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 trimer 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. Although current antiretroviral therapy decreases viral loads below detection levels, it does not eliminate latently infected cells that 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 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.

Th17 cell master transcription factor RORC2 regulates HIV-1 gene expression and viral outgrowth.

Among CD4+ T cells, T helper 17 (Th17) cells are particularly susceptible to HIV-1 infection and are depleted from mucosal sites, which causes damage to the gut barrier, resulting in a microbial translocation-induced systemic inflammation, a hallmark of disease progression. Furthermore, a proportion of latently infected Th17 cells persist long term in the gastrointestinal lymphatic tract where a low-level HIV-1 transcription is observed. This residual viremia contributes to chronic immune activation. Thus, Th17 cells are key players in HIV pathogenesis and viral persistence. It is, however, unclear why these cells are highly susceptible to HIV-1 infection. Th17 cell differentiation depends on the expression of the master transcriptional regulator RORC2, a retinoic acid-related nuclear hormone receptor that regulates specific transcriptional programs by binding to promoter/enhancer DNA. Here, we report that RORC2 is a key host cofactor for HIV replication in Th17 cells. We found that specific inhibitors that bind to the RORC2 ligand-binding domain reduced HIV replication in CD4+ T cells. The depletion of RORC2 inhibited HIV-1 infection, whereas its overexpression enhanced it. RORC2 was also found to promote HIV-1 gene expression by binding to the nuclear receptor responsive element in the HIV-1 long terminal repeats (LTR). In treated HIV-1 patients, RORC2+ CD4 T cells contained more proviral DNA than RORC2- cells. Pharmacological inhibition of RORC2 potently reduced HIV-1 outgrowth in CD4+ T cells from antiretroviral-treated patients. Altogether, these results provide an explanation as to why Th17 cells are highly susceptible to HIV-1 infection and suggest that RORC2 may be a cell-specific target for HIV-1 therapy.

COVID-19 vaccine humoral response in frequent platelet donors with plateletpheresis-associated lymphopenia.

BACKGROUND: Plateletpheresis involves platelet separation and collection from whole blood while other blood cells are returned to the donor. Because platelets are replaced faster than red blood cells, as many as 24 donations can be done annually. However, some frequent apheresis platelet donors (>20 donations annually) display severe plateletpheresis-associated lymphopenia; in particular, CD4+ T but not B cell numbers are decreased. COVID-19 vaccination thereby provides a model to assess whether lymphopenic platelet donors present compromised humoral immune responses. STUDY DESIGN AND METHODS: We assessed vaccine responses following 2 doses of COVID-19 vaccination in a cohort of 43 plateletpheresis donors with a range of pre-vaccination CD4+ T cell counts (76-1537 cells/µl). In addition to baseline T cell measurements, antibody binding assays to full-length Spike and the Receptor Binding Domain (RBD) were performed pre- and post-vaccination. Furthermore, pseudo-particle neutralization and antibody-dependent cellular cytotoxicity assays were conducted to measure antibody functionality. RESULTS: Participants were stratified into two groups: <400 CD4/µl (n = 27) and ≥ 400 CD4/µl (n = 16). Following the first dose, 79% seroconverted within the <400 CD4/µl group compared to 87% in the ≥400 CD4/µl group; all donors were seropositive post-second dose with significant increases in antibody levels. Importantly differences in CD4+ T cell levels minimally impacted neutralization, Spike recognition, and IgG Fc-mediated effector functions. DISCUSSION: Overall, our results indicate that lymphopenic plateletpheresis donors do not exhibit significant immune dysfunction; they have retained the T and B cell functionality necessary for potent antibody responses after vaccination.

Short-term antibody response after 1 dose of BNT162b2 vaccine in patients receiving hemodialysis.

