Biology subjects

Richard, J.

Publications and source records attributed to Richard, J..

At least 19 recordsLinked to original sources

bioRxiv2026

REV-ERBα/β Targeting Transcriptionally Reprograms HIV-Infected CD4+ T-Cells for Increased Viral Reactivation but Limited Virion Spread

The circadian clock repressors REV-ERB/{beta} control rhythmic gene expression and inhibit HIV-1 transcription. Whether REV-ERB/{beta} modulate the HIV-1 replication cycle beyond transcription remains unknown. Here, we demonstrate that memory CD4+ T-cells predominantly express the REV-ERB{beta} isoform ex vivo and that T-cell receptor (TCR) triggering downregulates both REV-ERB/{beta} expression, with levels of REV-ERB/{beta} mRNA being lower in ART-treated people with HIV (PWH) receiving antiretroviral therapy (ART) compared to people without HIV (PWoH) before/after TCR triggering. In single-round infection, the REV-ERB/{beta} antagonist SR8278 facilitated HIV-1 reverse transcription, integration, and intracellular HIV-p24 expression, but limited virion release. Moreover, SR8278 downregulated CCR5 mRNA expression, inhibited R5-tropic HIV-1 replication in vitro, and limited viral outgrowth in CD4+ T-cells from ART-treated PWH. Finally, genome-wide RNA-sequencing and functional validations revealed HIV-1 restriction/dependency factors that represent novel putative REV-ERB/{beta} targets. Thus, pharmacological inhibition of REV-ERB/{beta} uniquely combines a latency reversal activity with the inhibition of progeny virion spread.

immunology↗

bioRxiv2026

Nef and Vpu protect infected cells from ADCP mediated by plasma from people with HIV-1

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 presence of PWH plasma. This effect was further improved by increasing cell surface Env through IFN-induced BST-2 upregulation. IMPORTANCEDeveloping 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.

microbiology↗

bioRxiv2026

Predicting Head and Neck Squamous Cell Carcinoma outcomes using long-term Patient-Derived Tumor Organoids

Head and neck squamous cell carcinoma (HNSCC) remains associated with substantial morbidity and a 5-year overall survival rate of approximately 60%, reflecting persistent radio- and chemo-resistance and the lack of effective precision medicine strategies. Patient-Derived Tumor Organoids (PDTO) constitute promising functional models that may predict individual treatment response. In this study, we generated PDTO from surgically resected HNSCC of the oral cavity, oropharynx, larynx, and hypopharynx. A total of 20 long-term PDTO lines were established, maintaining growth over seven passages and successfully cryopreserved, capturing the molecular and clinical diversity of the patient cohort. These PDTO faithfully recapitulated histological features, major tumor marker expression, and the genomic and transcriptomic landscapes of their tumors of origin, with stability over time. Functional assays revealed heterogeneous responses to cisplatin and X-rays. Importantly, in vitro sensitivity of PDTO was associated with clinical outcome of patients at 24 months. Cisplatin response of PDTO predicted prognosis with 66.7% sensitivity and 100% specificity, while X-ray response showed 91.7% sensitivity and 75% specificity. Notably, all patients whose PDTO were classified as resistant to both cisplatin and X-rays experienced relapse and/or death within 24 months. Collectively, the successful long-term expansion and cryopreservation of HNSCC PDTO establish a stable and scalable preclinical resource that captures the molecular and clinical heterogeneity of the disease. This biobank provides a valuable platform for mechanistic studies and for the evaluation of innovative therapeutic strategies. This cohort represents one of the largest clinically annotated HNSCC PDTO collections to date, demonstrating a robust association between PDTO response to cisplatin and X-rays and patient prognosis. These findings support the predictive potential of PDTO-based functional assays and argue for their integration into standardized, rapid, and miniaturized precision oncology workflows for HNSCC.

cancer biology↗

bioRxiv2026

Reproductive history differentially shapes the neural response of middle-aged females to estradiol therapy after a metabolic challenge

