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Background Sex differences in human immunodeficiency virus (HIV) reservoir dynamics remain underexplored. Methods Longitudinal samples from virally suppressed midlife women (n = 59, median age 45 years) and age-matched men (n = 31) were analyzed retrospectively. At each time point, we measured sex hormones (by means of enzyme-linked immunosorbent assay) and cellular HIV DNA and RNA (by means of digital droplet polymerase chain reaction). Number of inducible HIV RNA+ cells, which provides an upper estimate of the replication-competent reservoir, was quantified longitudinally in a different subset of 14 women, across well-defined reproductive stages. Mixed-effects models included normalized reservoir outcomes and sex, time since antiretroviral therapy (ART) initiation, and the sex-by-time interaction as predictors. Results At ART initiation, women and men had median (interquartile range [IQR]) CD4+ T-cell counts of 204/μL (83–306/μL) versus 238/μL (120–284/μL), respectively; median ages of 45 (42–48) versus 47 (43–51) years; and median follow-up times of 79.2/μL (60.5–121.1/μL) versus 66.2/μL (43.2–80.6/μL) months. We observed a significant decline of total HIV DNA over time in both men and women (P Conclusions In contrast to men, in whom the HIV reservoir steadily declines with aging, the HIV reservoir in women is more dynamic. Total HIV DNA (including intact and defective genomes) declines more slowly in women than in men, while the inducible HIV RNA+ reservoir, which is highly enriched in replication-competent virus, increases in women after menopause.

Background Long-term studies of vaccine recipients are necessary to understand severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody durability and assess the impact of booster doses on antibody levels and protection from infection. The identification of vaccine breakthrough infections among fully vaccinated populations will be important in understanding vaccine efficacy and SARS-CoV-2 vaccine escape capacity. Methods SARS-CoV-2 spike (S) receptor-binding domain and nucleocapsid (N) immunoglobulin (Ig) G levels were measured in a longitudinal study of 1000 Chicago healthcare workers who were infection naive or previously infected and then vaccinated. Changes in S and N IgG were followed up through 14 months, and vaccine breakthrough infections were identified by increasing levels of N IgG. Results SARS-CoV-2 S IgG antibody levels among previously infected and previously noninfected individuals decreased steadily for 11 months after vaccination. Administration of a booster 8–11 months after vaccination increased S IgG levels >2-fold beyond those observed after 2 doses, resulting in S IgG levels that were indistinguishable between previously infected and uninfected individuals. Increases in N IgG identified vaccine breakthrough infections and showed >15% breakthrough infection rates during the Omicron wave starting in December 2021. Conclusions These results demonstrate SARS-CoV-2 antibody changes after vaccination and breakthrough infections and identify high levels of vaccine breakthrough infections during the Omicron wave, based on N IgG increases.

To examine associations between frailty and drug, alcohol, and tobacco use among a large diverse cohort of people with HIV (PWH) in clinical care in the current era.PWH at 7 sites across the United States completed clinical assessments of patient-reported measures and outcomes between 2016 and 2019 as part of routine care including drug and alcohol use, smoking, and other domains. Frailty was assessed using 4 of the 5 components of the Fried frailty phenotype and PWH were categorized as not frail, pre-frail, or frail. Associations of substance use with frailty were assessed with multivariate Poisson regression.Among 9336 PWH, 43% were not frail, 44% were prefrail, and 13% were frail. Frailty was more prevalent among women, older PWH, and those reporting current use of drugs or cigarettes. Current methamphetamine use (1.26: 95% CI 1.07-1.48), current (1.65: 95% CI 1.39-1.97) and former (1.21:95% CI 1.06-1.36) illicit opioid use, and former cocaine/crack use (1.17: 95% CI 1.01-1.35) were associated with greater risk of being frail in adjusted analyses. Current smoking was associated with a 61% higher risk of being frail vs. not frail (1.61: 95% CI 1.41-1.85) in adjusted analyses.We found a high prevalence of prefrailty and frailty among a nationally distributed cohort of PWH in care. This study identified distinct risk factors that may be associated with frailty among PWH, many of which, such as cigarette smoking and drug use, are potentially modifiable.

