Search

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., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: The authors declare no conflict of interest.

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.

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-1 CRF01_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-1 JR-FL , which do not transition to State 2A in the absence of ligands. These findings inform on the conformational dynamics of HIV-1 CRF01_AE Env, which are relevant for structure-based design of both synthetic inhibitors of receptor binding, and enhancers of ADCC as therapeutic alternatives., Competing Interests: Conflicts of Interest The authors declare no competing interests.

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.

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., Competing Interests: CONFLICT OF INTEREST The authors declare no competing interests.

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/mm 3 had lower levels of anti-RBD IgG antibodies compared to PLWH with a CD4 count > 250/mm 3 ( 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.

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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 (T H ) immunity. Unsupervised clustering of single-cell features reveals phenotypic and functional shifts over time and between cohorts. The third dose attenuates some features of T H 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 T H characteristics endure., Competing Interests: Declaration of interests C.T. serves as a consultant for Merck, Gilead, GSK, AstraZeneca, and Medicago., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: A.F. has filed a provisional patent application on the following monoclonal antibodies: CV3-1 and CV3-25. A.F., J.P., V.L. M.A.S., V.-P.L., and E.H. have filed a provisional patent application on the following monoclonal antibodies: EH3 and EH8., (© 2022 The Author(s).)

Due to the recrudescence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections worldwide, mainly caused by the Omicron variant of concern (VOC) and its sub-lineages, several jurisdictions are administering an mRNA vaccine boost. Here, we analyze humoral responses induced after the second and third doses of an mRNA vaccine in naive and previously infected donors who received their second dose with an extended 16-week interval. We observe that the extended interval elicits robust humoral responses against VOCs, but this response is significantly diminished 4 months after the second dose. Administering a boost to these individuals brings back the humoral responses to the same levels obtained after the extended second dose. Interestingly, we observe that administering a boost to individuals that initially received a short 3- to 4-week regimen elicits humoral responses similar to those observed in the long interval regimen. Nevertheless, humoral responses elicited by the boost in naive individuals do not reach those present in previously infected vaccinated individuals., Competing Interests: Declaration of interests A.R.G. is a consultant for Relation Therapeutics. E.J.W. is consulting for or is an advisor for Merck, Marengo, Janssen, Related Sciences, Synthekine, and Surface Oncology. E.J.W. is a founder of Surface Oncology, Danger Bio, and Arsenal Biosciences., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Although SARS-CoV-2 mRNA vaccination has been shown to be safe and effective in the general population, immunocompromised solid organ transplant recipients (SOTRs) were reported to have impaired immune responses after one or two doses of vaccine. In this study, we examined humoral responses induced after the second and the third dose of mRNA vaccine in different SOTR (kidney, liver, lung, and heart). Compared to a cohort of SARS-CoV-2 naïve immunocompetent health care workers (HCWs), the second dose induced weak humoral responses in SOTRs, except for the liver recipients. The third dose boosted these responses but they did not reach the same level as in HCW. Interestingly, although the neutralizing activity against Delta and Omicron variants remained very low after the third dose, Fc-mediated effector functions in SOTR reached similar levels as in the HCW cohort. Whether these responses will suffice to protect SOTR from severe outcome remains to be determined., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

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., (© 2022 AABB.)