BackgroundAdvancing age, the APOE{square}4 allele, and female sex are the top nonmodifiable risk factors for Alzheimers disease (AD). Female-specific experiences, such as parity and hormone therapy (HT) affect aging biomarkers such as metabolism and immune signaling and may even affect AD risk. Estradiol (E2), a common component of many HTs, affects cognition and brain health in aging females although this may vary depending on parity, genotype, and metabolic status. We hypothesized that prior parity influences brain and metabolic health, including response to E2, depending on APOE genotype. MethodsMiddle-aged female (10 month) wildtype (WT) or humanized (h) APOE{square}4 expressing rats, with different reproductive experience (nulliparous or primiparous) were fed a Western (WD) or standard diet (SD) for 2 months. In the second month, rats were given E2 or vehicle (oil) injections daily. Fear associative learning, plasma metabolic hormones, hippocampal inflammatory signalling, and neuroplasticity (neurogenesis, synaptic protein) were assessed. ResultsFemales fed a WD gained weight and displayed metabolic dysregulation, regardless of genotype. E2 treatment reduced WD-induced weight gain and reduced metabolic hormones, with stronger effects in WT rats. E2 treatment increased dorsal hippocampal inflammatory signalling selectively in primiparous hAPOE{square}4 females fed a WD. Previous parity increased neurogenesis and reduced certain cytokines in the hippocampus of middle-aged WT rats under a SD. Both E2 treatment and previous parity decreased dorsal neurogenesis in hippocampus of hAPOE{square}4 rats. In hAPOE{square}4 females, higher weight was associated with reduced contextual fear memory, an effect driven by primiparous females. In the cued fear conditioning task, hAPOE{square}4 females displayed better cued fear memory than WT, however, WD exposure reduced cued fear memory in this group. Together, this indicates that diet and weight gain may be more detrimental to associative memory in hAPOE{square}4 females and that E2 treatment has more favourable outcomes in WT rats. ConclusionsPrevious parity alters how females respond to E2 and metabolic stress in midlife. Primiparous hAPOE{square}4 females were especially vulnerable to the effects of WD and E2, exhibiting more inflammation, impaired memory, and reduced weight-loss. These findings highlight the importance of considering parity and genotype when evaluating midlife metabolic and cognitive risk. HighlightsO_LIEstradiol (E2) treatment reduced body weight gain under a Western diet (WD), with the most pronounced effects in wildtype primiparous rats. C_LIO_LIWD increased several metabolic hormones, and E2 treatment reduced several metabolic hormones after a WD in middle age, only in WT rats C_LIO_LIIncreased body weight impaired contextual associative memory in primiparous hAPOE{square}4 females. C_LIO_LIE2 treatment increased dorsal hippocampal inflammatory signalling in primiparous hAPOE{square}4 rats and the WD increased inflammatory signalling in primiparous WT rats. C_LIO_LIPrevious parity, but not E2 treatment, increased neurogenesis in WT rats only under standard diet conditions whereas a WD decreased neurogenesis based on genotype and previous parity C_LI Plain English SummaryAlzheimers disease (AD) causes general cognitive decline, and females are at higher risk than males, particularly those carrying the APOE{varepsilon}4 gene. Female-specific experiences, such as previous pregnancy (parity) and hormone therapy, as well as lifestyle factors like body weight and diet, may further influence AD risk. We examined how estradiol (E2), a hormone involved in the menstrual cycle, pregnancy, and some hormone therapies can affect memory, brain inflammation, synaptic plasticity (brain cell connectivity), and growth of new neurons (neurogenesis) in middle-aged female rats. We compared females with or without the APOE{varepsilon}4 gene and with or without prior parity, fed either a high-fat, high-sugar Western diet (WD) or a standard diet (SD). WD increased body weight and worsened metabolic health, but the strength of negative effects depended on genotype and pregnancy history. In females with the APOE{varepsilon}4 gene and prior parity, higher body weight was linked to poorer memory. hAPOE{varepsilon}4 females on SD showed better memory than non-carriers but this benefit disappeared on WD, suggesting WD and obesity are particularly detrimental for hAPOE{square}4 carriers with prior parity. WD-fed primiparous hAPOE{square}4 females treated with E2 also showed higher brain inflammation than other groups. Pregnancy history and E2 treatment were associated with more favourable outcomes in females without the genetic risk for AD and fed a standard diet. These findings highlight the importance of a personalized approach to hormone therapy and brain health in aging females. hAPOE{square}4 females with prior parity were most sensitive to the effects of diet. Together, this suggests genetic background, reproductive history, and diet interact to affect memory, inflammation, and neurogenesis, as well as the effects of E2 on these factors.

