and treat infection, reduce latent reservoirs, and/or mediate a functional cure. Clinical trials with "first-generation" bnAbs used alone or in combination show promising antiviral effects but also highlight that additional engineering of "enhanced" antibodies will be required for optimal clinical utility, while preserving or enhancing Current Good Manufacturing Practices (cGMP) manufacturing capability. Here, we report the engineering of an anti-CD4-binding site (CD4bs) bnAb, N49P9.3. Through a series of rational modifications, we produced a variant, N49P9.6-FR-LS, that demonstrates enhanced potency, superior antiviral activity in combination with other bnAbs, low polyreactivity, and longer circulating half-life. Additional engineering for manufacturing produced a final variant, eN49P9, with properties conducive to cGMP production. Overall, these efforts demonstrate the feasibility of developing enhanced anti-CD4bs bnAbs with greatly improved antiviral properties as well as potential translational value.

Designing metabolically stable peptides to target interactions of the tumor suppressor protein p53 with the two oncogenic proteins MDM2 and MDMX represents an attractive approach to harvesting "high-hanging fruits" often inaccessible to traditional anticancer drug discovery and development efforts. Here, we report the design of a proteolysis-resistant d-dodecapeptide, termed DPMI-ω (EFWYVEp-ClFEKLLR), capable of disrupting the p53-MDM2/MDMX complex by antagonizing MDM2 and MDMX. DPMI-ω, upon fabrication on gold nanoparticles, efficiently traversed tumor cells and killed them by reactivating the p53 signaling pathway. Further, DPMI-ω inhibited B16 melanoma growth in vivo and, when combined with an anti-PD1 antibody, powerfully augmented the efficacy of immunotherapy by expanding CD3+/CD8+ cytotoxic T cells and suppressing CD4+/CD25+ regulatory T cells. Our work validates the design of a therapeutically viable anticancer peptide, showcasing its potential in combination therapy to treat patients with tumors that are otherwise resistant or poorly responsive to antitumor immunotherapy.

BACKGROUND:While antiretroviral therapy (ART) efficiently suppresses viral replication, inflammation and immune dysfunction persist in some people living with HIV-1 (PLWH). HIV-1 soluble gp120 (sgp120) has been detected in PLWH plasma and its presence is linked to immune dysfunction. It was reported that sgp120 binding to CD4 on uninfected bystander CD4+ T cells sensitises them to cellular death via antibody-dependent cellular cytotoxicity (ADCC) mediated by non-neutralising anti-cluster A antibodies (Abs) present in PLWH plasma. METHODS:We included plasma from 520 PLWH on ART from three independent cohorts for measurements of anti-cluster A Abs and anti-CD4 binding site (anti-CD4BS) Abs. Associations between CD4+ T cell counts and anti-cluster A Abs was assessed using generalised least squares linear regression models, adjusting for potential confounders including age, sex, nadir CD4 and duration of ART. The role of anti-CD4BS Abs was evaluated using flow-cytometry based ADCC assays with primary CD4+ T cells. FINDINGS:We observed that non-neutralising anti-cluster A Abs are negatively associated with CD4+ T cell counts. Anti-CD4BS antibodies blocked the coating of uninfected bystander cells by sgp120, thereby preventing their elimination by ADCC. Supporting a protective role of anti-CD4BS antibodies, their presence in PLWH plasma abrogated the negative association between CD4 counts and anti-cluster A Abs. INTERPRETATION:Our results reveal that anti-cluster A Abs are associated with immune dysfunction in PLWH and anti-CD4BS antibodies might have a beneficial impact in these individuals. FUNDING:This study was supported by the Canadian Institutes of Health Research, the Canada Foundation for Innovation, the Fonds de Recherche du Québec-Santé, and the National Institutes of Health.

Fcγ receptors (FcγRs) are membrane-bound glycoproteins that bind to the fragment crystallizable (Fc) constant regions of IgG antibodies. Interactions between IgG immune complexes and FcγRs can initiate signal transduction that mediates important components of the immune response including activation of immune cells for clearance of opsonized pathogens or infected host cells. In humans, many studies have identified associations between FcγR gene polymorphisms and risk of infection, or progression of disease, suggesting a gene-level impact on FcγR-dependent immune responses. Rhesus macaques are an important translational model for most human health interventions, yet little is known about the breadth of rhesus macaque FcγR genetic diversity. This lack of knowledge prevents evaluation of the impact of FcγR polymorphisms on outcomes of preclinical studies performed in rhesus macaques. In this study we used long-read RNA sequencing to define the genetic diversity of FcγRs in 206 Indian-origin Rhesus macaques, Macaca mulatta. We describe the frequency of single nucleotide polymorphisms, insertions, deletions, frame-shift mutations, and isoforms. We also index the identified diversity using predicted and known rhesus macaque FcγR and Fc-FcγR structures. Future studies that define the functional significance of this genetic diversity will facilitate a better understanding of the correlation between human and macaque FcγR biology that is needed for effective translation of studies with antibody-mediated outcomes performed in rhesus macaques.

Environmental heat stress represents a pervasive threat to warfighters, athletes, and occupational workers, impacting performance and increasing the risk of injury. Exertional heat illness (EHI) is a spectrum of clinical disorders of increasing severity. While frequently predictable, EHI can occur unexpectedly and may be followed by long-term comorbidities, including cardiovascular dysfunction and exercise intolerance. The objective of this study was to assess genetic factors contributing to EHI. Whole-exome sequencing was performed in a cohort of 53 cases diagnosed with EHI. Rare variants in prioritized gene sets were analyzed and classified per published guidelines. Clinically significant pathogenic and potentially pathogenic variants were identified in 30.2% of the study cohort. Variants were found in 14 genes, including the previously known RYR1 and ACADVL genes and 12 other genes (CAPN3, MYH7, PFKM, RYR2, TRPM4, and genes for mitochondrial disorders) reported here for the first time in EHI. Supporting structural and functional studies of the TRPM4 p.Arg905Trp variant show that it impairs the thermal sensitivity of the TRPM4 channel, revealing a potentially new molecular mechanism contributing to EHI susceptibility. Our study demonstrates associations between EHI and genes implicated in muscle disorders, cardiomyopathies, thermoregulation, and oxidative phosphorylation deficiencies. These results expand the genetic heterogeneity of EHI and shed light on its molecular pathogenesis.