BACKGROUND AND AIMS: Despite mechanistic data implicating unresolving inflammation in stroke pathogenesis, data regarding circulating immune cell phenotypes – key determinants of inflammation propagation versus resolution - and incident stroke are lacking. Therefore, we aimed to comprehensively define associations of circulating immune phenotypes and activation profiles with incident stroke. METHODS: We investigated circulating leukocyte phenotypes and activation profiles with incident adjudicated stroke in 2104 diverse adults from the Multi-Ethnic Study of Atherosclerosis (MESA) followed over a median of 16.6 years. Cryopreserved cells from the MESA baseline examination were thawed and myeloid and lymphoid lineage cell subsets were measured using polychromatic flow cytometry and intracellular cytokine activation staining. We analyzed multivariable-adjusted associations of cell phenotypes, as a proportion of parent cell subsets, with incident stroke (overall) and ischemic stroke using Cox regression models. RESULTS: We observed associations of intermediate monocytes, early-activated CD4(+) T cells, and both CD4(+) and CD8(+) T cells producing interleukin-4 after cytokine stimulation (T(h2) and T(c2), respectively) with higher risk for incident stroke; effect sizes ranged from 35% to 62% relative increases in risk for stroke. Meanwhile, differentiated and memory T cell phenotypes were associated with lower risk for incident stroke. In sex-stratified analyses, positive and negative associations were especially strong among men but null among women. CONCLUSIONS: Circulating IL-4 producing T cells and intermediate monocytes were significantly associated with incident stroke over nearly two decades of follow-up. These associations were stronger among men and not among women. Further translational studies are warranted to define more precise targets for prognosis and intervention.

Many new TB vaccine candidates in the development pipeline need to be studied in people with HIV. People with HIV are at high risk of developing Mycobacterium tuberculosis (Mtb) infection and TB disease and tend to develop less robust vaccine induced immune responses. Many questions remain unanswered regarding priority vaccine indications, clinical trial design, measures of safety, immunogenicity, and efficacy considerations for people with HIV. To address these gaps, a roadmap for developing TB vaccines for people with HIV was developed through a series of symposia that posed framing questions to a panel of international experts for discussion. Framing questions specific to people with HIV included: 1) What is the use case or rationale for developing TB vaccines?; 2) What is the landscape of TB vaccines?; 3) Which vaccine candidates should be prioritized ?; 4) What are the TB vaccine trial design considerations?; 5) What is the role of immunological correlates of protection?; and 6) What are the gaps in preclinical models for studying TB vaccines? We provide a summary of our roadmap with the intention of informing TB vaccine development and the prioritization of clinical trials for inclusion of people with HIV.

Long COVID, a type of Post-Acute Sequelae of SARS CoV-2 infection (PASC), has been associated with sustained elevated levels of immune activation and inflammation. However, the pathophysiological mechanisms that drive this inflammation remain unknown. Inflammation during acute Coronavirus Disease 2019 (COVID-19) could be exacerbated by microbial translocation (from the gut and/or lung) to the blood. Whether microbial translocation contributes to inflammation during PASC is unknown. We found higher levels of fungal translocation – measured as β-glucan, a fungal cell wall polysaccharide – in the plasma of individuals experiencing PASC compared to those without PASC or SARS-CoV-2 negative controls. The higher β-glucan correlated with higher levels of markers of inflammation and elevated levels of host metabolites involved in activating N-Methyl-D-aspartate receptors (such as metabolites within the tryptophan catabolism pathway) with established neuro-toxic properties. Mechanistically, β-glucan can directly induce inflammation by binding to myeloid cells (via the Dectin-1 receptor) and activating Syk/NF-κB signaling. Using an in vitro Dectin-1/NF-κB reporter model, we found that plasma from individuals experiencing PASC induced higher NF-κB signaling compared to plasma from SARS-CoV-2 negative controls. This higher NF-κB signaling was abrogated by the Syk inhibitor Piceatannol. These data suggest a potential targetable mechanism linking fungal translocation and inflammation during PASC.

New tuberculosis vaccine candidates that are in the development pipeline need to be studied in people with HIV, who are at high risk of acquiring Mycobacterium tuberculosis infection and tuberculosis disease and tend to develop less robust vaccine-induced immune responses. To address the gaps in developing tuberculosis vaccines for people with HIV, a series of symposia was held that posed six framing questions to a panel of international experts: What is the use case or rationale for developing tuberculosis vaccines? What is the landscape of tuberculosis vaccines? Which vaccine candidates should be prioritised? What are the tuberculosis vaccine trial design considerations? What is the role of immunological correlates of protection? What are the gaps in preclinical models for studying tuberculosis vaccines? The international expert panel formulated consensus statements to each of the framing questions, with the intention of informing tuberculosis vaccine development and the prioritisation of clinical trials for inclusion of people with HIV.

