Characterization of the Genital Mucosa Immune Profile to Distinguish Phases of the Menstrual Cycle: Implications for HIV Susceptibility.

BACKGROUND: Inflammation and immune activation are key factors in sexual transmission of human immunodeficiency virus (HIV). We sought to define the impact of hormonal cycling on the mucosal immune environment and HIV risk in sex workers with a natural menstrual cycle. METHODS: We compared soluble mucosal immune factors and cervical mononuclear cells during hormone titer-defined phases of the menstrual cycle among 37 sex workers from Nairobi, Kenya. Systemic and mucosal samples were collected 14 days apart to distinguish the follicular and luteal phases of the menstrual cycle, and phases were confirmed by hormone measurements. Vaginal concentrations of 19 immune modulators and cervical T-cell activation markers were measured. RESULTS: The follicular phase signature was characterized by an elevated CCL2 level, decreased interleukin 1α and interleukin 1ß cervical concentrations, and a significant increase in the proportion of CD4+ T cells that expressed CD69. The genital concentration of CCL2 was the best marker to distinguish the follicular from the luteal phase in univariate and multivariate analyses and remained independent of elevated genital inflammation and bacterial vaginosis. CONCLUSION: The follicular phase of the menstrual cycle was associated with an elevated CCL2 level and retention of resident memory CD4+ T cells, which has implications for increased susceptibility to HIV infection.

Low-Dose Acetylsalicylic Acid Reduces T Cell Immune Activation: Potential Implications for HIV Prevention.

Introduction: Acetylsalicylic acid (ASA) is a well-known and safe anti-inflammatory. At low-dose, it is prescribed to prevent secondary cardiovascular events in those with pre-existing conditions and to prevent preeclampsia. Little is known about how low-dose ASA affects the immune response. In this study, we followed women to assess how ASA use modifies T cells immune phenotypes in the blood and at the genital tract. Methods: HIV uninfected women from Kenya were enrolled in this study and followed for one month to assess baseline responses including systemic/mucosal baseline immune activation. Participants then received 81mg of ASA daily for 6 weeks to assess changes to T cell immune activation (systemic and mucosal) relative to baseline levels. Results: The concentration of ASA measured in the blood was 58% higher than the level measured at the female genital tract. In the blood, the level of ASA was inversely correlated with the following: the proportion of Th17 expressing HLA-DR (p=0.04), the proportion of effector CD4+ T cells expressing CCR5 (p=0.03) and the proportion of CD8+Tc17 expressing CCR5 (p=0.04). At the genital tract, ASA use correlated with a decreased of activated CD4+T cells [CD4+CCR5+CD161+ (p=0.02) and CD4+CCR5+CD95+ (p=0.001)]. Conclusion: This study shows that ASA use impacts the immune response in both the systemic and genital tract compartments. This could have major implications for the prevention of infectious diseases such as HIV, in which the virus targets activated T cells to establish an infection. This could inform guidelines on ASA use in women. Clinical Trial Registration: ClinicalTrials.gov, identifier NCT02079077.

HIV-Exposed Seronegative Sex Workers Express Low T-Cell Activation and an Intact Ectocervical Tissue Microenvironment.

Immunological correlates of natural resistance to HIV have been identified in HIV-exposed seronegative (HESN) individuals and include a low-inflammatory genital mucosal status. The cervicovaginal epithelium has not been studied for such correlates despite constituting an important barrier against sexual HIV transmission. To fill this gap in knowledge, we collected samples of blood, cervical mononuclear cells, cervicovaginal lavage, and ectocervical tissue from Kenyan HESN sex workers (n = 29) and controls (n = 33). The samples were analyzed by flow cytometry, protein profiling, 16S rRNA gene sequencing, in situ image analysis, and tissue-based RNA sequencing. A significantly higher relative proportion of regulatory T cells in blood (B7+CD25hiFoxP3+CD127loCD4+ and B7+Helios+FoxP3+CD4+), and a significantly lower proportion of activated cervical T cells (CCR5+CD69+CD4+ and CCR5+CD69+CD8+), were found in the HESN group compared with the controls. In contrast, there were no statistically significant differences between the study groups in cervicovaginal protein and microbiome compositions, ectocervical epithelial thickness, E-cadherin expression, HIV receptor expression, and tissue RNA transcriptional profiles. The identification of an intact ectocervical microenvironment in HESN individuals add new data to current knowledge about natural resistance to sexual transmission of HIV.

