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We have previously shown that blood levels of B-cell Activating Factor (BAFF) rise relatively to disease progression status in the context of HIV-1 infection. Excess BAFF was concomitant with hyperglobulinemia and the deregulation of blood B-cell populations, notably with increased frequencies of a population sharing characteristics of transitional immature and marginal zone (MZ) B-cells, which we defined as marginal zone precursor-like” (MZp). In HIV-uninfected individuals, MZp present a B-cell regulatory (Breg) profile and function, which are lost in classic-progressors. Moreover, RNASeq analyses of blood MZp from classic-progressors depict a hyperactive state and signs of exhaustion, as well as an interferon signature similar to that observed in autoimmune disorders such as Systemic Lupus Erythematosus (SLE) and Sjögren Syndrome (SS), in which excess BAFF and deregulated MZ populations have also been documented. Based on the above, we hypothesize that excess BAFF may preclude the generation of HIV-1-specific IgG responses and drive polyclonal responses, including those from MZ populations, endowed with polyreactivity/autoreactivity. As such, we show that the quantity of HIV-1-specific IgG varies with disease progression status. In vitro, excess BAFF promotes polyclonal IgM and IgG responses, including those from MZp. RNASeq analyses reveal that blood MZp from classic-progressors are prone to Ig production and preferentially make usage of IGHV genes associated with some HIV broadly neutralizing antibodies (bNAbs), but also with autoantibodies, and whose impact in the battle against HIV-1 has yet to be determined.

IntroductionGrowth differentiation factor 15 (GDF-15) was originally described as a stress-induced cytokine, and a biomarker of aging and cardiovascular diseases. We hypothesized that circulating GDF-15 would be associated with COVID-19 disease severity. Herein, we explored this hypothesis in a large cohort of COVID-19 patients.MethodsBlood samples were collected from 926 COVID-19 adult patients and from 285 hospitalized controls from the Biobanque Québécoise de la COVID-19 (BQC19). COVID-19 severity was graded according to the WHO criteria. SOMAscan proteomics assay was performed on 50µL of plasma. ELISA were performed on 46 selected participants with left-over plasma to validate differences in plasma GDF-15 levels. Statistical analyses were conducted using GraphPad Prism 9.0 and SPSS. P values < 0.01 were considered significant.ResultsProteomics showed that plasma GDF-15 levels were higher in COVID-19 patients compared to hospitalized controls. GDF-15 levels increased with COVID-19 severity. COVID-19 patients presenting with comorbidities including diabetes, cancer, chronic obstructive pulmonary disease (COPD) and cardiovascular disease had higher GDF-15 levels. ELISA revealed significant elevation of GDF-15 until 30 days after hospitalization. Plasma GDF-15 elevation was correlated with older age. Moreover, GDF-15 levels correlated with pro-inflammatory cytokine interleukin-6 (IL-6) and inflammation marker C-reactive protein (CRP) as well as soluble levels of its putative receptor CD48. No association was established between anti-SARS-CoV-2 IgG levels and plasma GDF-15 levels.ConclusionsThis study confirms GDF-15 as a biomarker for COVID-19 severity. Clinical evaluation of GDF-15 levels could assist identification of persons at high-risk of progressing to severe disease, thus improving patient care.

Abstract Introduction HIV pre‐exposure prophylaxis (PrEP) has been recommended and partly subsidized in Québec, Canada, since 2013. We evaluated the population‐level impact of PrEP on HIV transmission among men who have sex with men (MSM) in Montréal, Québec's largest city, over 2013–2021. Methods We used an agent‐based mathematical model of sexual HIV transmission to estimate the fraction of HIV acquisitions averted by PrEP compared to a counterfactual scenario without PrEP. The model was calibrated to local MSM survey, surveillance, and cohort data and accounted for COVID‐19 pandemic impacts on sexual activity, HIV prevention, and care. PrEP was modelled from 2013 onwards, assuming 86% individual‐level effectiveness. The PrEP eligibility criteria were: any anal sex unprotected by condoms (past 6 months) and either multiple partnerships (past 6 months) or multiple uses of post‐exposure prophylaxis (lifetime). To assess potential optimization strategies, we modelled hypothetical scenarios prioritizing PrEP to MSM with high sexual activity (≥11 anal sex partners annually) or aged ⩽45 years, increasing coverage to levels achieved in Vancouver, Canada (where PrEP is free‐of‐charge), and improving retention. Results Over 2013–2021, the estimated annual HIV incidence decreased from 0.4 (90% credible interval [CrI]: 0.3–0.6) to 0.2 (90% CrI: 0.1–0.2) per 100 person‐years. PrEP coverage among HIV‐negative MSM remained low until 2015 (

Introduction In the COVID-19 pandemic, healthcare workers (HCWs) were at high risk of infection due to their exposure to COVID infections. HCWs were the backbone of our healthcare response to this pandemic; every HCW withdrawn or lost due to infection had a substantial impact on our capacity to deliver care. Primary prevention was a key approach to reduce infection. Vitamin D insufficiency is highly prevalent in Canadians and worldwide. Vitamin D supplementation has been shown to significantly decrease the risk of respiratory infections. Whether this risk reduction would apply to COVID-19 infections remained to be determined. This study aimed to determine the impact of high-dose vitamin D supplementation on incidence of laboratory-confirmed COVID-19 infection rate and severity in HCWs working in high COVID incidence areas.Methods and analysis PROTECT was a triple-blind, placebo-controlled, parallel-group multicentre trial of vitamin D supplementation in HCWs. Participants were randomly allocated in a 1:1 ratio in variable block size to intervention (one oral loading dose of 100 000 IU vitamin D3+10 000 IU weekly vitamin D3) or control (identical placebo loading dose+weekly placebo). The primary outcome was the incidence of laboratory-confirmed COVID-19 infection, documented by RT-qPCR on salivary (or nasopharyngeal) specimens obtained for screening or diagnostic purposes, as well as self-obtained salivary specimens and COVID-19 seroconversion at endpoint. Secondary outcomes included disease severity; duration of COVID-19-related symptoms; COVID-19 seroconversion documented at endpoint; duration of work absenteeism; duration of unemployment support; and adverse health events. The trial was terminated prematurely, due to recruitment difficulty.Ethics and dissemination This study involves human participants and was approved by the Research Ethics Board (REB) of the Centre hospitalier universitaire (CHU) Sainte-Justine serving as central committee for participating institutions (#MP-21-2021-3044). Participants provided written informed consent to participate in the study before taking part. Results are being disseminated to the medical community via national/international conferences and publications in peer-reviewed journals.Trial registration number https://clinicaltrials.gov/ct2/show/NCT04483635.