Previous reports demonstrated that severe acute respiratory syndrome coronavirus (SARS-CoV-2) binding immunoglobulin G levels did not increase significantly between the first and second doses of the BNT162b2 vaccine in previously infected individuals. We tested neutralizing antibodies (nAbs) against SARS-CoV-2 Delta and Omicron variants after the first and second doses of this vaccine in infection-naive and previously infected individuals. Delta, but not Omicron, nAb titers significantly increased from the first to the second dose in both groups of individuals. Importantly, we found that Omicron nAb titers were much lower than Delta nAb titers and that even after 2 doses of vaccine, 17 of 29 individuals in the infection-naive group and 2 of 27 in the previously infected group did not have detectable Omicron nAb titers. Infection history alone did not adequately predict whether a second dose resulted in adequate nAb. For future variants of concern, the discussion on the optimal number of vaccine doses should be based on studies testing for nAb against the specific variant.

Detection and epidemiological characterization of infectious disease outbreaks is key for early identification and response to potential pandemic threats. The rapid global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2020 highlighted the critical role of diagnostics early in the pandemic to understanding the epidemiology of the virus. As a natural extension of Abbott's work in diagnostics, virus discovery, and virus surveillance, the Abbott Pandemic Defense Coalition (APDC) was launched in early 2021. The APDC is a global, multi-sector scientific and public health partnership whose primary objective is the early detection and mitigation of infectious disease threats of pandemic potential. As of January 2022, the APDC network has partners on 5 continents including academic institutions, governmental and non-governmental organizations. A novel element of the APDC is the capacity for early development and rapid deployment of scalable, quality diagnostics targeting newly identified pathogens of pandemic potential.

Background A5350, a phase II, randomized, double-blind study, evaluated the safety and tolerability of the probiotic Visbiome Extra Strength (ES) over 24 weeks and measured effects on inflammation and intestinal barrier function. Methods The primary outcome was change in soluble CD14 (sCD14) levels; secondary outcomes included safety and tolerability, markers of inflammation and cellular activation, and microbiome. In a substudy, gut permeability was assessed by paired colonic biopsies measuring the area of lamina propria occupied by CD4+ cells, interleukin (IL)-17+ cells, and myeloperoxidase (MPO). Changes between arms were compared with the 2-sample t test with equal variance or the Wilcoxon rank-sum test. For safety, the highest graded adverse events (AEs) were compared between arms using the Fisher exact test. Results Overall, 93 participants enrolled: 86% male, median age 51 years, median CD4 count 712 cells/mm3. Visbiome ES was safe and well tolerated. There was no difference in mean change in sCD14 from baseline to week 25/26 between placebo (mean change, 92.3 µg/L; 95% CI, –48.5 to 233 µg/L) and Visbiome ES (mean change, 41.0 µg/L; 95% CI, –94.1 to 176.2 µg/L; P=.60). Similarly, no statistically significant differences between arms in inflammatory marker changes were identified. In substudy participants, no statistical differences between arms for change in cellular marker expression or gut permeability were observed (P>.05 for all). The microbiome demonstrated increased probiotic species and a significant decrease in Gammaproteobacteria (P=.044) in the Visbiome ES arm. Conclusions Visbiome ES was safe and altered the microbiome but demonstrated no effect on systemic inflammatory markers, pathology, or gut permeability in antiretroviral therapy–treated people with HIV.

Non-invasive biomarkers that predict HIV remission after antiretroviral therapy (ART) interruption are urgently needed. Such biomarkers can improve the safety of analytic treatment interruption (ATI) and provide mechanistic insights into the pathways involved in post-ART HIV control. We identified plasma glycomic and metabolic signatures of time-to-viral-rebound and probability-of-viral-rebound using samples from two independent cohorts. These samples include a large number of post-treatment controllers, a rare population demonstrating sustained virologic suppression after ART-cessation. The signatures remained significant after adjusting for key demographic and clinical confounders. We also confirmed a mechanistic link between biomarkers and HIV latency reactivation and myeloid inflammation in vitro. Finally, machine learning algorithms selected sets of biomarkers that predict time-to-viral-rebound with 74–76% capacity and probability-of-viral-rebound with 97.5% capacity. In summary, we fill a major gap in HIV cure research by identifying non-invasive biomarkers, with potential functional significance, that predict duration and probability of viral remission after treatment interruption.