BACKGROUND: Patients receiving in-centre hemodialysis are at high risk of exposure to SARS-CoV-2 and death if infected. One dose of the BNT162b2 SARS-CoV-2 vaccine is efficacious in the general population, but responses in patients receiving hemodialysis are uncertain. METHODS: We obtained serial plasma from patients receiving hemodialysis and health care worker controls before and after vaccination with 1 dose of the BNT162b2 mRNA vaccine, as well as convalescent plasma from patients receiving hemodialysis who survived COVID-19. We measured anti-receptor binding domain (RBD) immunoglobulin G (IgG) levels and stratified groups by evidence of previous SARS-CoV-2 infection. RESULTS: Our study included 154 patients receiving hemodialysis (135 without and 19 with previous SARS-CoV-2 infection), 40 controls (20 without and 20 with previous SARS-CoV-2 infection) and convalescent plasma from 16 patients. Among those without previous SARS-CoV-2 infection, anti-RBD IgG was undetectable at 4 weeks in 75 of 131 (57%, 95% confidence interval [CI] 47% to 65%) patients receiving hemodialysis, compared with 1 of 20 (5%, 95% CI 1% to 23%) controls (p < 0.001). No patient with nondetectable levels at 4 weeks developed anti-RBD IgG by 8 weeks. Results were similar in non-immunosuppressed and younger individuals. Three patients receiving hemodialysis developed severe COVID-19 after vaccination. Among those with previous SARS-CoV-2 infection, median anti-RBD IgG levels at 8 weeks in patients receiving hemodialysis were similar to controls at 3 weeks (p = 0.3) and to convalescent plasma (p = 0.8). INTERPRETATION: A single dose of BNT162b2 vaccine failed to elicit a humoral immune response in most patients receiving hemodialysis without previous SARS-CoV-2 infection, even after prolonged observation. In those with previous SARS-CoV-2 infection, the antibody response was delayed. We advise that patients receiving hemodialysis be prioritized for a second BNT162b2 dose at the recommended 3-week interval.

Daily variations of gut microbial translocation markers in ART-treated HIV-infected people.

BACKGROUND: Increased intestinal barrier permeability and subsequent gut microbial translocation are significant contributors to inflammatory non-AIDS comorbidities in people living with HIV (PLWH). Evidence in animal models have shown that markers of intestinal permeability and microbial translocation vary over the course of the day and are affected by food intake and circadian rhythms. However, daily variations of these markers are not characterized yet in PLWH. Herein, we assessed the variation of these markers over 24 h in PLWH receiving antiretroviral therapy (ART) in a well-controlled environment. METHODS: As in Canada, PLWH are predominantly men and the majority of them are now over 50 years old, we selected 11 men over 50 receiving ART with undetectable viremia for more than 3 years in this pilot study. Blood samples were collected every 4 h over 24 h before snacks/meals from 8:00 in the morning to 8:00 the next day. All participants consumed similar meals at set times, and had a comparable amount of sleep, physical exercise and light exposure. Plasma levels of bacterial lipopolysaccharide (LPS) and fungal (1→3)-ß-D-Glucan (BDG) translocation markers, along with markers of intestinal damage fatty acid binding protein (I-FABP) and regenerating islet-derived protein-3α (REG3α) were assessed by ELISA or the fungitell assay. RESULTS: Participants had a median age of 57 years old (range 50 to 63). Plasma levels of BDG and REG3α did not vary significantly over the course of the study. In contrast, a significant increase of LPS was detected between 12:00 and 16:00 (Z-score: - 1.15 ± 0.18 vs 0.16 ± 0.15, p = 0.02), and between 12:00 and 24:00 (- 1.15 ± 0.18 vs 0.89 ± 0.26, p < 0.001). The plasma levels of I-FABP at 16:00 (- 0.92 ± 0.09) were also significantly lower, compared to 8:00 the first day (0.48 ± 0.26, p = 0.002), 4:00 (0.73 ± 0.27, p < 0.001) or 8:00 on secondary day (0.88 ± 0.27, p < 0.001). CONCLUSIONS: Conversely to the fungal translocation marker BDG and the gut damage marker REG3α, time of blood collection matters for the proper evaluation for LPS and I-FABP as markers for the risk of inflammatory non-AIDS co-morbidities. These insights are instrumental for orienting clinical investigations in PLWH.

Division of labor among Mycobacterium smegmatis RNase H enzymes: RNase H1 activity of RnhA or RnhC is essential for growth whereas RnhB and RnhA guard against killing by hydrogen peroxide in stationary phase.

RNase H enzymes sense the presence of ribonucleotides in the genome and initiate their removal by incising the ribonucleotide-containing strand of an RNA:DNA hybrid. Mycobacterium smegmatis encodes four RNase H enzymes: RnhA, RnhB, RnhC and RnhD. Here, we interrogate the biochemical activity and nucleic acid substrate specificity of RnhA. We report that RnhA (like RnhC characterized previously) is an RNase H1-type magnesium-dependent endonuclease with stringent specificity for RNA:DNA hybrid duplexes. Whereas RnhA does not incise an embedded mono-ribonucleotide, it can efficiently cleave within tracts of four or more ribonucleotides in duplex DNA. We gained genetic insights to the division of labor among mycobacterial RNases H by deleting the rnhA, rnhB, rnhC and rnhD genes, individually and in various combinations. The salient conclusions are that: (i) RNase H1 activity is essential for mycobacterial growth and can be provided by either RnhC or RnhA; (ii) the RNase H2 enzymes RnhB and RnhD are dispensable for growth and (iii) RnhB and RnhA collaborate to protect M. smegmatis against oxidative damage in stationary phase. Our findings highlight RnhC, the sole RNase H1 in pathogenic mycobacteria, as a candidate drug discovery target for tuberculosis and leprosy.