Background Antiretroviral therapy (ART) halts HIV-1 replication, decreasing viremia to below the detection limit of clinical assays. However, some individuals experience persistent nonsuppressible viremia (NSV) originating from CD4+ T cell clones carrying infectious proviruses. Defective proviruses represent over 90% of all proviruses persisting during ART and can express viral genes, but whether they can cause NSV and complicate ART management is unknown.Methods We undertook an in-depth characterization of proviruses causing NSV in 4 study participants with optimal adherence and no drug resistance. We investigated the impact of the observed defects on 5′-leader RNA properties, virus infectivity, and gene expression. Integration-site specific assays were used to track these proviruses over time and among cell subsets.Results Clones carrying proviruses with 5′-leader defects can cause persistent NSV up to approximately 103 copies/mL. These proviruses had small, often identical deletions or point mutations involving the major splicing donor (MSD) site and showed partially reduced RNA dimerization and nucleocapsid binding. Nevertheless, they were inducible and produced noninfectious virions containing viral RNA, but lacking envelope.Conclusion These findings show that proviruses with 5′-leader defects in CD4+ T cell clones can give rise to NSV, affecting clinical care. Sequencing of the 5′-leader can help in understanding failure to completely suppress viremia.Funding Office of the NIH Director and National Institute of Dental and Craniofacial Research, NIH; Howard Hughes Medical Institute; Johns Hopkins University Center for AIDS Research; National Institute for Allergy and Infectious Diseases (NIAID), NIH, to the PAVE, BEAT-HIV, and DARE Martin Delaney collaboratories.

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.

While an important part of the world’s population is vaccinated against SARS-CoV-2, new variants continue to emerge. We observe that even after a fifth dose of the mRNA bivalent vaccine, most vaccinated individuals have antibodies that poorly neutralize several Omicron subvariants, including BQ.1.1, XBB, XBB.1.5, FD.1.1, and CH.1.1. However, Fc-effector functions remain strong and stable over time against new variants, which may partially explain why vaccines continue to be effective. We also observe that donors who have been recently infected have stronger antibody functional activities, including neutralization and Fc-effector functions, supporting the observations that hybrid immunity leads to better humoral responses.

Abstract Objectives: We evaluated the added value of infection control-guided, on demand, and locally performed severe acute respiratory coronavirus virus 2 (SARS-CoV-2) genomic sequencing to support outbreak investigation and control in acute-care settings. Design and setting: This 18-month prospective molecular epidemiology study was conducted at a tertiary-care hospital in Montreal, Canada. When nosocomial transmission was suspected by local infection control, viral genomic sequencing was performed locally for all putative outbreak cases. Molecular and conventional epidemiology data were correlated on a just-in-time basis to improve understanding of coronavirus disease 2019 (COVID-19) transmission and reinforce or adapt control measures. Results: Between April 2020 and October 2021, 6 outbreaks including 59 nosocomial infections (per the epidemiological definition) were investigated. Genomic data supported 7 distinct transmission clusters involving 6 patients and 26 healthcare workers. We identified multiple distinct modes of transmission, which led to reinforcement and adaptation of infection control measures. Molecular epidemiology data also refuted (n = 14) suspected transmission events in favor of community acquired but institutionally clustered cases. Conclusion: SARS-CoV-2 genomic sequencing can refute or strengthen transmission hypotheses from conventional nosocomial epidemiological investigations, and guide implementation of setting-specific control strategies. Our study represents a template for prospective, on site, outbreak-focused SARS-CoV-2 sequencing. This approach may become increasingly relevant in a COVID-19 endemic state where systematic sequencing within centralized surveillance programs is not available. Trial registration: clinicaltrials.gov identifier: NCT05411562

Introduction Chronic cytomegalovirus (CMV) infection is very frequent in people living with HIV (PLWH). High anti-CMV IgG titres, which may be linked to transient CMV replication, have been associated with earlier mortality, CD8 T-cell expansion, lower CD4/CD8 ratio and increased T-cell senescence. We previously showed that anti-CMV IgG titres correlated with gut permeability in PLWH on antiretroviral therapy (ART), which was associated with microbial translocation, systemic inflammation and non-infectious/non-AIDS comorbidities. Letermovir, a novel anti-CMV drug with a good safety profile, was recently approved for anti-CMV prophylaxis in allogeneic haematopoietic stem cell transplant recipients. A drastic and selective reduction of both low-grade replication and clinically significant CMV infections, combined with an improved immune reconstitution have been reported. In vitro, letermovir prevented CMV-induced epithelial disruption in intestinal tissues. Based on these findings, we aim to assess whether letermovir could inhibit CMV subclinical replication in CMV-seropositive PLWH receiving ART and, in turn, decrease CMV-associated gut damage and inflammation.Method and analysis We will conduct a multi-centre, open-label, randomised, controlled clinical trial, including a total of 60 CMV-seropositive ART-treated PLWH for at least 3 years, with a viral load 400 cells/µL. Forty participants will be randomised to receive letermovir for 14 weeks and 20 participants will receive standard of care (ART) alone. Plasma, pheripheral blood mononuclear cells (PBMCs), and stool samples will be collected. Colon biopsies will be collected in an optional substudy. We will assess the effect of letermovir on gut damage, microbial translocation, inflammation and HIV reservoir size.Ethics and dissemination The study was approved by Health Canada and the Research Ethics Boards of the McGill University Health Centre (MUHC-REB, protocol number: MP37-2022-8295). Results will be made available through publications in open access peer-reviewed journals and through the CIHR/CTN website.Trial registration number NCT05362916.

Summary: 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.

Summary: Spacing the first two doses of SARS-CoV-2 mRNA vaccines beyond 3–4 weeks raised initial concerns about vaccine efficacy. While studies have since shown that long-interval regimens induce robust antibody responses, their impact on B and T cell immunity is poorly known. Here, we compare SARS-CoV-2 naive donors B and T cell responses to two mRNA vaccine doses administered 3–4 versus 16 weeks apart. After boost, the longer interval results in a higher magnitude and a more mature phenotype of RBD-specific B cells. While the two geographically distinct cohorts present quantitative and qualitative differences in T cell responses at baseline and after priming, the second dose led to convergent features with overall similar magnitude, phenotype, and function of CD4+ and CD8+ T cell responses at post-boost memory time points. Therefore, compared to standard regimens, a 16-week interval has a favorable impact on the B cell compartment but minimally affects T cell immunity.

Summary: Background: Immune checkpoint blockade (ICB) partially reverses the dysfunctional state of antigen-specific T cell in chronic infections. However, its impact on the diverse subsets of CD4+ T cells in humans is largely unknown. Methods: We examined immune checkpoint (IC) expression and function in HIV-specific CD4+ T cells of viremic individuals (≥5000 vRNA cp/ml, n = 17) prior to ART and persons with spontaneous (n = 11) or therapy-induced (n = 16) viral suppression (

Summary: 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.

ObjectivesBesides CD4 regulatory T-cells (Tregs), immunosuppressor FoxP3+ CD8 T-cells are emerging as an important subset of Tregs, which contribute to immune dysfunction and disease progression in HIV infection. However, FoxP3+ CD8 T-cell dynamics in acute HIV infection and following early antiretroviral therapy (ART) initiation remain understudied.MethodsSubsets of FoxP3+ CD8 T-cells were characterized both prospectively and cross-sectionally in PBMCs from untreated acute (n=26) and chronic (n=10) HIV-infected individuals, early ART-treated in acute infection (n=10, median of ART initiation: 5.5 months post-infection), ART-treated in chronic infection (n=10), elite controllers (n=18), and HIV-uninfected controls (n=21).ResultsAcute and chronic infection were associated with increased total, effector memory, and terminally differentiated FoxP3+ CD8 T-cells, while early ART normalized only the frequencies of total FoxP3+ CD8 T-cells. We observed an increase in FoxP3+ CD8 T-cell immune activation (HLADR+/CD38+), senescence (CD57+/CD28-), and PD-1 expression during acute and chronic infection, which were not normalized by early ART. FoxP3+ CD8 T-cells in untreated participants expressed higher levels of immunosuppressive LAP(TGF-β1) and CD39 than uninfected controls, whereas early ART did not affect their expression. The expression of gut-homing markers CCR9 and Integrin-β7 by total FoxP3+ CD8 T-cells and CD39+ and LAP(TGF-β1)+ FoxP3+ CD8 T-cells increased in untreated individuals and remained higher than in uninfected controls despite early ART. Elite controllers share most of the FoxP3+ CD8 T-cell characteristics in uninfected individuals.ConclusionsAlthough early ART normalized total FoxP3+ CD8 T-cells frequencies, it did not affect the persistent elevation of the gut-homing potential of CD39+ and LAP(TGF-β1)+ FoxP3+ CD8 T-cell, which may contribute to immune dysfunction.