Non-invasive biomarkers that predict HIV remission after antiretroviral therapy (ART) interruption are urgently needed. Such biomarkers can improve the safety of analytic treatment interruption (ATI) and provide mechanistic insights into the pathways involved in post-ART HIV control. We identified plasma glycomic and metabolic signatures of time-to-viral-rebound and probability-of-viral-rebound using samples from two independent cohorts. These samples include a large number of post-treatment controllers, a rare population demonstrating sustained virologic suppression after ART-cessation. The signatures remained significant after adjusting for key demographic and clinical confounders. We also confirmed a mechanistic link between biomarkers and HIV latency reactivation and myeloid inflammation in vitro. Finally, machine learning algorithms selected sets of biomarkers that predict time-to-viral-rebound with 74-76% capacity and probability-of-viral-rebound with 97.5% capacity. In summary, we fill a major gap in HIV cure research by identifying non-invasive biomarkers, with potential functional significance, that predict duration and probability of viral remission after treatment interruption.

Background : Viral diversity presents an ongoing challenge for diagnostic tests, which need to accurately detect all circulating variants. The Abbott Global Surveillance program monitors severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants and their impact on diagnostic test performance. Objectives : To evaluate the capacity of Abbott molecular, antigen, and serologic assays to detect circulating SARS-CoV-2 variants, including all current variants of concern (VOC): B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma) and B.1.617.2 (delta). Study design : Dilutions of variant virus cultures (B.1.1.7, B.1.351, B.1.429, B.1.526.1, B.1.526.2, B.1.617.1, B.1.617.2, P.1, R.1 and control isolate WA1) and a panel of N = 248 clinical samples from patients with sequence confirmed variant infections (B.1.1.7, B.1.351, B.1.427, B.1.429, B.1.526, B.1.526.1, B.1.526.2, P.1, P.2, R.1) were evaluated on at least one assay: Abbott ID NOW COVID-19, m2000 RealTime SARS-CoV-2, Alinity m SARS-CoV-2, and Alinity m Resp-4-Plex molecular assays; the BinaxNOW COVID-19 Ag Card and Panbio COVID-19 Ag Rapid Test Device; and the ARCHITECT/Alinity i SARS-CoV-2 IgG and AdviseDx IgM assays, Panbio COVID-19 IgG assay, and ARCHITECT/Alinity i AdviseDx SARS-CoV-2 IgG II assay. Results : Consistent with in silico predictions, each molecular and antigen assay detected VOC virus cultures with equivalent sensitivity to the WA1 control strain. Notably, 100% of all tested variant patient specimens were detected by molecular assays (N = 197 m2000, N = 88 Alinity m, N = 99 ID NOW), and lateral flow assays had a sensitivity of >94% for specimens with genome equivalents (GE) per device above 4 log (85/88, Panbio; 54/57 Binax). Furthermore, Abbott antibody assays detected IgG and IgM in 94–100% of sera from immune competent B.1.1.7 patients 15–26 days after symptom onset. Conclusions : These data confirm variant detection for 11 SARS-CoV-2 assays, which is consistent with each assay target region being highly conserved. Importantly, alpha, beta, gamma, and delta VOCs were detected by molecular and antigen assays, indicating that these tests may be suitable for widescale use where VOCs predominate.