Transcription of lncRNA prt, clustered prt RNA sites for Mmi1 binding, and RNA polymerase II CTD phospho-sites govern the repression of pho1 gene expression under phosphate-replete conditions in fission yeast.

Expression of fission yeast Pho1 acid phosphatase is repressed during growth in phosphate-rich medium. Repression is mediated by transcription of the prt locus upstream of pho1 to produce a long noncoding (lnc) prt RNA. Repression is also governed by RNA polymerase II CTD phosphorylation status, whereby inability to place a Ser7-PO4 mark (as in S7A) derepresses Pho1 expression, and inability to place a Thr4-PO4 mark (as in T4A) hyper-represses Pho1 in phosphate replete cells. Here we find that basal pho1 expression from the prt-pho1 locus is inversely correlated with the activity of the prt promoter, which resides in a 110-nucleotide DNA segment preceding the prt transcription start site. CTD mutations S7A and T4A had no effect on the activity of the prt promoter or the pho1 promoter, suggesting that S7A and T4A affect post-initiation events in prt lncRNA synthesis that make it less and more repressive of pho1, respectively. prt lncRNA contains clusters of DSR (determinant of selective removal) sequences recognized by the YTH-domain-containing protein Mmi1. Altering the nucleobase sequence of two DSR clusters in the prt lncRNA caused hyper-repression of pho1 in phosphate replete cells, concomitant with increased levels of the prt transcript. The isolated Mmi1 YTH domain binds to RNAs with single or tandem DSR elements, to the latter in a noncooperative fashion. We report the 1.75 Å crystal structure of the Mmi1 YTH domain and provide evidence that Mmi1 recognizes DSR RNA via a binding mode distinct from that of structurally homologous YTH proteins that recognize m(6)A-modified RNA.

Vibrio cholerae O395 outer membrane vesicles modulate intestinal epithelial cells in a NOD1 protein-dependent manner and induce dendritic cell-mediated Th2/Th17 cell responses.

Like other Gram-negative pathogens, Vibrio cholerae, the causative agent of the diarrheal disease cholera, secretes outer membrane vesicles (OMVs). OMVs are complex entities composed of a subset of envelope lipid and protein components and play a role in the delivery of effector molecules to host cells. We previously showed that V. cholerae O395 cells secrete OMVs that are internalized by host cells, but their role in pathogenesis has not been well elucidated. In the present study, we evaluated the interaction of OMVs with intestinal epithelial cells. These vesicles induced expression of proinflammatory cytokines such as IL-8 and GM-CSF and chemokines such as CCL2, CCL20, and thymic stromal lymphopoietin in epithelial cells through activation of MAPK and NF-κB pathways in NOD1-dependent manner. Epithelial cells stimulated with OMVs activated dendritic cells (DCs) in a direct co-culture system. Activated DCs expressed high levels of co-stimulatory molecules; released inflammatory cytokines IL-1ß, IL-6, TNF-α, and IL-23 and chemokines CCL22 and CCL17; and subsequently primed CD4(+) T cells leading to IL-4, IL-13, and IL-17 expression. These results suggest that V. cholerae O395 OMVs modulate the epithelial proinflammatory response and activate DCs, which promote T cell polarization toward an inflammatory Th2/Th17 response.

Structural basis for c-di-GMP-mediated inside-out signaling controlling periplasmic proteolysis.