Continuous emergence of SARS-CoV-2 variants of concern (VOCs) is fueling the COVID-19 pandemic. Omicron (B.1.1.529) rapidly spread worldwide. The large number of mutations in its Spike raise concerns about a major antigenic drift that could significantly decrease vaccine efficacy and infection-induced immunity. A long interval between BNT162b2 mRNA doses elicits antibodies that efficiently recognize Spikes from different VOCs. Here, we evaluate the recognition of Omicron Spike by plasma from a cohort of SARS-CoV-2 naive and previously infected individuals who received their BNT162b2 mRNA vaccine 16 weeks apart. Omicron Spike is recognized less efficiently than D614G, Alpha, Beta, Gamma, and Delta Spikes. We compare with plasma activity from participants receiving a short (4 weeks) interval regimen. Plasma from individuals of the long-interval cohort recognize and neutralize better the Omicron Spike compared with those who received a short interval. Whether this difference confers any clinical benefit against Omicron remains unknown., Competing Interests: Declaration of interests A.F. has filed a provisional patent application on the anti-Spike monoclonal antibody CV3-25., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Emerging evidence indicates that both neutralizing and Fc-mediated effector functions of antibodies contribute to protection against SARS-CoV-2. It is unclear whether Fc-effector functions alone can protect against SARS-CoV-2. Here, we isolated CV3-13, a non-neutralizing antibody, from a convalescent individual with potent Fc-mediated effector functions. The cryoelectron microscopy structure of CV3-13 in complex with the SARS-CoV-2 spike reveals that the antibody binds from a distinct angle of approach to an N-terminal domain (NTD) epitope that only partially overlaps with the NTD supersite recognized by neutralizing antibodies. CV3-13 does not alter the replication dynamics of SARS-CoV-2 in K18-hACE2 mice, but its Fc-enhanced version significantly delays virus spread, neuroinvasion, and death in prophylactic settings. Interestingly, the combination of Fc-enhanced non-neutralizing CV3-13 with Fc-compromised neutralizing CV3-25 completely protects mice from lethal SARS-CoV-2 infection. Altogether, our data demonstrate that efficient Fc-mediated effector functions can potently contribute to the in vivo efficacy of anti-SARS-CoV-2 antibodies., Competing Interests: Declaration of interests L.S., A.T.M., and A.F. have filed a provisional patent application on the following monoclonal antibodies: CV3-1, CV3-13, and CV3-25., (Copyright © 2022. Published by Elsevier Inc.)

The rapid emergence of SARS-CoV-2 variants is fueling the recent waves of the COVID-19 pandemic. Here, we assessed ACE2 binding and antigenicity of Mu (B.1.621) and A.2.5 Spikes. Both these variants carry some mutations shared by other emerging variants. Some of the pivotal mutations such as N501Y and E484K in the receptor-binding domain (RBD) detected in B.1.1.7 (Alpha), B.1.351 (Beta) and P.1 (Gamma) are now present within the Mu variant. Similarly, the L452R mutation of B.1.617.2 (Delta) variant is present in A.2.5. In this study, we observed that these Spike variants bound better to the ACE2 receptor in a temperature-dependent manner. Pseudoviral particles bearing the Spike of Mu were similarly neutralized by plasma from vaccinated individuals than those carrying the Beta (B.1.351) and Delta (B.1.617.2) Spikes. Altogether, our results indicate the importance of measuring critical parameters such as ACE2 interaction, plasma recognition and neutralization ability of each emerging variant.

The standard regimen of the BNT162b2 mRNA vaccine for SARS-CoV-2 includes two doses administered three weeks apart. However, some public health authorities spaced these doses, raising questions about efficacy. We analyzed longitudinal humoral responses against the D614G strain and variants of concern for SARS-CoV-2 in a cohort of SARS-CoV-2-naive and previously infected individuals who received the BNT162b2 mRNA vaccine with sixteen weeks between doses. While administering a second dose to previously infected individuals did not significantly improve humoral responses, these responses significantly increased in naive individuals after a 16-week spaced second dose, achieving similar levels as in previously infected individuals. Comparing these responses to those elicited in individuals receiving a short (4-week) dose interval showed that a 16-week interval induced more robust responses among naive vaccinees. These findings suggest that a longer interval between vaccine doses does not compromise efficacy and may allow greater flexibility in vaccine administration., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (Copyright © 2021 the Author(s). Published by PNAS.)