Infection with the human immunodeficiency virus (HIV), when left untreated, typically leads to disease progression towards acquired immunodeficiency syndrome. Some people living with HIV (PLWH) control their virus to levels below the limit of detection of standard viral load assays, without treatment. As such, they represent examples of a functional HIV cure. These individuals, called Elite Controllers (ECs), are rare, making up

Monkeypox is a zoonotic Orthopoxvirus infection usually present in regions of Africa. Recent outbreaks of Monkeypox infection have been reported in non endemic region and human-to-human contact is believed to be the main driver for propagation. While the disease is usually self-contained, severe complications, such as neurological and ocular involvements may arise. We report the case of a 34-year-old male who presented with myocarditis and concurrent genital Monkeypox infection. Other usual causes of myocardial injury were ruled out. We believe it to be the first documented case of myocarditis secondary to Monkeypox. We report a new complication of the disease and the possible underlying mechanisms. Our case report raises awareness about possible unknown complications of Monkeypox as outbreaks continue to happen around the world.

The Omicron BQ.1.1 variant is now the major SARS-CoV-2 circulating strain in many countries. Because of the many mutations present in its Spike glycoprotein, this variant is resistant to humoral responses elicited by monovalent mRNA vaccines. With the goal to improve immune responses against Omicron subvariants, bivalent mRNA vaccines have recently been approved in several countries. In this study, we measure the capacity of plasma from vaccinated individuals, before and after a fourth dose of mono- or bivalent mRNA vaccine, to recognize and neutralize the ancestral (D614G) and the BQ.1.1 Spikes. Before and after the fourth dose, we observe a significantly better recognition and neutralization of the ancestral Spike. We also observe that fourth-dose vaccinated individuals who have been recently infected better recognize and neutralize the BQ.1.1 Spike, independently of the mRNA vaccine used, than donors who have never been infected or have an older infection. Our study supports that hybrid immunity, generated by vaccination and a recent infection, induces higher humoral responses than vaccination alone, independently of the mRNA vaccine used.

Human cytomegalovirus (CMV) infection drives the expansion and differentiation of natural killer (NK) cells with adaptive-like features. We investigated whether age and time on antiretroviral therapy (ART) influenced adaptive NK cell frequency and functionality. Flow cytometry was used to evaluate the frequency of adaptive and conventional NK cells in 229 CMV+ individuals of whom 170 were people living with HIV (PLWH). The frequency of these NK cell populations producing CD107a, CCL4, IFN-γ or TNF-α was determined following a 6-h antibody dependent (AD) stimulation. Though ART duration and age were correlated, longer time on ART was associated with a reduced frequency of adaptive NK cells. In general, the frequency and functionality of NK cells following AD stimulation did not differ significantly between treated CMV+PLWH and CMV+HIV- persons, suggesting that HIV infection, per se, did not compromise AD NK cell function. AD activation of adaptive NK cells from CMV+PLWH induced lower frequencies of IFN-γ or TNF-α secreting cells in older persons, when compared with younger persons.

Chronic inflammation persists in people living with HIV (PLHIV) despite antiretrovial therapy (ART) and is involved in their premature development of cardiovascular diseases (CVD) such as atherosclerosis. We have previously reported that an excess of “B-cell activating factor” (BAFF), an important molecule for the selection and activation of first-line Marginal Zone (MZ) B-cell populations, is associated with deregulations of precursor-like MZ (MZp), whose potent B-cell regulatory (Breg) capacities are altered in PLHIV, early on and despite 1–2 years of ART. Based on these observations, and growing evidence that MZ populations are involved in atherosclerosis control, we designed a cross sectional study to explore the associations between BAFF and its analogue “A proliferation-inducing ligand” (APRIL) with subclinical CVD in long-time-treated individuals of the Canadian HIV and Aging Cohort Study (CHACS) imaging sub-study group. We also characterized the Breg profile of MZp from the blood of these individuals. Results were correlated with the total volume of atherosclerotic plaques (TPV) and with CVD risk factors and biomarkers. TPV was measured using cardiac computerised tomography angiography, and presence of CVD was defined as TPV > 0. We report that blood levels of BAFF are elevated and correlate positively with CVD and its risk factors in PLHIV from the CHACS, in contrast to APRIL levels, which correlate negatively with these factors. The expression levels of Breg markers such as NR4A3, CD39, CD73 and CD83 are significantly lower in PLHIV when compared to those of HIV-uninfected controls. In vitro experiments show that APRIL upregulates the expression of Breg markers by blood MZp from HIV-uninfected individuals, while this modulation is dampened by the addition of recombinant BAFF. Altogether, our observations suggest that strategies viewed to modulate levels of BAFF and/or APRIL could eventually represent a potential treatment target for CVD in PLHIV.

We have reported excess B-cell activating factor (BAFF) in the blood of HIV-infected progressors, which was concomitant with increased frequencies of precursor-like marginal zone (MZp) B-cells, early on and despite antiretroviral therapy (ART). In controls, MZp possess a strong B-cell regulatory (Breg) potential. They highly express IL-10, the orphan nuclear receptors (NR)4A1, NR4A2 and NR4A3, as well as the ectonucleotidases CD39 and CD73, all of which are associated with the regulation of inflammation. Furthermore, we have shown MZp regulatory function to involve CD83 signaling. To address the impact of HIV infection and excessive BAFF on MZp Breg capacities, we have performed transcriptomic analyses by RNA-seq of sorted MZp B-cells from the blood of HIV-infected progressors. The Breg profile and function of blood MZp B-cells from HIV-infected progressors were assessed by flow-cytometry and light microscopy high-content screening (HCS) analyses, respectively. We report significant downregulation of NR4A1, NR4A2, NR4A3 and CD83 gene transcripts in blood MZp B-cells from HIV-infected progressors when compared to controls. NR4A1, NR4A3 and CD83 protein expression levels and Breg function were also downregulated in blood MZp B-cells from HIV-infected progressors and not restored by ART. Moreover, we observe decreased expression levels of NR4A1, NR4A3, CD83 and IL-10 by blood and tonsillar MZp B-cells from controls following culture with excess BAFF, which significantly diminished their regulatory function. These findings, made on a limited number of individuals, suggest that excess BAFF contributes to the alteration of the Breg potential of MZp B-cells during HIV infection and possibly in other situations where BAFF is found in excess.