A living mapping review for COVID-19 funded research projects: final (27 month) update.

BACKGROUND: The coronavirus disease 2019 (COVID-19) has resulted in an unprecedented research response, demonstrating exceptional examples of rapid research and collaboration. There has however been an ongoing need for greater coordination, with limited resources for research and the shifting global pandemic. METHODS: The UK Collaborative on Development Research (UKCDR) and Global Research Collaboration for Infectious Disease Preparedness (GloPID-R), two funder coordination groups have collaborated to develop a live database of funded research projects across the world relating to COVID-19. Drawing data continually from their members and further global funding bodies, as of 15th October 2022 the database contains 20,006 projects, funded by 351 funders, taking place across 157 countries representing an investment of at least $7.4 billion. To our knowledge it is one of the most comprehensive databases. The database is aligned to the World Health Organisation and GloPID-R Global Research Roadmap: 2019 Novel Coronavirus and the UN Research Roadmap for the COVID-19 Recovery. It is being used by the WHO, governments and further policy makers, research funders and researchers. This living mapping review aims to supplement the database by providing an open, accessible, and frequently updated resource summarising the characteristics of the COVID-19 funded research portfolio. Both descriptive and thematic analyses are presented and updated frequently to aid interpretation of the global COVID-19 funded research portfolio. RESULTS: In this final version ten analysis, we provide an updated detailed descriptive analysis of the database (on data from three months after version nine) and focus our thematic analysis on research gaps, research areas in need of coordination, study populations, and research locations (with a focus on resource-limited countries). CONCLUSIONS: As the global research response to COVID-19 plateaus, this living mapping review has helped both funders and researchers to prioritise resources and review investments.

Looking for Evidence of Public Health's Role for Long-Term Evacuees.

Many Canadians have had personal experience of a major emergency or disaster at some point in their lifetime and close to a third of those affected were evacuated from their homes or communities. Most evacuations have lasted less than 2 weeks, but in some instances, people have been displaced for months or years. For example, hundreds of residents evacuated following flooding in Lake St. Martin, Manitoba in 2011, remain displaced today. In order to learn more about the roles and responses of public health for long-term evacuees (LTEs) in Canada, we conducted a narrative review of published English-language documents, beginning with literature specific to Canada and then expanding to include literature on other high-income countries. We found that while researchers have explored public health considerations in emergency preparedness, acute disaster management, and resettlement in these contexts there is a dearth of published evidence regarding the public health implications of prolonged evacuation and the public health responses to long-term evacuation in Canada and in other high-income countries. Because the public health needs of diverse populations of LTEs have not been fully investigated, it is likely that they are neither well-understood nor adequately addressed in public health policy and practice.

Increased Cervical CD4+CCR5+ T Cells Among Kenyan Sex Working Women Using Depot Medroxyprogesterone Acetate.

Depot medroxyprogesterone acetate (DMPA) is the most common hormonal contraceptive used by women in sub-Saharan Africa, however, it has been epidemiologically associated with HIV infections. To assess whether DMPA has an effect on the number and activation of HIV target cells, this study assessed the levels and phenotype of blood- and mucosal-derived HIV target cells among women using DMPA. Thirty-five HIV uninfected women from the Pumwani Sex Worker cohort from Nairobi, Kenya were enrolled in the study (15 using DMPA and 20 not using hormonal contraception). Blood (plasma and peripheral blood mononuclear cells) and cervicovaginal (lavage, cervical cells, and ectocervical biopsies) samples were collected. Cellular phenotype and activation status were determined by flow cytometry, cytokine levels were assessed by bead array and image analysis assessed cell number and phenotype in situ. In blood, the proportion of HIV target cells and activated T cells was lower in DMPA users versus those not using hormonal contraceptives. However, analysis of cervical mononuclear cells showed that DMPA users had elevated levels of activated T cells (CD4+CD69+) and expressed lower levels of the HIV co-receptor CCR5 on a per cell basis, while tissue samples showed that in the ectocervix, DMPA users had a higher proportion of CD4+CCR5+ T cells. This study demonstrates that DMPA users had higher levels of activated T cells and HIV target cells in the genital tract. The increased pool of mucosal HIV target cells provides new biological information about the potential impact of DMPA on HIV susceptibility.