Towards the end of 2020, multiple variants of concern (VOCs) and variants of interest (VOIs) have arisen from the original SARS-CoV-2 Wuhan-Hu-1 strain. Mutations in the Spike protein are highly scrutinized for their impact on transmissibility, pathogenesis and vaccine efficacy. Here, we contribute to the growing body of literature on emerging variants by evaluating the impact of single mutations on the overall antigenicity of selected variants and their binding to the ACE2 receptor. We observe a differential contribution of single mutants to the global variants phenotype related to ACE2 interaction and antigenicity. Using biolayer interferometry, we observe that enhanced ACE2 interaction is mostly modulated by a decrease in off-rate. Finally, we made the interesting observation that the Spikes from tested emerging variants bind better to ACE2 at 37°C compared to the D614G variant. Whether improved ACE2 binding at higher temperature facilitates emerging variants transmission remain to be demonstrated., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., Competing Interests: Competing interests: None declared., (© 2021 CMA Joule Inc. or its licensors.)

Background: Several types of extracellular vesicles (EVs) secreted by various immune and non-immune cells are present in the human plasma. We previously demonstrated that EV abundance and microRNA content change in pathological conditions, such as HIV infection. Here, we investigated daily variations of large and small EVs, in terms of abundance and microRNA contents in people living with HIV (PLWH) receiving antiretroviral therapy (HIV+ART) and uninfected controls (HIV-)., Methods: Venous blood samples from n = 10 HIV+ART and n = 10 HIV- participants were collected at 10:00 and 22:00 the same day. Large and small plasma EVs were purified, counted, and the mature miRNAs miR-29a, miR-29b, miR-92, miR-155, and miR-223 copies were measured by RT-PCR., Results: Large EVs were significantly bigger in the plasma collected at 10:00 versus 22:00 in both groups. There was a significant day-night increase in the quantity of 5 miRNAs in HIV- large EVs. In HIV+ART, only miR-155 daily variation has been observed in large EVs. Finally, EV-miRNA content permits to distinguish HIV- to HIV+ART in multivariate analysis., Conclusion: These results point that plasma EV amount and microRNA contents are under daily variation in HIV- people. This new dynamic measure is disrupted in PLWH despite viral-suppressive ART. This study highlights a significant difference concerning EV abundance and their content measured at 22:00 between both groups. Therefore, the time of blood collection must be considered in the future for the EV as biomarkers.

The frequency and functions of Th17-polarized CCR6 + RORyt + CD4 + T cells are rapidly compromised upon HIV infection and are not restored with long-term viral suppressive antiretroviral therapy (ART). In line with this, Th17 cells represent selective HIV-1 infection targets mainly at mucosal sites, with long-lived Th17 subsets carrying replication-competent HIV-DNA during ART. Therefore, novel Th17-specific therapeutic interventions are needed as a supplement of ART to reach the goal of HIV remission/cure. Th17 cells express high levels of peroxisome proliferator-activated receptor gamma (PPARy), which acts as a transcriptional repressor of the HIV provirus and the rorc gene, which encodes for the Th17-specific master regulator RORyt. Thus, we hypothesized that the pharmacological inhibition of PPARy will facilitate HIV reservoir reactivation while enhancing Th17 effector functions. Consistent with this prediction, the PPARy antagonist T0070907 significantly increased HIV transcription (cell-associated HIV-RNA) and RORyt-mediated Th17 effector functions (IL-17A). Unexpectedly, the PPARy antagonism limited HIV outgrowth from cells of ART-treated people living with HIV (PLWH), as well as HIV replication in vitro . Mechanistically, PPARy inhibition in CCR6 + CD4 + T cells induced the upregulation of transcripts linked to Th17-polarisation (RORyt, STAT3, BCL6 IL-17A/F, IL-21) and HIV transcription (NCOA1-3, CDK9, HTATIP2). Interestingly, several transcripts involved in HIV-restriction were upregulated (Caveolin-1, TRIM22, TRIM5α, BST2, miR-29), whereas HIV permissiveness transcripts were downregulated (CCR5, furin), consistent with the decrease in HIV outgrowth/replication. Finally, PPARy inhibition increased intracellular HIV-p24 expression and prevented BST-2 downregulation on infected T cells, suggesting that progeny virion release is restricted by BST-2-dependent mechanisms. These results provide a strong rationale for considering PPARy antagonism as a novel strategy for HIV-reservoir purging and restoring Th17-mediated mucosal immunity in ART-treated PLWH., Competing Interests: DP, AF, YZ, JPG, JR, AF, MJR, LRM, DC, HC, TRWS, and AG declare no financial or non-financial competing interests to disclose. EAC is a member of the Scientific Advisory Board of Theratechnologies. JPR performed contract research and/or served on Advisory Boards for Gilead Sciences Canada Inc., Merck Canada Inc., Abbvie Corp., ViiV Healthcare, Bristol Myers Squibb, Janssen Inc., Argos Pharmaceuticals from InnaVirVax, and Theravectys. NC received research funding from EMD Serono and served on Advisory Boards for Gilead Sciences. PA's laboratory receives research funding from Glaxo Smith Klein/NeoMed for projects different from the present study. PA served as a Consultant at Merck Canada Inc., (© Pathogens and Immunity 2020.)