Summary: The crosstalk between intestinal epithelial cells (IECs) and Th17-polarized CD4+ T cells is critical for mucosal homeostasis, with HIV-1 causing significant alterations in people living with HIV (PLWH) despite antiretroviral therapy (ART). In a model of IEC and T cell co-cultures, we investigated the effects of IL-17A, the Th17 hallmark cytokine, on IEC ability to promote de novo HIV infection and viral reservoir reactivation. Our results demonstrate that IL-17A acts in synergy with TNF to boost IEC production of CCL20, a Th17-attractant chemokine, and promote HIV trans-infection of CD4+ T cells and viral outgrowth from reservoir cells of ART-treated PLWH. Importantly, the Illumina RNA-sequencing revealed an IL-17A-mediated pro-inflammatory and pro-viral molecular signature, including a decreased expression of type I interferon (IFN-I)-induced HIV restriction factors. These findings point to the deleterious features of IL-17A and raise awareness for caution when designing therapies aimed at restoring the paucity of mucosal Th17 cells in ART-treated PLWH.

Background: HIV infection promotes the expansion of immunosuppressive regulatory T-cells (Tregs), contributing to immune dysfunction, tissue fibrosis and disease progression. Early antiretroviral treatment (ART) upon HIV infection improves CD4 count and decreases immune activation. However, Treg dynamics and their epigenetic regulation following early ART initiation remain understudied. Methods: Treg subsets were characterized by flow cytometry in 103 individuals, including untreated HIV-infected participants in acute and chronic phases, ART-treated in early infection, elite controllers (ECs), immunological controllers (ICs), and HIV-uninfected controls. The methylation status of six regulatory regions of the foxp3 gene was assessed using MiSeq technology. Findings: Total Treg frequency increased overtime during HIV infection, which was normalized in early ART recipients. Tregs in untreated individuals expressed higher levels of activation and immunosuppressive markers (CD39, and LAP(TGF-β1)), which remained unchanged following early ART. Expression of gut migration markers (CCR9, Integrin-β7) by Tregs was elevated during untreated HIV infection, while they declined with the duration of ART but not upon early ART initiation. Notably, gut-homing Tregs expressing LAP(TGF-β1) and CD39 remained higher despite early treatment. Additionally, the increase in LAP(TGF-β1)+ Tregs overtime were consistent with higher demethylation of conserved non-coding sequence (CNS)-1 in the foxp3 gene. Remarkably, LAP(TGF-β1)-expressing Tregs in ECs were significantly higher than in uninfected subjects, while the markers of Treg activation and gut migration were not different. Interpretation: Early ART initiation was unable to control the levels of immunosuppressive Treg subsets and their gut migration potential, which could ultimately contribute to gut tissue fibrosis and HIV disease progression. Funding: This study was funded by the Canadian Institutes of Health Research (CIHR, grant MOP 142294) and in part by the AIDS and Infectious Diseases Network of the Réseau SIDA et maladies infectieuses du Fonds de recherche du Québec-Santé (FRQ-S).

Summary: With the recent approval of highly effective coronavirus disease 2019 (COVID-19) vaccines, functional and lasting immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently under investigation as antibody levels in plasma were shown to decline during convalescence. Since the absence of antibodies does not equate to absence of immune memory, we evaluate the presence of SARS-CoV-2-specific memory B cells in convalescent individuals. Here, we report a longitudinal assessment of humoral immune responses on 32 donors up to 8 months post-symptom onset. Our observations indicate that anti-Spike and anti-receptor binding domain (RBD) immunoglobulin M (IgM) in plasma decay rapidly, whereas the reduction of IgG is less prominent. Neutralizing activity also declines rapidly when compared to Fc-effector functions. Concomitantly, the frequencies of RBD-specific IgM+ B cells wane significantly when compared to RBD-specific IgG+ B cells, which remain stable. Our results add to the current understanding of immune memory following SARS-CoV-2 infection, which is critical for secondary infection prevention and vaccine efficacy.

Abstract Background The aim of this study was to establish the prevalence of lipodystrophy and its association to cumulative exposure to antiretroviral drugs. Method We conducted a cross sectional study in all HIV- infected patients attending the HIV clinic in the Centre hospitalier universitaire de Montréal (CHUM) with DEXA scan. Lipodystrophy was defined as a trunk/limb fat ratio ≥ 1.5. Association between cumulative exposure to antiretroviral (measured in years of use) with trunk/limb fat ratio (coded as a continuous variable) was assessed using univariate and multivariate linear regression for each antiretroviral drug with at least 40 exposed patients. Results One hundred sixty-six patients were included. Seventy-five percent were male, median age was 56 years, 67% were Caucasian. Overall, prevalence of lipodystrophy was 47%, with a mean trunk/limb fat ratio of 1.87, SD = 1.03, min = 0.6 and max = 5.87. Each 10-year increase in age and HIV infection duration was associated with an average increase of 0.24 and 0.34 for the trunk/limb fat ratio respectively. (p = 0.003, p = 0.002, respectively) Patients classified as lipodystrophic were more likely to be diabetic (50 vs. 28%, p = 0.07) and to have dyslipidemia (47 vs. 19%, p = 0.01). According to viral load at DEXA test, each one log increase was associated with less probability (0.7) of lipodystrophy. (p = 0.01) Among ARV drugs tested, there was an association between years of use of d4T, ritonavir and raltegravir and higher trunk/limb fat ratio (indicating more lipodystrophy) (p

Background: Extracellular vesicles (EVs) are intercellular messengers with epigenetic potential since they can shuttle microRNA (miRNA). EVs and miRNA play a role in human immunodeficiency virus (HIV) infection immunopathogenesis. Chronic immune activation and systemic inflammation during HIV infection despite effective antiretroviral therapy (ART) are associated with non-acquired immunodeficiency syndrome (AIDS) comorbidities in people living with HIV (PLWH). Analysis of plasma EVs and their miRNA content may be useful as immune activation or inflammatory biomarkers in PLWH receiving ART. In this study, we hypothesized that the number, size, and miRNA of large and small EVs could reflect immune activation associated with an elevated CD8 T-cell count or a low CD4/CD8 ratio in PLWH. Methods: Plasma EVs subtype purified from PLWH and uninfected controls were sized using dynamic light scattering and quantified using flow cytometry and acetylcholine esterase (AChE) activity. Expression of mature miRNAs miR-92, miR-155, miR-223 was measured by quantitative reverse-transcriptase polymerase chain reaction in EVs and leucocytes. Results: HIV infection induces increased production of small EVs in plasma. EV subtypes were differentially enriched in miR-92, miR-155, and miR-223. Positive correlations between CD8 T-cell count and large EVs abundance and small EVs AChE activity were observed. CD4/CD8 ratio was negatively correlated with small EV AChE activity, and miRNA-155 level per small EV was negatively correlated with CD8 T-cell count. Conclusions: These findings suggest that quantifying large or small EVs and profiling miRNA content per EV might provide new functional biomarkers of immune activation and inflammation.