Background Women with vaginal microbial community state types (CST) with high diversity and a paucity of Lactobacillus crispatus have increased risk of HIV acquisition. Identifying host genetic factors associated with the vaginal microbial composition may aid in elucidating the biological mechanisms regulating these microbial traits and inter-individual variations in associated diseases. Methods We conducted genome-wide associations studies (GWASs) on vaginal microbiome traits on 171 Kenyan women. Study participants were genotyped using the Infinium Global Screening Array and 16S rRNA gene amplicon sequencing was performed to characterize the vaginal microbiome. Linear and logistic regression were performed, adjusting for age and principal components of genetic ancestry, to evaluate the association between L. crispatus, L. iners, G. vaginalis, Shannon diversity index, and CST with host genetic single nucleotide polymorphisms (SNPs). Pathway enrichment analyses were performed to identify biological processes putatively associated with the vaginal microbiome traits. Results At baseline, the median age of study participants was 22 years (IQR: 22 – 25) with 22% having Bacterial vaginosis (Nugent score 7-10). L. crispatus and L. iners were present in 24% and 83% of samples with a mean relative abundance of 31% and 45%, respectively. The most significant SNPs associated were: rs73330467 located between LOC101927488-GRAMD2B ( P =4.79x10 -6 ) for L. crispatus ; rs527430 in the FOXD2-TRABD2B ( P =6.98x10 -7 ) region for L. iners ; rs1229660 in the SNX10-LOC441204 ( P =4.65x10 -6 ) region for G. vaginalis; rs972741 in the ZKSCAN2-HS3ST4 ( P =8.52x10 -7 ) region for Shannon diversity index; and rs2302902 in ELK3 ( P =3.09x10 -6 ) for CST. During pathway enrichment analysis, Toll-like receptors, cytokine production, and other components of innate immune response were associated with L. crispatus, L. iners, and CST. Multiple genomic loci were replicated, including IL-8 (Shannon, CST), TIRAP ( L. iners , Shannon), TLR2 (Shannon, CST), MBL2 ( L. iners , G. vaginalis , CST), and MYD88 ( L. iners, Shannon). Conclusions We identified numerous genetic loci on several pathways related to host immunity and infection that were associated with vaginal microbiome traits, providing insight into potential host genetic influences on vaginal microbiome composition. This new information should guide larger longitudinal studies, with genetic and functional comparison across microbiome sites within individuals, and across populations.

Our microbial cotravelers have increasingly apparent roles in both maintaining health and causing disease in several organ systems. Investigators gather annually at the National Institutes of Health to present new discoveries regarding the role of the microbiome in human health and a special focus on persons living with HIV. Here, we summarize the discussions from the third annual Virology Education workshop on the microbiome in HIV, which took place in October of 2017.

Recent developments in molecular techniques have allowed researchers to identify previously uncultured organisms, which has propelled a vast expansion of our knowledge regarding our commensal microbiota. Interest in the microbiome specific to HIV grew from earlier findings suggesting that bacterial translocation from the intestines is the cause of persistent immune activation despite effective viral suppression with antiretroviral therapy (ART). Studies of SIV infected primates have demonstrated that Proteobacteria preferentially translocate and that mucosal immunity can be restored with probiotics. Pathogenic SIV infection results in a massive expansion of the virome, whereas non-pathogenic SIV infection does not. Human HIV infected cohorts have been shown to have microbiota distinctive from that of HIV negative controls and efforts to restore the intestinal microbiome via probiotics have often had positive results on host markers. The microbiota of the genital tract may play a significant role in acquisition and transmission of HIV. Modification of commensal microbial communities likely represents an important therapeutic adjunct to treatment of HIV. Here we review the literature regarding human microbiome in HIV infection.

Infection with the human immunodeficiency virus (HIV) is associated with a progressive decrease in CD4 T-cell number and a consequent impairment in host immune defenses. Analysis of T cells from patients infected with HIV, or of T cells infected in vitro with HIV, demonstrates a significant fraction of both infected and uninfected cells dying by apoptosis. The many mechanisms that contribute to HIV-associated lymphocyte apoptosis include chronic immunologic activation; gp120/160 ligation of the CD4 receptor; enhanced production of cytotoxic ligands or viral proteins by monocytes, macrophages, B cells, and CD8 T cells from HIV-infected patients that kill uninfected CD4 T cells; and direct infection of target cells by HIV, resulting in apoptosis. Although HIV infection results in T-cell apoptosis, under some circumstances HIV infection of resting T cells or macrophages does not result in apoptosis; this may be a critical step in the development of viral reservoirs. Recent therapies for HIV effectively reduce lymphoid and peripheral T-cell apoptosis, reduce viral replication, and enhance cellular immune competence; however, they do not alter viral reservoirs. Further understanding the regulation of apoptosis in HIV disease is required to develop novel immune-based therapies aimed at modifying HIV-induced apoptosis to the benefit of patients infected with HIV.