neuroscience↗

bioRxiv2025

The HIV-1 envelope cytoplasmic tail protects infected cells from ADCC by downregulating CD4

HIV-1-mediated CD4 downregulation is a well-known mechanism that protects infected cells from antibody-dependent cellular cytotoxicity (ADCC). While CD4 downregulation by HIV-1 Nef and Vpu proteins has been extensively studied, the contribution of the HIV-1 envelope glycoprotein (Env) in this mechanism is less understood. While Env is known to retain CD4 in the endoplasmic reticulum (ER) through its CD4-binding site (CD4bs), little is known about the mechanisms underlying this process. Here we show that the cytoplasmic tail of Env is a major determinant in CD4 downregulation. This function is highly conserved as it was observed with nine different infectious molecular clones from four clades. The small but significant accumulation of CD4 at the surface of cells infected with Env cytoplasmic tail deleted viruses is sufficient to trigger Env to adopt a more "open" conformation. This prompted recognition of such HIV-1-infected cells by plasma from people living with HIV (PLWH) and several families of CD4-induced (CD4i) antibodies, leading to the elimination of these cells by ADCC. While cytoplasmic tail truncations are known to enhance Env expression at the cell surface, this did not fully explain the increased recognition of infected cells by CD4i antibodies and plasma from PLWH. Introduction of the CD4bs D368R mutation, which abrogates CD4 interaction, decreased Env recognition and ADCC. Overall, our results show that CD4 downregulation by the cytoplasmic tail of Env contributes to the protection of infected cells from ADCC. IMPORTANCEHIV-1-mediated CD4 downregulation is a central mechanism involved in the protection of infected cells from antibody-dependent cellular cytotoxicity. CD4 downregulation prevents the premature interaction between HIV-1 envelope glycoproteins (Env) and CD4, which would otherwise "open" Env and expose vulnerable epitopes recognized by CD4-induced antibodies present in the plasma from people living with HIV. While the mechanisms of CD4 downregulation by the viral accessory proteins Nef and Vpu have been elucidated, the function of Env in this process is less clear. Here we show that the cytoplasmic tail of Env plays an important role, thus contributing to the protection of infected cells from ADCC.

microbiology↗

bioRxiv2025

Conjugated GLP-1 and Estradiol Treatment as a Novel Treatment for Age-related Cognitive Decline in Males and Females

Middle age represents a critical window for metabolic and cognitive health, particularly in the context of rising obesity and diabetes rates. Glucagon-like peptide-1 (GLP-1)-based therapies, which regulate blood glucose and body weight, show sex-specific effects, with estradiol potentiating their metabolic benefits. However, research on GLP-1s cognitive and neuroprotective roles has largely been conducted in males. Here, we investigated the effects of GLP-1 conjugated to estradiol (GE2) on metabolism, cognition, cytokine levels and neurogenesis in the dentate gyrus of middle-aged male and female rats fed a standard (SD) or Western (WD) diet. In both sexes, WD increased body weight and plasma leptin levels, regardless of sex. GE2 treatment led to weight loss, enhanced cued and contextual fear memory, reduced cytokine levels in the hippocampus in SD rats, and increased neurogenesis in the dorsal dentate gyrus (DG), regardless of sex. Sex-specific differences were observed in fat distribution, glucose regulation, central cytokine levels, and neuroplasticity after WD and GE2 treatment. In females only, GE2 reduced visceral (gonadal) fat, reduced cytokines in the dorsal hippocampus, and improved basal blood glucose in response to a WD. In males only, GE2 restored neurogenesis in the DG after WD exposure, and reduced cytokine levels in the amygdala. These findings suggest that although WD increased body weight and GE2 improved associative learning in both sexes, both WD and GE2 had differential affects on metabolic hormones, insulin regulation, cytokine levels and neuroplasticity. Our findings underscore the importance of sex-specific approaches in metabolic and neuroprotective therapeutics in middle age.