Purpose: To assess the reproducibility of different epicardial fat measurement and their association with other adiposity measurements in HIV-infected and non-HIV-infected patients. Methods and materials: In this cross-sectional study, 167 HIV-infected and 58 non-HIV-infected consecutive participants (200 males; mean age 56 years) with low/intermediate cardiovascular risk were recruited between 2012 and 2017 from a large prospective cohort and underwent non-contrast cardiac CT. Two independent observers measured epicardial fat volume, area and thickness in all participants. For intra-observer agreement, one observer did a second assessment in a subset of 40 patients. Agreement was assessed with the intraclass correlation coefficient (ICC). Pearson's correlation was estimated to assess the association between epicardial fat, body-mass index (BMI) and dual-energy x-ray absorptiometry (DEXA) derived percentage of body fat. Results: Inter-observer agreement was excellent for epicardial fat volume (ICC 0.75) and area (ICC 0.95) and good for epicardial fat thickness (ICC near the left anterior descending artery (LAD) 0.64, ICC near right coronary artery (RCA) 0.64). Intra-observer agreement was excellent for epicardial fat volume (ICC 0.97), area (ICC 0.99), thickness at LAD (ICC 0.71) and good for epicardial fat thickness at RCA (ICC 0.68). Epicardial fat volume had a better correlation to total body fat (r = 0.28, p < 0.001) and trunk fat (r = 0.37, p < 0.001), in comparison to other epicardial fat indices. Conclusion: Assessment of epicardial fat volume is highly reproducible in both HIV-infected and non-HIV-infected patients and shows a superior correlation with DEXA-based body and trunk fat measurements. Epicardial fat volume should be considered over other CT assessment methods when quantifying epicardial fat in HIV patients.

ABSTRACT In the absence of effective vaccines and with limited therapeutic options, convalescent plasma is being collected across the globe for potential transfusion to coronavirus disease 2019 (COVID-19) patients. The therapy has been deemed safe, and several clinical trials assessing its efficacy are ongoing. While it remains to be formally proven, the presence of neutralizing antibodies is thought to play a positive role in the efficacy of this treatment. Indeed, neutralizing titers of ≥1:160 have been recommended in some convalescent plasma trials for inclusion. Here, we performed repeated analyses at 1-month intervals on 31 convalescent individuals to evaluate how the humoral responses against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike glycoprotein, including neutralization, evolve over time. We observed that the levels of receptor-binding-domain (RBD)-specific IgG and IgA slightly decreased between 6 and 10 weeks after the onset of symptoms but that RBD-specific IgM levels decreased much more abruptly. Similarly, we observed a significant decrease in the capacity of convalescent plasma to neutralize pseudoparticles bearing wild-type SARS-CoV-2 S or its D614G variant. If neutralization activity proves to be an important factor in the clinical efficacy of convalescent plasma transfer, our results suggest that plasma from convalescent donors should be recovered rapidly after resolution of symptoms. IMPORTANCE While waiting for an efficient vaccine to protect against SARS-CoV-2 infection, alternative approaches to treat or prevent acute COVID-19 are urgently needed. Transfusion of convalescent plasma to treat COVID-19 patients is currently being explored; neutralizing activity in convalescent plasma is thought to play a central role in the efficacy of this treatment. Here, we observed that plasma neutralization activity decreased a few weeks after the onset of the symptoms. If neutralizing activity is required for the efficacy of convalescent plasma transfer, our results suggest that convalescent plasma should be recovered rapidly after the donor recovers from active infection.

Antibody dependent (AD) functions such as AD cellular cytotoxicity (ADCC) were associated with lower viral load (VL) in untreated HIV progressors and protection from HIV infection in the modestly protective RV144 HIV vaccine trial. Target cells used to measure ADCC, AD complement deposition (ADCD), and AD cellular trogocytosis (ADCT) have been either HIV envelope (Env) gp120-coated CEM.NKr.CCR5 cells or HIV infected cell cultures. In HIV infected cell cultures, uninfected bystander cells take up gp120 shed from infected cells. Both gp120-coated and gp120+ bystander cells expose CD4 induced (CD4i) epitopes, which are normally hidden in native trimeric Env expressed by genuinely HIV infected cells since Nef and Vpu downmodulate cell surface CD4. Antibody dependent assays using either of these target cells probe for CD4i Abs that are abundant in HIV+ plasma but that do not recognize HIV-infected cells. Here, we examined ADCC, ADCD, and ADCT functions using a target cell line, sorted HIV-infected cell line cells, whose HIV infection frequency nears 100% and that expresses HIV Env in a native trimeric closed conformation. Using sorted HIV-infected cells (siCEM) as targets, we probed the binding and AD functions of anti-gp120/Env Abs in plasma from HIV-infected untreated progressor (UTP, n = 18) and treated (TP, n = 24) subjects, compared to that in Elite controllers (EC, n = 37) and Viral Controllers (VC, n = 16), which are rare subsets of HIV-infected individuals who maintain undetectable or low VL, respectively, without treatment. Gp120-coated beads were used to measure AD cellular phagocytosis. Equivalent concentrations of input IgG in plasma from UTPs, ECs, and VCs supported higher levels of all AD functions tested than plasma from TPs. When AD activities were normalized to the concentration of anti-gp120/Env-specific Abs, between-group differences largely disappeared. This finding suggests that the anti-gp120/Env Abs concentrations and not their potency determined AD functional levels in these assays. Elite controllers did differ from the other groups by having AD functions that were highly polyfunctional and highly correlated with each other. PCR measurement of HIV reservoir size showed that ADCC activity was higher in ECs and VCs with a reservoir size below the limit of detection compared to those having a measurable HIV reservoir size.

ABSTRACT Measuring Envelope (Env)-specific antibody (Ab)-dependent cellular cytotoxicity (ADCC)-competent Abs in HIV+ plasma is challenging because Env displays distinctive epitopes when present in a native closed trimeric conformation on infected cells or in a CD4-bound conformation on uninfected bystander cells. We developed an ADCC model which distinguishes Env-specific ADCC-competent Abs based on their capacity to eliminate infected, bystander, or Env rgp120-coated cells as a surrogate for shed gp120 on bystander cells. A panel of monoclonal Abs (MAbs), used to opsonize these target cells, showed that infected cells were preferentially recognized/eliminated by MAbs to CD4 binding site, V3 loop, and viral spike epitopes whereas bystander/coated cells were preferentially recognized/eliminated by Abs to CD4-induced (CD4i) epitopes. In HIV-positive (HIV+) plasma, Env-specific Abs recognized and supported ADCC of infected cells, though a majority were directed toward CD4i epitopes on bystander cells. For ADCC activity to be effective in HIV control, ADCC-competent Abs need to target genuinely infected cells. IMPORTANCE HIV Env-specific nonneutralizing Abs (NnAbs) able to mediate ADCC have been implicated in protection from HIV infection. However, Env-specific NnAbs have the capacity to support ADCC of both HIV-infected and HIV-uninfected bystander cells, potentially leading to misinterpretations when the assay used to measure ADCC does not distinguish between the two target cell types present in HIV cultures. Using a novel ADCC assay, which simultaneously quantifies the killing activity of Env-specific Abs on both infected and uninfected bystander cells, we observed that only a minority of Env-specific Abs in HIV+ plasma mediated ADCC of genuinely HIV-infected cells displaying Env in its native closed conformation. This assay can be used for the development of vaccine strategies aimed at eliciting Env-specific Ab responses capable of controlling HIV infection.