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)-β-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.

Fission yeast phosphate homeostasis entails transcriptional induction of genes encoding phosphate-mobilizing proteins under conditions of phosphate starvation. Transcription factor Pho7, a member of the Zn 2 Cys 6 family of fungal transcription regulators, is the central player in the starvation response. The DNA binding sites in the promoters of phosphate-responsive genes have not been defined, nor have any structure-function relationships been established for the Pho7 protein. Here we narrow this knowledge gap by (i) delineating an autonomous DNA-binding domain (DBD) within Pho7 that includes the Zn 2 Cys 6 module, (ii) deploying recombinant Pho7 DBD in DNase I footprinting and electrophoretic mobility shift assays (EMSAs) to map the Pho7 recognition sites in the promoters of the phosphate-regulated pho1 and tgp1 genes to a 12-nucleotide sequence motif [5'-TCG(G/C)(A/T)xxTTxAA], (iii) independently identifying the same motif as a Pho7 recognition element via in silico analysis of available genome-wide ChIP-seq data, (iv) affirming that mutations in the two Pho7 recognition sites in the pho1 promoter efface pho1 expression in vivo , and (v) establishing that the zinc-binding cysteines and a pair of conserved arginines in the DBD are essential for Pho7 activity in vivo IMPORTANCE Fungi respond to phosphate starvation by inducing the transcription of a set of phosphate acquisition genes that comprise a phosphate regulon. Pho7, a member of the Zn 2 Cys 6 family of fungal transcription regulators, is the central player in the phosphate starvation response in fission yeast. The present study identifies a 12-nucleotide Pho7 DNA binding motif [5'-TCG(G/C)(A/T)xxTTxAA] in the promoters of phosphate-regulated genes, pinpoints DNA and protein features important for Pho7 binding to DNA, and correlates them with Pho7-dependent gene expression in vivo The results highlight distinctive properties of Pho7 vis-a-vis other fungal zinc binuclear cluster transcription factors as well as the divergent cast of transcription factors deployed for phosphate homeostasis in fission yeast versus budding yeast., (Copyright © 2017 Schwer et al.)

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., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., (© 2016 Chatterjee et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Stable surface adhesion of cells is one of the early pivotal steps in bacterial biofilm formation, a prevalent adaptation strategy in response to changing environments. In Pseudomonas fluorescens, this process is regulated by the Lap system and the second messenger cyclic-di-GMP. High cytoplasmic levels of cyclic-di-GMP activate the transmembrane receptor LapD that in turn recruits the periplasmic protease LapG, preventing it from cleaving a cell surface-bound adhesin, thereby promoting cell adhesion. In this study, we elucidate the molecular basis of LapG regulation by LapD and reveal a remarkably sensitive switching mechanism that is controlled by LapD's HAMP domain. LapD appears to act as a coincidence detector, whereby a weak interaction of LapG with LapD transmits a transient outside-in signal that is reinforced only when cyclic-di-GMP levels increase. Given the conservation of key elements of this receptor system in many bacterial species, the results are broadly relevant for cyclic-di-GMP- and HAMP domain-regulated transmembrane signaling., (Copyright © 2014, Chatterjee et al.)