The lower genital tract is a unique mucosal environment and further studies are needed to define the types of immune responses that occur during genital infections. We investigated the expression of antimicrobial proteins/peptides (AMP) and cytokines produced in response to Trichomonas vaginalis (TV) infection. A cohort of women (30 TV+, 30 TV-) aged 18-45 that was at high risk of acquiring HIV was studied. mRNA was isolated from cervical cytobrush samples, reverse transcribed, and real time PCR used to determine AMP and cytokine expression. Cytokines were measured by ELISA in cervical vaginal lavage (CVL). TV+ samples had significantly higher mRNA levels of S100A7, S100A8, S100A9 and Muc1 than TV- samples. In contrast, lactoferrin was lower in the TV+ group. For defensins (HBDs), mRNA for HBD4 was significantly higher in TV+ samples, while HBD103 was lower in TV+ samples, but there was no difference in HBD1. Cathelicidin was not different between TV+ and TV- samples. mRNA for RegIIIγ was detected in only four samples. Although a role in inducing expression of AMPs has been shown for IL-22 and IL-17A, these two cytokines, as well as IFNγ, were not different in TV+ and TV- groups. However, IL-17 protein, but not IL-22 was higher in TV+ CVL samples (p < 0.05). Our data identify AMPs that have altered expression in the cervix during TV infection suggesting that these AMPs might have an important role in immunity against TV infection.

Identification of the mechanisms of natural control of HIV replication in elite controllers (EC) may inform approaches for prevention of HIV. We investigated whether EC produce envelope-specific serum IgA responses of greater magnitude, avidity and function compared to subjects on combination antiretroviral therapy (cART). Serum was obtained from age- and race-matched HIV-uninfected women and infected women classified as EC or cART controllers (HC) or noncontrollers (HN). The EC and HC had undetectable viremia and CD4 T cell counts > 450/µl. The HN had high viremia and CD4 T cells < 250/µl. Concentrations and avidity of IgA and IgG against Clade B consensus gp120 and gp41BAL proteins were determined using ELISA. THP-1 monocytes and fluorescent beads coated with gp120 or gp41 were used to evaluate phagocytosis in whole serum and in IgA- or IgG-depleted serum. The magnitude of IgA and IgG responses to gp120 and gp41 was not found to differ between groups. Also, no differences were observed for avidity and phagocytic activity of gp120-specific antibodies. However, avidity and phagocytic activity of the anti-gp41 IgA in EC was significantly greater (p = 0.0186 or p = 0.0054 compared to HC or HN; p = 0.0474 compared to HC, respectively). These data indicate that, in EC, affinity maturation of IgA antibodies to gp41 may occur to a greater extent, resulting in gp41-specific IgA with greater antiviral function. Induction of gp41-specific IgA should be considered in vaccine design.

Evidence for HIV-1 infection of trophoblasts is discordant. Utilizing highly purified full-term trophoblasts, we demonstrate that full-term trophoblasts express CXCR4 but are negative for CCR5 and CD4 cell surface proteins. Full-term trophoblasts were refractory to infection by HIV-1 IIIB and primary isolates of HIV-1. However, full-term trophoblasts could be infected by a CD4-independent, CXCR4-utilizing HIV-1 strain, as demonstrated by substantial p24 (5.5 ng/ml) levels and HIV-1 gag/pol DNA content (3050 copies/microg) 7 days postinfection. These data illustrate that trophoblasts express the essential host factors for productive HIV-1 infection and that the block to HIV-1 infection may be at the level of entry. In additional, our data suggest that CD4-independent mechanisms of infection may play a role in promoting in utero HIV-1 transmission.

SIV/HIV infection is associated with B cell dysfunction, the significance of which is not known. We compared the B cell dynamics in blood, lymph nodes (LNs) and gut during SIVsab pathogenic infection of pigtailed macaques (PTMs) and nonprogressive SIVsab infection of African green monkeys (AGMs) by monitoring the B cell subsets, B regulatory cells (Bregs), CD4+ T follicular helper (Tfh) cells, B cell immune activation, apoptosis, exhaustion and homing to the intestine. Binding anti-gp41 and neutralizing antibodies (Nabs) were also measured. Acute SIV infection induced a transient but significant loss of total circulating B cells in PTMs only. In AGMs, total B cell from LNs increased during chronic infection. B cells transiently increased in the gut and naïve subset decreased in all three compartments in both species. Memory B cell subsets showed a different dynamic for both species. Bregs increased in PTM, correlated with IL-10 expression. B cell proliferation occurred in both species. PTMs expressed high baseline levels of α4β7, high degree of B cell activation and apoptosis and high frequency of Tfh. The dynamics of Ab response was similar in both species, but rapid progressor PTMs lacked the ability to mount anti-gp41. Our data show that B cell dysfunction only occurs in pathogenic SIV infection. While B cell changes were not correlated with production of Nabs, rapid disease progression associated a severe incapacity to mount an anti-SIV Ab response.