neuroscience↗

bioRxiv2024

The asymmetric opening of HIV-1 Env by a potent CD4 mimetic enables anti-coreceptor binding site antibodies to mediate ADCC

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. Structural and conformational analyses reveal that CJF-III-288, in combination with 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.

microbiology↗

bioRxiv2024

The combination of three CD4-induced antibodies targeting highly conserved Env regions with a small CD4-mimetic achieves potent ADCC activity

The majority of naturally-elicited antibodies against the HIV-1 envelope glycoproteins (Env) are non-neutralizing (nnAbs), because they are unable to recognize the Env timer in its native "closed" conformation. Nevertheless, it has been shown that nnAbs have the potential to eliminate HIV-1-infected cells by Antibody-Dependent Cellular Cytotoxicity (ADCC) provided that Env is present on the cell surface in its "open" conformation. This is because most nnAbs recognize epitopes that become accessible only after Env interaction with CD4 and the exposure of epitopes that are normally occluded in the closed trimer. HIV-1 limits this vulnerability by downregulating CD4 from the surface of infected cells, thus preventing a premature encounter of Env with CD4. Small CD4-mimetics (CD4mc) sensitize HIV-1-infected cells to ADCC by opening the Env glycoprotein and exposing CD4-induced (CD4i) epitopes. There are two families of CD4i nnAbs, termed anti-cluster A and anti-CoRBS Abs, which are known to mediate ADCC in the presence of CD4mc. Here, we performed Fab competition experiments and found that anti-gp41 cluster I antibodies comprise a major fraction of the plasma ADCC activity in people living with HIV (PLWH). Moreover, addition of gp41 cluster I antibodies to cluster A and CoRBS antibodies greatly enhanced ADCC mediated cell killing in the presence of a potent indoline CD4mc, CJF-III-288. This cocktail outperformed broadly-neutralizing antibodies and even showed activity against HIV-1 infected monocyte-derived macrophages. Thus, combining CD4i antibodies with different specificities achieves maximal ADCC activity, which may be of utility in HIV cure strategies. IMPORTANCEThe elimination of HIV-1-infected cells remains an important medical goal. While current antiretroviral therapy decreases viral loads below detection levels, it does not eliminate latently infected cells which form the viral reservoir. Here, we developed a cocktail of non-neutralizing antibodies targeting highly conserved Env regions and combined it with a potent indoline CD4mc. This combination exhibited very potent ADCC activity against HIV-1-infected primary CD4+ T cells as well as monocyte-derived macrophages, suggesting its potential utility in decreasing the size of the viral reservoir.

microbiology↗

bioRxiv2024

CD4 downregulation precedes Env expression and protects HIV-1-infected cells from ADCC mediated by non-neutralizing antibodies