3D-printed alternatives to standard flocked swabs were rapidly developed to provide a response to the unprecedented and sudden need for an exponentially growing amount of diagnostic tools to fight the COVID-19 pandemic. In light of the anticipated shortage, a hospital-based 3D-printing platform was implemented in our institution for the production of swabs for nasopharyngeal and oropharyngeal sampling based on the freely available, open-source design provided to the community by University of South Florida’s Health Radiology and Northwell Health System teams as a replacement for locally used commercial swabs. Validation of our 3D-printed swabs was performed with a head-to-head diagnostic accuracy study of the 3D-printed “Northwell model” with the cobas PCR Media® swab sample kit. We observed an excellent concordance (total agreement 96.8%, Kappa 0.936) in results obtained with the 3D-printed and flocked swabs, indicating that the in-house 3D-printed swab could be used reliably in the context of a shortage of flocked swabs. To our knowledge, this is the first study to report on autonomous hospital-based production and clinical validation of 3D-printed swabs.

Background: CXCL13 is preferentially secreted by Follicular Helper T cells (TFH) to attract B cells to germinal centers. Plasma levels of CXCL13 have been reported to be elevated during chronic HIV-infection, however there is limited data on such elevation during early phases of infection and on the effect of ART. Moreover, the contribution of CXCL13 to disease progression and systemic immune activation have been partially defined. Herein, we assessed the relationship between plasma levels of CXCL13 and systemic immune activation.Methods: Study samples were collected in 114 people living with HIV (PLWH) who were in early (EHI) or chronic (CHI) HIV infection and 35 elite controllers (EC) compared to 17 uninfected controls (UC). A subgroup of 11 EHI who initiated ART and 14 who did not were followed prospectively. Plasma levels of CXCL13 were correlated with CD4 T cell count, CD4/CD8 ratio, plasma viral load (VL), markers of microbial translocation [LPS, sCD14, and (1→3)-β-D-Glucan], markers of B cell activation (total IgG, IgM, IgA, and IgG1-4), and inflammatory/activation markers like IL-6, IL-8, IL-1β, TNF-α, IDO-1 activity, and frequency of CD38+HLA-DR+ T cells on CD4+ and CD8+ T cells.Results: Plasma levels of CXCL13 were elevated in EHI (127.9 ± 64.9 pg/mL) and CHI (229.4 ± 28.5 pg/mL) compared to EC (71.3 ± 20.11 pg/mL), and UC (33.4 ± 14.9 pg/mL). Longitudinal analysis demonstrated that CXCL13 remains significantly elevated after 14 months without ART (p < 0.001) and was reduced without normalization after 24 months on ART (p = 0.002). Correlations were observed with VL, CD4 T cell count, CD4/CD8 ratio, LPS, sCD14, (1→3)-β-D-Glucan, total IgG, TNF-α, Kynurenine/Tryptophan ratio, and frequency of CD38+HLA-DR+ CD4 and CD8 T cells. In addition, CMV+ PLWH presented with higher levels of plasma CXCL13 than CMV- PLWH (p = 0.005).Conclusion: Plasma CXCL13 levels increased with HIV disease progression. Early initiation of ART reduces plasma CXCL13 and B cell activation without normalization. CXCL13 represents a novel marker of systemic immune activation during early and chronic HIV infection and may be used to predict the development of non-AIDS events.

Extracellular vesicles (EVs) and their contents (proteins, lipids, messenger RNA, microRNA, and DNA) are viewed as intercellular signals, cell-transforming agents, and shelters for viruses that allow both diagnostic and therapeutic interventions. EVs circulating in the blood of individuals infected with human immunodeficiency virus (HIV-1) may provide insights into pathogenesis, inflammation, and disease progression. However, distinguishing plasma membrane EVs from exosomes, exomeres, apoptotic bodies, virions, and contaminating proteins remains challenging. We aimed at comparing sucrose and iodixanol density and velocity gradients along with commercial kits as a means of separating EVs from HIV particles and contaminating protein like calprotectin; and thereby evaluating the suitability of current plasma EVs analysis techniques for identifying new biomarkers of HIV-1 immune activation. Multiple analysis have been performed on HIV-1 infected cell lines, plasma from HIV-1 patients, or plasma from HIV-negative individuals spiked with HIV-1. Commercial kits, the differential centrifugation and density or velocity gradients to precipitate and separate HIV, EVs, and proteins such as calprotectin, have been used. EVs, virions, and contaminating proteins were characterized using Western blot, ELISA, RT-PCR, hydrodynamic size measurement, and enzymatic assay. Conversely to iodixanol density or velocity gradient, protein and virions co-sedimented in the same fractions of the sucrose density gradient than AChE-positive EVs. Iodixanol velocity gradient provided the optimal separation of EVs from viruses and free proteins in culture supernatants and plasma samples from a person living with HIV (PLWH) or a control and revealed a new population of large EVs enriched in microRNA miR-155 and mitochondrial DNA. Although EVs and their contents provide helpful information about several key events in HIV-1 pathogenesis, their purification and extensive characterization by velocity gradient must be investigated thoroughly before further use as biomarkers. By revealing a new population of EVs enriched in miR-155 and mitochondrial DNA, this study paves a way to increase our understanding of HIV-1 pathogenesis.

Human immunodeficiency virus type 1 (HIV-1) has evolved a sophisticated strategy to conceal conserved epitopes of its envelope glycoproteins (Env) recognized by antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies. These antibodies, which are present in the sera of most HIV-1-infected individuals, preferentially recognize Env in its CD4-bound conformation. Accordingly, recent studies showed that small CD4-mimetics (CD4mc) able to “push” Env into this conformation sensitize HIV-1-infected cells to ADCC mediated by HIV+ sera. Here we test whether CD4mc also expose epitopes recognized by anti-cluster A monoclonal antibodies such as A32, thought to be responsible for the majority of ADCC activity present in HIV+ sera and linked to decreased HIV-1 transmission in the RV144 trial. We made the surprising observation that CD4mc are unable to enhance recognition of HIV-1-infected cells by this family of antibodies in the absence of antibodies such as 17b, which binds a highly conserved CD4-induced epitope overlapping the co-receptor binding site (CoRBS). Our results indicate that CD4mc initially open the trimeric Env enough to allow the binding of CoRBS antibodies but not anti-cluster A antibodies. CoRBS antibody binding further opens the trimeric Env, allowing anti-cluster A antibody interaction and sensitization of infected cells to ADCC. Therefore, ADCC responses mediated by cluster A antibodies in HIV-positive sera involve a sequential opening of the Env trimer on the surface of HIV-1-infected cells. The understanding of the conformational changes required to expose these vulnerable Env epitopes might be important in the design of new strategies aimed at fighting HIV-1.