The bacterial second messenger bis-(3'-5') cyclic dimeric guanosine monophosphate (c-di-GMP) has emerged as a central regulator for biofilm formation. Increased cellular c-di-GMP levels lead to stable cell attachment, which in Pseudomonas fluorescens requires the transmembrane receptor LapD. LapD exhibits a conserved and widely used modular architecture containing a HAMP domain and degenerate diguanylate cyclase and phosphodiesterase domains. c-di-GMP binding to the LapD degenerate phosphodiesterase domain is communicated via the HAMP relay to the periplasmic domain, triggering sequestration of the protease LapG, thus preventing cleavage of the surface adhesin LapA. Here, we elucidate the molecular mechanism of autoinhibition and activation of LapD based on structure-function analyses and crystal structures of the entire periplasmic domain and the intracellular signaling unit in two different states. In the absence of c-di-GMP, the intracellular module assumes an inactive conformation. Binding of c-di-GMP to the phosphodiesterase domain disrupts the inactive state, permitting the formation of a trans-subunit dimer interface between adjacent phosphodiesterase domains via interactions conserved in c-di-GMP-degrading enzymes. Efficient mechanical coupling of the conformational changes across the membrane is realized through an extensively domain-swapped, unique periplasmic fold. Our structural and functional analyses identified a conserved system for the regulation of periplasmic proteases in a wide variety of bacteria, including many free-living and pathogenic species.

Conformational dynamics of the HIV-1 envelope glycoprotein from CRF01_AE is associated with susceptibility to antibody-dependent cellular cytotoxicity.

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 Env 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 binding of antibodies targeting this site. 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 towards more "open" conformations. In this work, through adaptation of an established smFRET imaging approach, we report that the conformational dynamics of native, unliganded HIV-1CRF01_AE Env indicates frequent sampling of 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 HIV-1CRF01_AE Env, which are relevant for structure-based design of both synthetic inhibitors of receptor binding, and enhancers of ADCC as therapeutic alternatives.

NTB-A and 2B4 Natural Killer Cell Receptors Modulate the Capacity of a Cocktail of Non-Neutralizing Antibodies and a Small CD4-Mimetic to Eliminate HIV-1-Infected Cells by Antibody-Dependent Cellular Cytotoxicity.

Natural Killer (NK) cells have the potential to eliminate HIV-1-infected cells by antibody-dependent cellular cytotoxicity (ADCC). NK cell activation is tightly regulated by the engagement of its inhibitory and activating receptors. The activating receptor CD16 drives ADCC upon binding to the Fc portion of antibodies; NK cell activation is further sustained by the co-engagement of activating receptors NTB-A and 2B4. During HIV-1 infection, Nef and Vpu accessory proteins contribute to ADCC escape by downregulating the ligands of NTB-A and 2B4. HIV-1 also evades ADCC by keeping its envelope glycoproteins (Env) in a "closed" conformation which effectively masks epitopes recognized by non-neutralizing antibodies (nnAbs) which are abundant in the plasma of people living with HIV. To achieve this, the virus uses its accessory proteins Nef and Vpu to downregulate the CD4 receptor, which otherwise interacts with Env and exposes the epitopes recognized by nnAbs. Small CD4-mimetic compounds (CD4mc) have the capacity to expose these epitopes, thus sensitizing infected cells to ADCC. Given the central role of NK cell co-activating receptors NTB-A and 2B4 in Fc-effector functions, we studied their contribution to CD4mc-mediated ADCC. Despite the fact that their ligands are partially downregulated by HIV-1, we found that both co-activating receptors significantly contribute to CD4mc sensitization of HIV-1-infected cells to ADCC.

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 suggests 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. IMPORTANCE: Antibody-dependent cellular cytotoxicity (ADCC) represents an effective immune response for clearing virally infected cells, making ADCC-mediating antibodies promising therapeutic candidates for HIV-1 cure strategies. Broadly neutralizing antibodies (bNAbs) target epitopes present on the native "closed" envelope glycoprotein (Env), while non-neutralizing antibodies (nnAbs) recognize epitopes exposed upon Env-CD4 interaction. Here, we provide evidence that env mRNA is predominantly expressed by productively infected cells that have already downmodulated cell-surface CD4. This indicates that CD4 downmodulation by HIV-1 precedes Env expression, making productively infected cells resistant to ADCC mediated by nnAbs but sensitive to those mediated by bnAbs. These findings offer critical insights for the development of immunotherapy-based strategies aimed at targeting and eliminating productively infected cells in people living with HIV.

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.

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.

Humoral Responses Elicited by SARS-CoV-2 mRNA Vaccine in People Living with HIV.