HIV-1 envelope glycoprotein (Env) conformation substantially impacts antibody-dependent cellular cytotoxicity (ADCC). Envs from primary HIV-1 isolates adopt a prefusion "closed" conformation, which is targeted by broadly-neutralizing antibodies (bnAbs). CD4 binding drives Env into more "open" conformations, which are recognized by non-neutralizing Abs (nnAbs). To better understand Env-Ab and Env-CD4 interaction in CD4+ T cells infected with HIV-1, we simultaneously measured antibody binding and HIV-1 mRNA expression using multiparametric flow cytometry and RNA-flow fluorescent in situ hybridization (FISH) techniques. We observed that env mRNA is almost exclusively expressed by HIV-1 productively-infected cells that already downmodulated CD4. This suggest that CD4 downmodulation precedes env mRNA expression. Consequently, productively-infected cells express "closed" Envs on their surface, which renders them resistant to nnAbs. Cells recognized by nnAbs were all env mRNA negative, indicating Ab binding through shed gp120 or virions attached to their surface. Consistent with these findings, treatment of HIV-1 infected humanized mice with the ADCC mediating nnAb A32 failed to lower viral replication or reduce the size of the viral reservoir. These findings confirm the resistance of productively-infected CD4+ T cells to nnAbs-mediated ADCC and question the rationale of immunotherapy approaches using this strategy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/592003v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1c34d91org.highwire.dtl.DTLVardef@11975c0org.highwire.dtl.DTLVardef@81b5beorg.highwire.dtl.DTLVardef@1c2e0b2_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Metformin Enhances Antibody-Mediated Recognition of HIV-Infected CD4+ T-Cells by Decreasing Viral Release

The mechanistic target of rapamycin (mTOR) positively regulates multiple steps of the HIV-1 replication cycle. We previously reported that a 12-weeks supplementation of antiretroviral therapy (ART) with metformin, an indirect mTOR inhibitor used in type-2 diabetes treatment, reduced mTOR activation and HIV transcription in colon-infiltrating CD4+ T-cells, together with systemic inflammation in nondiabetic people with HIV-1 (PWH). Herein, we investigated the antiviral mechanisms of metformin. In a viral outgrowth assay performed with CD4+ T-cells from ART-treated PWH, and upon infection in vitro with replication-competent and VSV-G-pseudotyped HIV-1, metformin decreased virion release, but increased the frequency of productively infected CD4lowHIV-p24+ T-cells. These observations coincided with increased BST2/Tetherin (HIV release inhibitor) and Bcl-2 (pro-survival factor) expression, and improved recognition of productively infected T-cells by HIV-1 Envelope antibodies. Thus, metformin exerts pleiotropic effects on post-transcription/translation steps of the HIV-1 replication cycle and may be used to accelerate viral reservoir decay in ART-treated PWH. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/580166v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@a17eeborg.highwire.dtl.DTLVardef@939d9aorg.highwire.dtl.DTLVardef@1502a62org.highwire.dtl.DTLVardef@e8ec2_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

immunology↗

bioRxiv2023

Optical control of cardiac rhythm by in vivo photoactivation of an ERG channel peptide inhibitor

RATIONALECardiac rhythm, conduction and synchronization of electrical activity require the coordinated action of different types of ion channels that differ according to transmural and regional specificities. Classical pharmacology affects these ion channels in a non-regionalized way which explains why treating arrhythmias, that often occur in specific foci, has often limited efficacy in addition to negative side-effects on non-targeted organs. Photopharmacology is an emergent technology that has the potential to counteract all the negative aspects of classical pharmacology by restricting drug activity in a spatio-temporal manner. OBJECTIVEWe tested the potential of photopharmacology in specifically regulating heart activity by using a caged derivative of a natural peptide inhibitor of the ERG channel, BeKm1. The peptide was uncaged and activity monitored in vitro on a cell line expressing the hERG channel, on human cardiomyocytes derived from iPS cells, and ex vivo and in vivo on zebrafish larvae and rat hearts. METHODS AND RESULTSCaged BeKm-1 is inactive and fully active upon uncaging. Uncaging of the peptide on human iPS-derived cardiomyocytes enlarges the action potential duration and triggers arrhythmias. Uncaging also triggers bradycardia and disturbs cardiac conduction within the atria in perfused rat hearts upon illumination. The potency of photopharmacology for cardiac electrical modulation was further validated in zebrafish larvae where illumination of the caged compound induces bradycardia and atrio-ventricular desynchrony. Finally, in anesthetized rats, illumination of the caged peptide in the right atria, containing the sino-atrial node, leads to bradycardia without arrhythmia. CONCLUSIONSThis report demonstrates that photopharmacology, using the caged peptide strategy, can be used for dynamically regulating cardiac electrical activity in vivo and that spatial illumination restriction can dissociate the bradycardic effect from the arrhythmic one. The technology is applicable to all kinds of cardiac ion channels and regions of interest to create arrhythmogenic models or investigate new clinical applications.