Elite controllers (ECs) are a rare subset of HIV-1 slow progressors characterized by prolonged viremia suppression. HLA alleles B27 and B57 promote the cytotoxic T lymphocyte (CTL)-mediated depletion of infected cells in ECs, leading to the emergence of escape mutations in the viral capsid (CA). Whether those mutations modulate CA detection by innate sensors and effectors is poorly known. Here, we investigated the targeting of CA from B27/B57+ individuals by cytosolic antiviral factors Mx2 and TRIM5α. Toward that aim, we constructed chimeric HIV-1 vectors using CA isolated from B27/B57+ or control subjects. HIV-1 vectors containing B27/B57+-specific CA had increased sensitivity to TRIM5α but not to Mx2. Following exposure to those vectors, cells showed increased resistance against both TRIM5α-sensitive and -insensitive HIV-1 strains. Induction of the antiviral state did not require productive infection by the TRIM5α-sensitive virus, as shown using chemically inactivated virions. Depletion experiments revealed that TAK1 and Ubc13 were essential to the TRIM5α-dependent antiviral state. Accordingly, induction of the antiviral state was accompanied by the activation of NF-κB and AP-1 in THP-1 cells. Secretion of IFN-I was involved in the antiviral state in THP-1 cells, as shown using a receptor blocking antibody. This work identifies innate activation pathways that are likely to play a role in the natural resistance to HIV-1 progression in ECs.

BACKGROUND:Although reverse sequence algorithms (RSA) for syphilis screening are performing well, they still have to rely on treponemal confirmatory tests at least for sera reactive by enzyme immunoassay/chemiluminescence immunoassay (EIA/CIA) and unreactive by rapid plasma reagin (RPR). Quebec's laboratory network previously showed that 3.3% of EIA/CIA reactive and weakly-reactive RPR samples (RPR titer of 1 to 4) would have been misclassified as syphilis cases if a treponemal confirmatory test had not been performed. OBJECTIVES:To correlate the magnitude of signal-to-cutoff (S/CO) ratios of the 4 most used commercial first-line EIA/CIA kits in Quebec with syphilis confirmation results and establish a S/CO value above which treponemal confirmation would not be required. METHODS:Serum samples from previously undiagnosed individuals (n = 7 404) obtained between January 2014 and February 2017 that were reactive by EIA/CIA and either negative by RPR or reactive with a low titer (1 to 4) were included in the study. All samples were tested with Treponema pallidum particle agglutination (TP-PA) and, if negative or inconclusive, with a line immunoassay (LIA). Syphilis infection confirmation was defined by a reactive TP-PA or LIA. Logistic regression analysis was used to determine S/CO values (95% CI lower bound = 0.98) above which confirmation would not be required. The four kits studied were Architect TP, BioPlex IgG, Syphilis EIA II, and Trep-Sure. RESULTS:Of 2609 reactive EIA/CIA specimens tested for the determination of S/CO values, 1730 (66%) were confirmed as true syphilis cases. Confirmation rate was significantly higher in samples with low-titer positive RPR (92%) than with negative RPR samples (54%); p

Female sex workers (FSWs) continue to carry a heavy burden of sexually transmitted infections (STI). For prevention purposes, there is a need to identify most-at-risk subgroups among them. The objective of this longitudinal cohort study conducted at Dispensaire IST, Cotonou, Benin, was to assess Neisseria gonorrhoeae (NG) / Chlamydia trachomatis (CT) incidence and determinants; and HIV incidence among FSWs in presence of STI/HIV risk reduction activities. Overall, 319 adult FSWs were followed quarterly from September 2008 to March 2012. NG/CT were detected from endocervical swabs by Amplified DNA Assays employing Strand displacement amplification technology. HIV testing was done on capillary blood using two consecutive rapid diagnostic tests. Anderson-Gill proportional hazard models (HR) were used to determine factors independently associated with NG/CT incidence. The majority of FSWs were HIV-negative (188, 58.9%). There were 6 HIV seroconversions among these 188 HIV-negative women. HIV incidence (95% Confidence interval, CI) was 1.41 (0.28-2.54) seroconversions per 100 person-years at risk (PYAR): 6 events / 425.1 PYAR. Sixty-two out of 319 women experienced 83 new episodes of NG/CT for an overall incidence rate (95% CI) of 10.8 (8.17-13.88) events / 100 PYAR. From month-24 onwards, HIV-positive women (treated: HR (95%CI): 4.2 (1.60-10.77); untreated: HR (95%CI): 4.2 (1.59-11.49) were more likely to acquire NG/CT compared to HIV-negative FSWs. Longer duration in sex work (>2 years: HR; 95%CI: 0.4 (0.22-0.72)) was protective against NG/CT. Refusal by clients (55.8%) was the main reason for non-condom use. Enrolling women from one clinic (Dispensaire IST) may have impaired generalizability of the findings. New NG/CT/HIV infections were observed among FSWs notwithstanding ongoing prevention interventions. To eliminate HIV transmission among FSWs, STI/HIV control programs need to promote women's empowerment and address vulnerability to infection of HIV-positive FSWs.

Previously, we showed that Killer Immunoglobulin-like Receptor (KIR)3DS1 homozygotes (hmz) are more frequent in HIV exposed seronegative (HESN) than in recently HIV infected (HIV+) individuals. KIR3DS1 encodes an activating Natural Killer (NK) cell receptor (NKR). The link between KIR genotype and HIV outcomes likely arises from the function that NK cells acquire through expression of particular NKRs. An initial screen of 97 HESN and 123 HIV+ subjects for the frequency of KIR region gene carriage observed between-group differences for several telomeric KIR region loci. In a larger set of up to 106 HESN and 439 HIV+ individuals, more HESN than HIV+ subjects were KIR3DS1 homozygotes, lacked a full length KIR2DS4 gene and carried the telomeric group B KIR haplotype motif, TB01. TB01 is characterized by the presence of KIR3DS1, KIR2DL5A, KIR2DS3/5 and KIR2DS1, in linkage disequilibrium with each other. We assessed which of the TB01 encoded KIR gene products contributed to NK cell responsiveness by stimulating NK cells from 8 HIV seronegative KIR3DS1 and TB01 motif homozygotes with 721.221 HLA null cells and evaluating the frequency of KIR3DS1+/-KIR2DL5+/-, KIR3DS1+/-KIR2DS1+/-, KIR3DS1+/-KIR2DS5+/- NK cells secreting IFN-γ and/or expressing CD107a. A higher frequency of NK cells expressing, versus not, KIR3DS1 responded to 721.221 stimulation. KIR2DL5A+, KIR2DS1+ and KIR2DS5+ NK cells did not contribute to 721.221 responses or modulate those by KIR3DS1+ NK cells. Thus, of the TB01 KIR gene products, only KIR3DS1 conferred responsiveness to HLA-null stimulation, demonstrating its ligation can activate ex vivo NK cells.

Quantifying HIV Envelope (Env)-specific antibodies in HIV+ plasma is useful for interpreting antibody dependent cellular cytotoxicity assay results. HIV Env, the only viral protein expressed on the surface of infected cells, has a native trimeric closed conformation on cells infected with wild-type HIV. However, CD4+ uninfected bystander cells in HIV+ cell cultures bind gp120 shed from HIV+ cells exposing CD4-induced epitopes normally hidden in native Env. We used flow-cytometry based assays to quantify antibodies in HIV+ plasma specific for native trimeric Env or gp120/CD4 conjugates using CEM.NKr.CCR5 (CEM) cells infected with HIV (iCEM) or coated with recombinant gp120 (cCEM), as a surrogate for gp120+ HIV- bystander cells. Results from both assays were compared to those of a plate-based ELISA to monomeric gp120. The levels of Env-specific antibodies to cCEM and iCEM, measured by flow cytometry, and to gp120 by ELISA were positively correlated. More antibodies in HIV+ plasma recognized the gp120 conformation exposed on cCEM than on iCEM. Comparisons of plasma from untreated progressors, treated progressors, and elite controllers revealed that antibodies to Env epitopes were the lowest in treated progressors. Plasma from elite controllers and untreated progressors had similarly high levels of Env-specific antibodies, despite elite controllers having undetectable HIV viral loads, while untreated progressors maintained high viral loads.