TLR2-signaling may contribute to B-cell activation during HIV infection leading to HIV-NHL genesis; a common malignancy in anti-retroviral therapy (ART) treated patients. TLR-signaling and anti-tumor immunity inhibitory regulatory B cells (Bregs) contribute to lymphomagenesis but their roles in HIV-NHL genesis remain unexplored. We investigated, if in HIV- donors, TLR-activated B cells mimic an activated/germinal center (GC)-like phenotype aberrantly expressed in B-cell lymphomas. Further, in HIV+ART subjects: (cases =

IL22 and IL17 have important antimicrobial and epithelial repair functions. However their role in the female lower genital tract has not been well defined. We determined the frequency and phenotype of cells producing these cytokines at this mucosal site, because we postulated that they contribute to resistance to sexually transmitted infections. Cervical tissue was obtained from benign hysterectomies. Cells were obtained by collagenase digestion, stimulated with PMA/ionomycin/Monensin for 4 hrs and stained for flow cytometry. On average in endocervical tissue, the CD3+CD45+ cells were 71% (CD4+ ~50%) and CD3- CD45+ cells were 24%. 20% of CD45+ cells were Lin- (negative for CD3, CD14, CD16, CD19, CD20 and CD56). Unstimulated CD4+ T-cells were < 0.05% IL22+, IL17+ and IL22+IL17+, while after stimulation the percentages were 1.9%, 5.5% and 1.6% respectively. Unstimulated CD3- cells in cervical tissue were 2.3% IL22+, 3% IL17+ and 4.6% of IL22+IL17+, and the percentages after stimulation were 1.6%, 6% and 1.9% positive respectively. Our data show that in cervical tissue, there are CD3+ and CD3- cells capable of making IL22 and IL17. This is important because this is the first study to show what types of cells in the genital tract are making these cytokines, and suggests that these cells could induce antimicrobial peptide production by cervical epithelial cells in response to sexually transmitted pathogens.

Fc receptor-like 4 (FcRL4) attenuates B-cell receptor signaling (BCR) while enhancing TLR-responsiveness. FcRL4-mediated BCR dampening is dependent on tyrosine phosphatases SHP1/2. However, regulation of FcRL4 expression and mechanisms underlying FcRL4 amplification of TLR-signaling remain unknown. We find that exposure of human B cells to TLR-2-7 -9 ligands leads to upregulated FcRL4 expression. In FcRL4 transfected B-cell lines, TLR9-induced FcRL4 upregulation was associated with increased HcK expression. Aditionally, p38 inhibition suppressed upregulation of FcRL4 and CD23 but not HcK expression. HcK is an enhancer of TLR-signaling, likely activated by SHP-1/2 and we found that HcK or SHP1/2 inhibition, significantly suppressed TLR9-induced upregulation of CD23. To determine an in vivo relevance for our findings we assessed TLR-stimulated purified FcRL4+ B cells and compared to FcRL4- B cells, FcRL4+ B cells expressed significantly higher levels of the pro-inflammatory cytokine IL-6. Evaluating B cells of HIV+ viremic subjects showed that ex vivo unstimulated FcRL4hi B cell subsets expressed high levels of IL-6. Further, FcRL4+ B cells exhibited high levels of HcK and phosphorylated MAPkinases p38 and Erk .Our data indicate that TLR-activation upregulates FcRL4 expression, and FcRL4 recruited SHP1/2 likely activates HcK thereby amplifying TLR-signaling. Finally, in HIV+ viremic subjects TLR-activated FcRL4+ B cells likely contribute to inflammation via IL-6 expression.

Circulating cytokine levels can provide valuable information about immune status, but often require high-sensitivity assays. Multiplex assays hold great promise, especially when limited sample volumes are available. Four high-sensitivity cytokine multiplex assays (BioRad, BioSource/Invitrogen, Linco/Millipore, Meso Scale Discovery [MSD]) were evaluated for ability to detect circulating levels of cytokines, and for lot and laboratory variability. Assays were performed in six different laboratories utilizing archived serum from HIV-uninfected and -infected subjects from the Multicenter AIDS Cohort Study (MACS) and the Women’s Interagency HIV Study (WIHS), and commercial plasma samples spanning initial HIV viremia. In a majority of serum samples IL6, IL8, IL10, and TNFα were detectable with at least three kits, while IL1β was clearly detected with only one kit. No single multiplex panel detected all cytokines, and there were highly significant differences (p