pharmacology and toxicology↗

bioRxiv2023

Piperidine CD4-mimetic compounds expose vulnerable Env epitopes sensitizing HIV-1-infected cells to ADCC

The ability of HIV-1 accessory proteins Nef and Vpu to decrease CD4 levels contributes to the protection of infected cells from antibody-dependent cellular cytotoxicity (ADCC) by preventing the exposure of Env vulnerable epitopes. Small-molecule CD4 mimetics (CD4mc) based on the indane and piperidine scaffolds such as (+)-BNM-III-170 and (S)-MCG-IV-210 sensitize HIV-1 infected cells to ADCC by exposing CD4-induced (CD4i) epitopes recognized by non-neutralizing antibodies abundantly present in plasma from people living with HIV. Here, we characterize a new family of CD4mc, (S)-MCG-IV-210 derivatives, based on the piperidine scaffold which engage the gp120 within the Phe43 cavity by targeting the highly-conserved Asp368 Env residue. We utilized structure-based approaches and developed a series of piperidine analogs with improved activity to inhibit infection of difficult-to-neutralize tier-2 viruses and sensitize infected cells to ADCC mediated by HIV+ plasma. Moreover, the new analogs formed an H-bond with the -carboxylic acid group of Asp368, opening a new avenue to enlarge the breadth of this family of anti-Env small molecules. Overall, the new structural and biological attributes of these molecules make them good candidates for strategies aimed at the elimination HIV-1-infected cells.

immunology↗

bioRxiv2023

Cellular and molecular signatures of motherhood in the adult and ageing brain

Pregnancy is marked by brain changes to volume, structure, connectivity, some of which are long-lasting. Few studies have examined possible mechanisms of these changes or the effects of multiple pregnancies. Here, we characterized various cellular and molecular signatures of parity (nulliparous, primiparous, biparous) in the hippocampus, an important area for cognitive and emotional regulation, and in plasma. We investigated density of neural stems cells (Sox2) and microglia (Iba-1), and levels of the postsynaptic density protein (PSD-95), cell signalling pathways, hippocampal and peripheral inflammation and the tryptophan-kynurenine (TRP-KYN) pathway, at 1 week after weaning (7 months) and in middle-age (13 months). Parity increased PSD-95 levels in both age groups and prevented the age-related decrease in neural stem cell density observed in nulliparous rats. Biparity increased cell signalling phosphoproteins (pp706sk, S6RP) and number of microglia in the dentate gyrus, regardless of age. Parity resulted in transient changes to the TRP-KYN system and peripheral inflammation. Thus, parity has lasting effects on synaptic plasticity and alters the trajectory of hippocampal aging. Highlights- Parity increased the postsynaptic protein PSD-95 in the hippocampus, regardless of age. - Biparity increased microglial density and cell signalling in the hippocampus, regardless of age. - Parity prevented the age-related decline in hippocampal neural stem cells. - Parity transiently increased tryptophan-kynurenine pathway metabolites. - Aging reduced plasma cytokine levels, an effect more prominent with nulliparity.

neuroscience↗

bioRxiv2023

Indoline CD4-mimetic Compounds Mediate Potent and Broad HIV-1 Inhibition and Sensitization to Antibody-dependent Cellular Cytotoxicity