Using safe, affordable and accessible non-steroidal anti-inflammatory drugs to reduce the number of HIV target cells in the blood and at the female genital tract.

INTRODUCTION: At its basic level, HIV infection requires a replication-competent virus and a susceptible target cell. Elevated levels of vaginal inflammation has been associated with the increased risk of HIV infection as it brings highly activated HIV target cells (CCR5+CD4+ T cells; CCR5+CD4+CD161+ Th17 T cells) to the female genital tract (FGT) where they interact with HIV. Decreased HIV risk has been associated with a phenotype of decreased immune activation, called immune quiescence, described among Kenyan female sex workers who were intensely exposed to HIV yet remain uninfected. Current prevention approaches focus on limiting viral access. We took the novel HIV prevention approach of trying to limit the number of HIV target cells in the genital tract by reducing inflammation using safe, affordable and globally accessible anti-inflammatory drugs. METHODS: We hypothesized that the daily administration of low doses of acetylsalicylic acid (ASA 81 mg) or hydroxychloroquine (HCQ 200 mg) would reduce inflammation thereby decreasing HIV target cells at the FGT. Low-risk HIV seronegative women from Nairobi, Kenya were randomized for six weeks therapy of ASA (n = 37) or HCQ (n = 39) and tested to determine the impact on their systemic and mucosal immune environment. RESULTS: The results showed that HCQ use was associated with a significant reduction in the proportion of systemic T cells that were CCR5+CD4+ (p = 0.01) and Th17 (p = 0.01). In the ASA arm, there was a 35% and 28% decrease in the proportion of genital T cells that were CD4+CCR5+ (p = 0.017) and Th17 (p = 0.04) respectively. Proteomic analyses of the cervical lavage showed ASA use was associated with significantly reduced amount of proteins involved in the inflammatory response and cell recruitment at the mucosa, although none of the individual proteins passed multiple comparison correction. These changes were more apparent in women with Lactobacillus dominant microbiomes. CONCLUSION: Together, these data indicate that taking low-dose ASA daily was associated with significant reduction in HIV target cells at the FGT. This study provides proof-of-concept for a novel HIV-prevention approach that reducing inflammation using safe, affordable and globally accessible non-steroidal anti-inflammatory agents is associated with significant reduction in the proportion of HIV-target cells at the FGT.

DC-SIGN and DC-SIGNR genetic diversity among different ethnic populations: potential implications for pathogen recognition and disease susceptibility.

Dendritic cell-specific intracellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and DC-SIGNR are C-type lectins that serve both as cell adhesion and pathogen recognition receptors. Because of the essential role of the these molecules in the immune response, the implication of their alleles in human disease states, and the possible genetic variation at these loci among ethnically diverse populations, we undertook a study to analyze the full extent of DC-SIGN and DC-SIGNR polymorphisms in Caucasian Canadian and indigenous African populations. We report several novel nucleotide variants within regulatory 5'- and 3'-untranslated regions of the genes that could affect their transcription and translation. There were significant differences in the distribution of DC-SIGN and DC-SIGNR alleles among African and non-African populations. Finally, our study clearly demonstrates that Africans show greater genetic diversity at these two closely-related immune loci than observed in other major population groups. The differences may reflect evolutionary pressures generated by environmental factors, such as prevalent pathogens in these geographically distinct regions. Further studies will be needed to determine the net impact of DC-SIGN and DC-SIGNR genetic variants on the expression, translation, and function of the proteins and to understand how these functional polymorphisms may affect immune responses or immune escape.

CD161 identifies polyfunctional Th1/Th17 cells in the genital mucosa that are depleted in HIV-infected female sex workers from Nairobi, Kenya.