While mRNA SARS-CoV-2 vaccination elicits strong humoral responses in the general population, humoral responses in people living with HIV (PLWH) remain to be clarified. Here, we conducted a longitudinal study of vaccine immunogenicity elicited after two and three doses of mRNA SARS-CoV-2 vaccine in PLWH stratified by their CD4 count. We measured the capacity of the antibodies elicited by vaccination to bind the Spike glycoprotein of different variants of concern (VOCs). We also evaluated the Fc-mediated effector functions of these antibodies by measuring their ability to eliminate CEM.NKr cells stably expressing SARS-CoV-2 Spikes. Finally, we measured the relative capacity of the antibodies to neutralize authentic SARS-CoV-2 virus after the third dose of mRNA vaccine. We found that after two doses of SARS-CoV-2 mRNA vaccine, PLWH with a CD4 count < 250/mm3 had lower levels of anti-RBD IgG antibodies compared to PLWH with a CD4 count > 250/mm3 (p < 0.05). A third dose increased these levels and importantly, no major differences were observed in their capacity to mediate Fc-effector functions and neutralize authentic SARS-CoV-2. Overall, our work demonstrates the importance of mRNA vaccine boosting in immuno-compromised individuals presenting low levels of CD4.

Identification of aryl hydrocarbon receptor as a barrier to HIV-1 infection and outgrowth in CD4+ T cells.

The aryl hydrocarbon receptor (AhR) regulates Th17-polarized CD4+ T cell functions, but its role in HIV-1 replication/outgrowth remains unknown. Genetic (CRISPR-Cas9) and pharmacological inhibition reveal AhR as a barrier to HIV-1 replication in T cell receptor (TCR)-activated CD4+ T cells in vitro. In single-round vesicular stomatitis virus (VSV)-G-pseudotyped HIV-1 infection, AhR blockade increases the efficacy of early/late reverse transcription and subsequently facilitated integration/translation. Moreover, AhR blockade boosts viral outgrowth in CD4+ T cells of people living with HIV-1 (PLWH) receiving antiretroviral therapy (ART). Finally, RNA sequencing reveals genes/pathways downregulated by AhR blockade in CD4+ T cells of ART-treated PLWH, including HIV-1 interactors and gut-homing molecules with AhR-responsive elements in their promoters. Among them, HIC1, a repressor of Tat-mediated HIV-1 transcription and a tissue-residency master regulator, is identified by chromatin immunoprecipitation as a direct AhR target. Thus, AhR governs a T cell transcriptional program controlling viral replication/outgrowth and tissue residency/recirculation, supporting the use of AhR inhibitors in "shock and kill" HIV-1 remission/cure strategies.

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 exist, 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 with CD4+ T cells. However, treatment of infected macrophages with small CD4 mimetics exposes vulnerable CD4-induced Env epitopes and sensitizes them to ADCC.

Spike recognition and neutralization of SARS-CoV-2 Omicron subvariants elicited after the third dose of mRNA vaccine.

Several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants have recently emerged, becoming the dominant circulating strains in many countries. These variants contain a large number of mutations in their spike glycoprotein, raising concerns about vaccine efficacy. In this study, we evaluate the ability of plasma from a cohort of individuals that received three doses of mRNA vaccine to recognize and neutralize these Omicron subvariant spikes. We observed that BA.4/5 and BQ.1.1 spikes are markedly less recognized and neutralized compared with the D614G and other Omicron subvariant spikes tested. Also, individuals who have been infected before or after vaccination present better humoral responses than SARS-CoV-2-naive vaccinated individuals, thus indicating that hybrid immunity generates better humoral responses against these subvariants.

Molecular basis for antiviral activity of two pediatric neutralizing antibodies targeting SARS-CoV-2 Spike RBD.

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.

A third SARS-CoV-2 mRNA vaccine dose in people receiving hemodialysis overcomes B cell defects but elicits a skewed CD4+ T cell profile.

Cellular immune defects associated with suboptimal responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccination in people receiving hemodialysis (HD) are poorly understood. We longitudinally analyze antibody, B cell, CD4+, and CD8+ T cell vaccine responses in 27 HD patients and 26 low-risk control individuals (CIs). The first two doses elicit weaker B cell and CD8+ T cell responses in HD than in CI, while CD4+ T cell responses are quantitatively similar. In HD, a third dose robustly boosts B cell responses, leads to convergent CD8+ T cell responses, and enhances comparatively more T helper (TH) immunity. Unsupervised clustering of single-cell features reveals phenotypic and functional shifts over time and between cohorts. The third dose attenuates some features of TH cells in HD (tumor necrosis factor alpha [TNFα]/interleukin [IL]-2 skewing), while others (CCR6, CXCR6, programmed cell death protein 1 [PD-1], and HLA-DR overexpression) persist. Therefore, a third vaccine dose is critical to achieving robust multifaceted immunity in hemodialysis patients, although some distinct TH characteristics endure.