Binding to the host cell receptors, CD4 and CCR5/CXCR4, triggers large-scale conformational changes in the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer [(gp120/gp41)3] that promote virus entry into the cell. CD4-mimetic compounds (CD4mcs) comprise small organic molecules that bind in the highly conserved CD4-binding site of gp120 and prematurely induce inactivating Env conformational changes, including shedding of gp120 from the Env trimer. By inducing more "open," antibody-susceptible Env conformations, CD4mcs also sensitize HIV-1 virions to neutralization by antibodies and infected cells to antibody-dependent cellular cytotoxicity (ADCC). Here, we report the design, synthesis and evaluation of novel CD4mcs based on an indoline scaffold. Compared with our current lead indane scaffold CD4mc, BNM-III-170, several indoline CD4mcs exhibit increased potency and breadth against HIV-1 variants from different geographic clades. Viruses that were selected for resistance to the lead indane CD4mc, BNM-III-170, are susceptible to inhibition by the indoline CD4mcs. The indoline CD4mcs also potently sensitize HIV-1-infected cells to ADCC mediated by plasma from HIV-1-infected individuals. Crystal structures indicate that the indoline CD4mcs gain potency compared to the indane CD4mcs through more favorable {pi}-{pi} overlap from the indoline pose and by making favorable contacts with the vestibule of the CD4-binding pocket on gp120. The rational design of indoline CD4mcs thus holds promise for further improvements in antiviral activity, potentially contributing to efforts to treat and prevent HIV-1 infection.

microbiology↗

bioRxiv2022

Molecular basis for antiviral activity of pediatric neutralizing antibodies targeting SARS-CoV-2 Spike receptor binding domain

Neutralizing antibodies (NAbs) hold great promise for clinical interventions against SARS-CoV- 2 variants of concern (VOCs). Understanding NAb epitope-dependent antiviral mechanisms is crucial for developing vaccines and therapeutics against VOCs. Here we characterized two potent NAbs, EH3 and EH8, isolated from an unvaccinated pediatric patient with exceptional plasma neutralization activity. EH3 and EH8 cross-neutralize the early VOCs and mediate strong Fc-dependent effector activity in vitro. Structural analyses of EH3 and EH8 in complex with the receptor-binding domain (RBD) revealed the molecular determinants of the epitope-driven protection and VOC-evasion. While EH3 represents the prevalent IGHV3-53 NAb whose epitope substantially overlaps with the ACE2 binding site, EH8 recognizes a narrow epitope exposed in both RBD-up and RBD-down conformations. When tested in vivo, a single-dose prophylactic administration of EH3 fully protected stringent K18-hACE2 mice from lethal challenge with Delta VOC. Our study demonstrates that protective NAbs responses converge in pediatric and adult SARS-CoV-2 patients.

microbiology↗

bioRxiv2022

Small CD4 mimetics sensitize HIV-1-infected macrophages to antibody-dependent cellular cytotoxicity

HIV-1 Envelope (Env) conformation determines the susceptibility of infected CD4+ T cells to Antibody Dependent Cellular Cytotoxicity (ADCC). Upon interaction with CD4, Env adopts more "open" conformations, exposing ADCC epitopes. HIV-1 limits Env-CD4 interaction and protects infected cells against ADCC by downregulating CD4 via Nef, Vpu and Env. Limited data exists however of the role of these proteins in downmodulating CD4 on infected macrophages and how this impacts Env conformation. While Nef, Vpu and Env are all required to efficiently downregulate CD4 on infected CD4+T cells, we show here that any one of these proteins is sufficient to downmodulate most CD4 from the surface of infected macrophages. Consistent with this finding, Nef and Vpu have a lesser impact on Env conformation and ADCC sensitivity in infected macrophages compared to CD4+T cells. However, treatment of infected macrophages with small CD4-mimetics expose vulnerable CD4-induced Env epitopes and sensitize them to ADCC.

microbiology↗

bioRxiv2022

An S1 subunit vaccine and combination adjuvant (COVAC-1) elicits robust protection against SARS-CoV-2 challenge in African green monkeys