HIV-1 tropism assays play a crucial role in determining the response to CCR5 receptor antagonists. Initially, phenotypic tests were used, but limited access to these tests prompted the development of alternative strategies. Recently, genotyping tropism has been validated using a Canadian technology in clinical trials investigating the use of maraviroc in both experienced and treatment-naive patients. The present guidelines review the evidence supporting the use of genotypic assays and provide recommendations regarding tropism testing in daily clinical management.

Necrotizing fasciitis is a serious disease characterized by the necrosis of the subcutaneous tissues and fascia. E. coli as the etiologic agent of necrotizing fasciitis is a rare occurrence. A 66-year-old woman underwent total abdominal hysterectomy with bilateral salpingo-oophorectomy. She rapidly developed necrotizing fasciitis which led to her death 68 hours following surgery. An E. coli strain was isolated from blood and fascia cultures. DNA microarray revealed the presence of 20 virulence genes.

This is the first Canadian case of Chikungunya virus (CHIKV) infection reported in a traveller returning from the Caribbean. Following multiple mosquito bites in Martinique Island in January 2014, the patient presented with high fever, headaches, arthralgia on both hands and feet, and a rash on the trunk upon his return to Canada. Initial serological testing for dengue virus infection was negative. Support therapy with nonsteroidal anti-inflammatory drugs was administered. The symptoms gradually improved 4 weeks after onset with residual arthralgia and morning joint stiffness. This clinical feature prompted the clinician to request CHIKV virus serology which was found to be positive for the presence of IgM and neutralizing antibodies. In 2014, over four hundred confirmed CHIKV infection cases were diagnosed in Canadian travellers returning from the Caribbean and Central America. Clinical suspicion of CHIKV or dengue virus infections should be considered in febrile patients with arthralgia returning from the recently CHIKV endemic countries of the Americas.

BACKGROUND:Accurate and practical biologic tools to estimate HIV incidence is crucial to better monitor the epidemic and evaluate the effectiveness of HIV prevention and treatment programs. METHODS:We evaluated two avidity assays to measure recent HIV infection: the Sedia HIV-1 LAg-Avidity EIA (Sedia Biosciences, Portland) and the Centers for Disease Control and Prevention (CDC)-modified Bio-Rad-Avidity assay (Bio-Rad Laboratories, Mississauga, ON). Longitudinal specimens (n = 473) obtained from 123 treatment-naive seroconverted individuals enrolled in the Primary HIV-1 Infection (PHI) cohort of Quebec were used to determine the average time an individual is considered to be recently infected (mean duration of recent infection; MDRI), for the two avidity assays alone and in combination using a nonparametric survival method analysis. A total of 420 specimens from individuals with established HIV infection (90 individuals from the PHI cohort of Quebec and 330 individuals from the Laboratoire de santé publique du Quebec (LSPQ) serobank) were also tested to investigate false recency rate (FRR). RESULTS:The CDC-modified Bio-Rad-Avidity gave an estimated MDRI of 234 days (95% CI 220-249) at the avidity index cutoff of 30% while the Sedia-LAg-Avidity assay gave an estimated MDRI of 120 days (95% CI 109-132) at the normalized optical density (ODn) cutoff of 1.5. The FRR among individuals with established HIV infection was 10.2% (7.5%-13.5%) with the CDC-modified Bio-Rad-Avidity assay as compared to 6.0% (3.9%-8.7%) with the Sedia-LAg-Avidity assay. When optimizing a multiassay algorithm (MAA) that includes sequentially the CDC-modified Bio-Rad-Avidity assay then the Sedia-LAg-Avidity assay EIA (avidity index/ODn: 30%/1.7), the MDRI was 136 days (95% CI 123-148) and the FRR, 3.3% (95% CI 1.8-5.6). CONCLUSION:Multiassay algorithms that include the CDC-modified Bio-Rad-Avidity assay and the Sedia-LAg-Avidity assay performed better than each avidity assay alone. Such 2-assay algorithm that starts with the CDC-modified Bio-Rad-Avidity assay followed by the Sedia-LAg-Avidity assay allowed a better classification of HIV-1 infections.

Legionella is found in natural and man-made aquatic environments, such as cooling towers and hot water plumbing infrastructures. Legionella pneumophila serogroup 1 (Lp1) is the most common etiological agent causing waterborne disease in the United States and Canada. This study reports the molecular characterization of Lp strains during a 10 year period. We conducted sequence-based typing (SBT) analysis on a large set of Lp isolates (n = 284) to investigate the province of Quebec sequence types (STs) distribution in order to identify dominant clusters. From 2005 to 2015, 181 clinical Lp isolates were typed by SBT (141 sporadic cases and 40 outbreak related cases). From the same period of time, 103 environmental isolates were also typed. Amongst the 108 sporadic cases of Lp1 typed, ST-62 was the most frequent (16.6%), followed by ST-213 (10.2%), ST-1 (8.3%) and ST-37 (8.3%). Amongst other serogroups (SG), ST-1327 (SG5) (27.3%) and ST-378 (SG10) (12.2%) were the most frequent. From the environmental isolates, ST-1 represent the more frequent SBT type (26.5%). Unweighted pair group method with arithmetic mean (UPGMA) dendrogram from the 108 sporadic cases of SG1 contains 4 major clusters (A to D) of related STs. Cluster B contains the majority of the strains (n = 61) and the three most frequent STs in our database (ST-62, ST-213 and ST-1). During the study period, we observed an important increase in the incidence rate in Quebec. All the community associated outbreaks, potentially or confirmed to be associated with a cooling tower were caused by Lp1 strains, by opposition to hospital associated outbreaks that were caused by serogroups of Lp other than SG1. The recent major Quebec City outbreak caused by ST-62, and the fact that this genotype is the most common in the province supports whole genome sequencing characterization of this particular sequence type in order to understand its evolution and associated virulence factors.

The management and treatment of HIV and hepatitis B virus (HBV)-coinfected patients present specific challenges for clinicians. The morbidity and mortality related to these concomitant infections are growing concerns, while the use of antiviral drugs effective against both viruses complicates therapeutic decision making. The present document provides guidelines for physicians regarding care and treatment of patients coinfected with HIV and HBV. Primary prevention of HBV in HIV-positive patients is achieved through appropriate vaccination schedules. Follow-up before treatment of HBV may include liver biopsy, screening for hepatocellular carcinoma and testing for esophageal varicies in cases of cirrhosis. In HBV-infected patients requiring treatment, recommendations regarding initiation, duration and choice of first-line drugs are made. Finally, in the case of resistance, appropriate alternative therapies are necessary.

BACKGROUND AND OBJECTIVES: A Canadian group, consisting of six physicians and an HIV researcher with significant experience and knowledge in HIV management, reviewed the available data and developed guidelines for Canadian health care providers (who treat HIV infection) on the appropriate use of maraviroc (UK-427,857) in HIV-infected adults.