CD161 identifies a subset of circulating Th17 cells that are depleted in the blood and gut of HIV-infected individuals. In the female reproductive tract (FRT), the pattern of CD161 expression on CD4+ cells remains unknown. Here, we characterized CD161 expression in the FRT of Kenyan female sex workers (FSW). Compared to the blood, CD161+CD4+ T cells were enriched in the FRT of uninfected FSWs. These cells were depleted in FRT of HIV-infected FSWs. Cervical CD161+ cells harboured an activated phenotype (CD69, CD95, HLA-DR) with elevated expression of tissue-homing markers (CCR6, ß7 integrin) and HIV co-receptor (CCR5). Mitogen-stimulated production of IL-17 confirmed the Th17 commitment of CD161+CD4+ T cells in the FRT with a predominance of polyfunctional Th1/Th17 cells. Here, we showed that the expression of CD161 on CD4+T cells is modulated at the FRT, but still identified a highly activated cellular subset, which differentiates into pro-inflammatory Th1/Th17 cells, expresses multiple HIV susceptibility markers and are depleted in HIV-infected individuals. The use of CD161 as a biomarker of HIV targets in the FRT reduces the need for functional assessment of cells and could have important implications in better understanding HIV pathogenesis and Th17 fate in the FRT of high-risk women.

The Impact of Sex Work Interruption on Blood-Derived T Cells in Sex Workers from Nairobi, Kenya.

BACKGROUND: Unprotected sexual intercourse exposes the female genital tract (FGT) to semen-derived antigens, which leads to a proinflammatory response. Studies have shown that this postcoital inflammatory response can lead to recruitment of activated T cells to the FGT, thereby increasing risk of HIV infection. OBJECTIVE: The purpose of this study was to evaluate the impact of sex work on activation and memory phenotypes of peripheral T cells among female sex workers (FSW) from Nairobi, Kenya. SUBJECTS: Thirty FSW were recruited from the Pumwani Sex Workers Cohort, 10 in each of the following groups: HIV-exposed seronegative (at least 7 years in active sex work), HIV positive, and New Negative (HIV negative, less than 3 years in active sex work). Blood was obtained at three different phases (active sex work, abstinence from sex work-sex break, and following resumption of sex work). Peripheral blood mononuclear cells were isolated and stained for phenotypic markers (CD3, CD4, CD8, and CD161), memory phenotype markers (CD45RA and CCR7), activation markers (CD69, HLA-DR, and CD95), and the HIV coreceptor (CCR5). T-cell populations were compared between groups. RESULTS: In HIV-positive women, CD8+CCR5+ T cells declined at the sex break period, while CD4+CD161+ T cells increased when returning to sex work. All groups showed no significant changes in systemic T-cell activation markers following the interruption of sex work, however, significant reductions in naive CD8+ T cells were noted. For each of the study points, HIV positives had higher effector memory and CD8+CD95+ T cells and lower naive CD8+ T cells than the HIV-uninfected groups. CONCLUSIONS: Interruption of sex work had subtle effects on systemic T-cell memory phenotypes.

Improving Adherence to Post-Cervical Biopsy Sexual Abstinence in Kenyan Female Sex Workers.

PROBLEM: Cervical biopsies offer a unique opportunity for studying local immune response. To investigate hormonally induced immune fluctuations in cervical tissues of Kenyan female sex workers, we improved biopsy sampling protocol safety. Here, we report on steps taken to minimize exposure to HIV following two cervical biopsies. METHODS OF STUDY: Women were asked to abstain from vaginal intercourse to limit HIV exposure during wound healing with financial compensation. A comprehension tool for informed consent, on-site detection of prostate-specific antigens indicating unprotected intercourse within 48 hr, and bi-weekly text message reminders were implemented. RESULTS: The implemented methods improved compliance with post-procedure abstinence by two times (P = 0.013). Fourteen days following a cervical biopsy, no sign of genital inflammation or change in HIV T-cell target proportion were observed. CONCLUSIONS: This study provides new tools for limiting HIV exposure in studies requiring biopsy sampling among women at risk of acquiring HIV.

Collection, isolation, and flow cytometric analysis of human endocervical samples.

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

Naturally-occurring genetic variants in human DC-SIGN increase HIV-1 capture, cell-transfer and risk of mother-to-child transmission.