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-1β, 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.

Vibrio cholerae induces acute inflammatory response at intestinal epithelial surface; the underlying cellular immune mechanisms for such effects are largely unexplored. Mucosal immune response is controlled by crosstalk between the intestinal epithelial cells (ECs) and dendritic cells (DCs). An EC-derived cytokine thymic stromal lymphopoietin (TSLP) has been found a critical regulator of several human inflammatory conditions. TSLP is highly elevated in ECs stimulated with V. cholerae and its recombinant flagellin (rFlaA). V. cholerae treated human ECs produce DC-attracting chemokine MIP-3α (CCL20). Flagellin, a potent V. cholerae factor was responsible for maximum stimulation of epithelial CCL20 production and subsequent DC activation. Activated DCs express high levels of costimulatory molecules and secrete inflammatory cytokines TNF-α, IL-6 and IL-1β. Bacteria stimulated ECs conditioned DCs to produce Th2 cell-attracting chemokines CCL17 and CCL22. TSLP and other mediators present in the V. cholerae stimulated EC-culture filtrate potently activated DCs, which subsequently primed CD4(+)T cells to differentiate into T helper type 2 (Th2) cells that produce high amounts of IL-4, IL-13 and TNF-α and low IFN-γ. TSLP-induced proinflammatory response in DCs involved the transcriptional mechanisms, MAPKs (ERK1/2, p38 and JNK) and STAT3 activation. This study suggests TSLP and other mediators released from ECs in response to V. cholerae colonization actively influence DCs in initiating inflammatory response., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The bacterial dinucleotide second messenger c-di-GMP has emerged as a central molecule in regulating bacterial behavior, including motility and biofilm formation. Proteins for the synthesis and degradation of c-di-GMP and effectors for its signal transmission are widely used in the bacterial domain. Previous work established the GGDEF-EAL domain-containing receptor LapD as a central switch in Pseudomonas fluorescens cell adhesion. LapD senses c-di-GMP inside the cytosol and relays this signal to the outside by the differential recruitment of the periplasmic protease LapG. Here we identify the core components of an orthologous system in Legionella pneumophila. Despite only moderate sequence conservation at the protein level, key features concerning the regulation of LapG are retained. The output domain of the LapD-like receptor from L. pneumophila, CdgS9, binds the LapG ortholog involving a strictly conserved surface tryptophan residue. While the endogenous substrate for L. pneumophila LapG is unknown, the enzyme processed the corresponding P. fluorescens substrate, indicating a common catalytic mechanism and substrate recognition. Crystal structures of L. pneumophila LapG provide the first atomic models of bacterial proteases of the DUF920 family and reveal a conserved calcium-binding site important for LapG function.

Biofilm formation by Pseudomonas fluorescens Pf0-1 requires the cell surface adhesin LapA. We previously reported that LapG, a periplasmic cysteine protease of P. fluorescens, cleaves the N terminus of LapA, thus releasing this adhesin from the cell surface and resulting in loss of the ability to make a biofilm. The activity of LapG is regulated by the inner membrane-localized cyclic-di-GMP receptor LapD via direct protein-protein interactions. Here we present chelation and metal add-back studies demonstrating that calcium availability regulates biofilm formation by P. fluorescens Pf0-1. The determination that LapG is a calcium-dependent protease, based on in vivo and in vitro studies, explains the basis of this calcium-dependent regulation. Based on the crystal structure of LapG of Legionella pneumophila in the accompanying report by Chatterjee and colleagues (D. Chatterjee et al., J. Bacteriol. 194:4415-4425, 2012), we show that the calcium-binding residues of LapG, D134 and E136, which are near the critical C135 active-site residue, are required for LapG activity of P. fluorescens in vivo and in vitro. Furthermore, we show that mutations in D134 and E136 result in LapG proteins no longer able to interact with LapD, indicating that calcium binding results in LapG adopting a conformation competent for interaction with the protein that regulates its activity. Finally, we show that citrate, an environmentally relevant calcium chelator, can impact LapG activity and thus biofilm formation, suggesting that a physiologically relevant chelator of calcium can impact biofilm formation by this organism.