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent responsible for the ongoing global pandemic. With over 500 million cases and more than 6 million deaths reported globally, the need for access to effective vaccines is clear. An ideal SARS-CoV-2 vaccine will prevent pathology in the lungs and prevent virus replication in the upper respiratory tract, thus reducing transmission. Here, we assessed the efficacy of an adjuvanted SARS-CoV-2 S1 subunit vaccine, called COVAC-1, in an African green monkey (AGM) model. AGMs immunized and boosted with COVAC-1 were protected from SARS-CoV-2 challenge compared to unvaccinated controls based on reduced pathology and reduced viral RNA levels and infectious virus in the respiratory tract. Both neutralizing antibodies and antibodies capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC) were observed in vaccinated animals prior to the challenge. COVAC-1 induced effective protection, including in the upper respiratory tract, thus supporting further development and utility for determining the mechanism that confers this protection. AUTHOR SUMMARYVaccines that can prevent the onward transmission of SARS-CoV-2 and prevent disease are highly desirable. Whether this can be accomplished without mucosal immunization by a parenterally administered subunit vaccine is not well established. Here we demonstrate that following two vaccinations, a protein subunit vaccine containing the S1 portion of the SARS-CoV-2 spike glycoprotein and the novel adjuvant TriAdj significantly reduces the amount of virus in the lungs and also mediates rapid clearance of the virus from the upper respiratory tract. Further support of the effectiveness of COVAC-1 was the observation of reduced pathology in the lungs and viral RNA being largely absent from tissues, blood, and rectal swabs. Thus COVAC-1 appears promising at mediating protection in both the upper and lower respiratory tract and may be capable of reducing subsequent transmission of SARS-CoV-2. Further investigation into the mechanism of protection in the upper respiratory tract and the initial immune response that supports this would be warranted.

microbiology↗

bioRxiv2022

Characterization of human immunodeficiency virus (HIV-1) envelope glycoprotein variants selected for resistance to a CD4-mimetic compound

Binding to host cell receptors, CD4 and CCR5/CXCR4, triggers conformational changes in the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer that promote virus entry. CD4 binding allows the gp120 exterior Env to bind CCR5/CXCR4 and induces a pre-hairpin intermediate conformation in the gp41 transmembrane Env. Small-molecule CD4-mimetic compounds (CD4mcs) bind within the conserved Phe-43 cavity of gp120, near the binding site for CD4. CD4mcs inhibit HIV-1 infection by competing with CD4 and by prematurely activating Env, leading to irreversible inactivation. BNM-III-170 is a CD4mc that inhibits the infection of approximately 70% of HIV-1 strains at micromolar concentrations. We selected and analyzed variants of the primary HIV-1AD8 strain resistant to BNM-III-170. Two changes (S375N and I424T) in gp120 residues that flank the Phe-43 cavity each conferred [~]5-fold resistance to BNM- III-170 with minimal fitness cost. A third change (E64G) in Layer 1 of the gp120 inner domain resulted in [~]100-fold resistance to BNM-III-170, [~]2-3-fold resistance to soluble CD4-Ig, and a moderate decrease in viral fitness. The gp120 changes additively or synergistically contributed to BNM-III-170 resistance. The sensitivity of the Env variants to BNM-III-170 inhibition of virus entry correlated with their sensitivity to BNM-III-170- induced Env activation and shedding of gp120. The S375N and I424T changes, but not the E64G change, conferred resistance to BMS-806, a potent HIV-1 entry inhibitor that blocks Env conformational transitions. These studies identify pathways whereby HIV-1 can develop resistance to CD4mcs and BMS-806 conformational blockers, two classes of entry inhibitors that target the conserved gp120 Phe-43 cavity. IMPORTANCECD4-mimetic compounds (CD4mcs) and BMS-806 are small-molecule inhibitors of human immunodeficiency virus (HIV-1) entry into host cells. Although CD4mcs and BMS-806 inhibit HIV-1 entry by different mechanisms, they both target a pocket on the viral envelope glycoprotein (Env) spike that is used for binding to the receptor, CD4, and is highly conserved among HIV-1 strains. Our study identifies changes near this pocket that can confer various levels of resistance to the antiviral effects of both a CD4mc and BMS-806. We relate the antiviral potency of a CD4mc against this panel of HIV-1 variants to the ability of the CD4mc to activate changes in Env conformation and to induce the shedding of the gp120 exterior Env from the spike. These findings will guide efforts to improve the potency and breadth of small-molecule HIV-1 entry inhibitors.

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