BACKGROUND: Mother-to-child transmission (MTCT) is the main cause of HIV-1 infection in children worldwide. Dendritic cell-specific ICAM-3 grabbing-nonintegrin (DC-SIGN, also known as CD209) is an HIV-1 receptor that enhances its transmission to T cells and is expressed on placental macrophages. METHODS AND FINDINGS: We have investigated the association between DC-SIGN genetic variants and risk of MTCT of HIV-1 among Zimbabwean infants and characterized the impact of the associated mutations on DC-SIGN expression and interaction with HIV-1. DC-SIGN promoter (p-336C and p-201A) and exon 4 (198Q and 242V) variants were all significantly associated with increased risk of intrauterine (IU) HIV-1 infection. Promoter variants decreased DC-SIGN expression both in vitro and in placental CD163(+) macrophages (Hofbauer cells) of HIV-1 unexposed infants but not of HIV-1 exposed infants. The exon 4 protein-modifying mutations increased HIV-1 capture and transmission to T cells in vitro. CONCLUSION: This study provides compelling evidence to support an important role of DC-SIGN in IU HIV-1 infection.

Functional genetic variants in DC-SIGNR are associated with mother-to-child transmission of HIV-1.

BACKGROUND: Mother-to-child transmission (MTCT) is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related (DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin (L-SIGN)), can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes (H1-H1, H1-H3, H3-H3) had a 3.6-fold increased risk of in utero (IU) (P = 0.013) HIV-1 infection and a 5.7-fold increased risk of intrapartum (IP) (P = 0.025) HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms (SNPs) in promoter region (p-198A) and intron 2 (int2-180A) that were associated with increased risk of both IU (P = 0.045 and P = 0.003, respectively) and IP (P = 0.025, for int2-180A) HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers (P = 0.011). CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission.

Molecular characteristics of pbp1a and pbp2b in clinical Streptococcus pneumoniae isolates in Quebec, Canada.

OBJECTIVES: To investigate the nature of the amino acid motifs found in penicillin-binding protein (PBP) 2b and PBP1a of penicillin-resistant Streptococcus pneumoniae isolates across Quebec (Canada), and to obtain preliminary information regarding the prevalence of these alterations. METHODS: DNA sequences of pbp2b (codons 210-675) and pbp1a (codons 310-682) transpeptidase domains were determined and compared in 48 clinical isolates comprising 17 penicillin-susceptible (PSSP), 19 penicillin-intermediate (PISP) and 12 penicillin-resistant (PRSP) pneumococci. RESULTS: The degree of diversity within PBP1a and PBP2b correlated with increased resistance to beta-lactam antibiotics. There were an average of 0.6 +/- 0.4 and 2.9 +/- 0.2 mutations in PSSP, 16.8 +/- 1.4 and 36.3 +/- 5.2 in PISP, and 18.7 +/- 2.5 and 51.4 +/- 1.3 in PRSP isolates compared with control penicillin-susceptible R6-PBP2b and R6-PBP1a sequences, respectively. At least seven PBP2b and six PBP1a distinct amino acid profiles were identified among intermediate or resistant strains isolated in Quebec. The pattern of distribution of the PBPs' altered amino acids differs from that of other countries, with pneumococci isolates from Quebec showing a unique genetic signature. CONCLUSION: This study will serve as a basis for future monitoring of genetic changes associated with the emergence and spread of beta-lactam resistance in Quebec, Canada.

Genetic analysis of pbp2x in clinical Streptococcus pneumoniae isolates in Quebec, Canada.

OBJECTIVES: To investigate the nature of the amino acid motifs found in penicillin-binding protein (PBP) 2x of penicillin-resistant Streptococcus pneumoniae isolates across the province of Quebec (Canada), and to obtain preliminary information regarding the prevalence of these alterations. METHODS: The pbp2x genomic region encompassing codons 178-703, which includes the entire region of the transpeptidase domain, was sequenced and compared for 52 clinical isolates comprising 20 penicillin-susceptible (PSSP), 20 penicillin-intermediate (PISP) and 12 penicillin-resistant (PRSP) pneumococci. RESULTS: The degree of diversity within PBP2x correlated with increased resistance to beta-lactam antibiotics. There were an average of 5.0 +/- 1.8 mutations in PSSP, 37.9 +/- 4.4 in PISP, and 63.0 +/- 2.0 in PRSP isolates when compared with the control penicillin-susceptible strain R6. At least six distinct amino acid profiles were identified among PISP strains isolated in Quebec. In contrast, all PRSP isolates shared a similar pattern of altered amino acids compared with the sequence from susceptible strains. CONCLUSIONS: These data will be useful in future studies to monitor the genetic changes associated with the emergence and spread of beta-lactam resistance in Quebec.