Cholera toxin (CT) is the major virulence factor of pathogenic Vibrio cholerae. The present study demonstrates that a fraction of CT is associated with the outer membrane vesicles (OMVs) released by V. cholerae. Atomic force microscopy (AFM) and also transmission electron microscopy (TEM) of purified OMVs from toxigenic V. cholerae O395 revealed spherical shaped vesicles of size range 20-200 nm. Immunoblotting of purified OMVs with polyclonal anti-CT antibody and GM1-ganglioside dependent ELISA suggest that CT is associated with OMVs. CHO cell assay indicated that OMV associated CT is physiologically active. OMVs labeled with fluorescent dye interacted with intestinal epithelial cells via the CT-receptor and were internalized increasing the cAMP level. Thus OMVs may represent an important vehicle in delivering CT to epithelial cells., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

This communication describes the development of a thiamin sensor based on the bacterial thiamin binding protein. A triple mutant (C48S, C50S, S62C) of TbpA was labeled on C62 with N-[2-(L-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC). Thiamin binding to this protein reduced the coumarin fluorescence giving a thiamin sensor with low nanomolar sensitivity.

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., Competing Interests: The authors have declared that no competing interests exist.

A community-based cross-sectional study was conducted in brothel-based sex workers of West Bengal, Eastern India, to determine their oncogenic human papillomavirus (HPV) status and the presence of pre-cancerous lesions. A total of 229 sex workers from three districts of West Bengal participated in the study. All the study participants were interviewed with the aid of a pre-tested questionnaire to determine their sociodemographics, risk behaviour and risk perceptions after obtaining informed verbal consent. The interview was followed by collection of cervical cells from all participants using a disposable vaginal speculum and cervical cytobrush. Oncogenic HPV DNA was detected by real-time polymerase chain reaction (PCR). A simultaneous Papanicolaou test ('Pap smear') was performed to detect cervical cytological abnormalities. Overall, the prevalence of oncogenic HPV was found to be 25% (58/229) among the studied population. A subset (n=112) of the sample was tested separately to determine the existence and magnitude of HPV genotypes 16 and 18. The results showed that genotype 16 was prevalent in 10% (11/112), genotype 18 in 7% (8/112) and both genotype 16 and 18 in 7% (8/112). The HPV prevalence rate showed a decreasing trend with age, being 71.4% in the 10-19 years age group, 32.3% in the 20-29 years age group, 18.3% in the 30-39 years age group and 2.5% in the >or=40 years age group (statistically significant differences, P1 year, respectively. This difference was found to be statistically significant both by univariate and multivariate analysis. In this study, it was observed that sex workers with an average number of daily clients of six or more had an HPV prevalence of 67% (n=6), those with four to five clients had a prevalence of 45% (n=9), those with two to three clients had a prevalence of 30% (n=34) and those with one or less clients had a prevalence of 10% (n=9) (statistically significant differences, P=0.00003). Multivariate analysis showed a statistical association only with a duration of sex work of or=101 (OR=2.5; 95% CI 1.3-5). Regarding pre-cancerous lesions, 2 of 229 sex workers showed the presence of a low-grade squamous intraepithelial lesion along with high-risk HPV. Thus, 1% of the studied population suffer from a pre-cancerous lesion caused by high-risk HPV. This study concludes that young sex workers are particularly vulnerable to high-risk HPV, similar to human immunodeficiency virus (HIV). The observation of older sex workers relatively free from HPV supports the view of acquired immunity against HPV, which needs to be studied in-depth further. There is a need for a suitable community-based intervention programme targeted towards sex workers, with special reference to younger sex workers, for control and prevention of HPV and cervical cancer. Vaccination against HPV for newly entrant sex workers may be an important component for a successful intervention programme., (Copyright (c) 2008 King Saud Bin Abdulaziz University for Health Sciences. All rights reserved.)