The Pumwani Sexworker Cohort in Nairobi, Kenya has enrolled >2100 women from 1985 to 2002. A small subset of women enrolled during this period remain HIV uninfected despite high-risk sex work exposures. This study investigated the expression of genes involved in leukocyte movement and migration and their role in HIV resistance. Whole blood was collected from 75 HIV uninfected women: 38 HIV resistant women (average of 15.39 negative years) and 37 susceptible new enrollees (average of 1.027 negative years). The mRNA expression of 84 genes was analyzed using a custom RT2 Profiler PCR-array and compared between HIV resistant women and HIV negative susceptible controls. The function and influence of genes with mRNA significantly differentially expressed were analyzed using Ingenuity Pathway Analysis (QIAGEN) and Pathway Studio (Elsevier). Eighteen genes were significantly up-expressed in HIV resistant women. Among them, 16 genes are involved in the leukocyte movement and migration (p < 0.00001). Among them, 4 genes (CTNNB1, ITGB1, PIK3CA and PTPN11) are involved in transendothelial leukocyte migration signaling (p < 0.00001), 8 genes (CTNNB1, DPP4, ITGB1, PIK3CA, PTPN11, TICAM1, TIMP1, VCAN) are involved in cellular infiltration by leukocytes (p < 0.00001), and 6 genes (CTNNB1, ITGB1, MAP3K4, PIK3CA, PTPN11, TIMP1) are involved in the Leukocyte Extravasation Signaling (p < 0.00001). We propose that the HIV resistant women of the PSC may exhibit a unique leukocyte migratory response in dealing with pathogen infection. The potential mechanisms could contribute to the unique leukocyte migratory response: modifications to the PI3K/AKT signaling, extracellular matrix remodeling, integrin structure and function, and activation of T-cell receptors.

HIV-1 remains a global health crisis 1 , highlighting the need to identify new targets for therapies. Here, given the disproportionate HIV-1 burden and marked human genome diversity in Africa 2 , we assessed the genetic determinants of control of set-point viral load in 3,879 people of African ancestries living with HIV-1 participating in the international collaboration for the genomics of HIV 3 . We identify a previously undescribed association signal on chromosome 1 where the peak variant associates with an approximately 0.3 log 10 -transformed copies per ml lower set-point viral load per minor allele copy and is specific to populations of African descent. The top associated variant is intergenic and lies between a long intergenic non-coding RNA ( LINC00624 ) and the coding gene CHD1L , which encodes a helicase that is involved in DNA repair 4 . Infection assays in iPS cell-derived macrophages and other immortalized cell lines showed increased HIV-1 replication in CHD1L- knockdown and CHD1L- knockout cells. We provide evidence from population genetic studies that Africa-specific genetic variation near CHD1L associates with HIV replication in vivo. Although experimental studies suggest that CHD1L is able to limit HIV infection in some cell types in vitro, further investigation is required to understand the mechanisms underlying our observations, including any potential indirect effects of CHD1L on HIV spread in vivo that our cell-based assays cannot recapitulate.

Some people remain healthier throughout life than others but the underlying reasons are poorly understood. Here we hypothesize this advantage is attributable in part to optimal immune resilience (IR), defined as the capacity to preserve and/or rapidly restore immune functions that promote disease resistance (immunocompetence) and control inflammation in infectious diseases as well as other causes of inflammatory stress. We gauge IR levels with two distinct peripheral blood metrics that quantify the balance between (i) CD8 + and CD4 + T-cell levels and (ii) gene expression signatures tracking longevity-associated immunocompetence and mortality-associated inflammation. Profiles of IR metrics in ~48,500 individuals collectively indicate that some persons resist degradation of IR both during aging and when challenged with varied inflammatory stressors. With this resistance, preservation of optimal IR tracked (i) a lower risk of HIV acquisition, AIDS development, symptomatic influenza infection, and recurrent skin cancer; (ii) survival during COVID-19 and sepsis; and (iii) longevity. IR degradation is potentially reversible by decreasing inflammatory stress. Overall, we show that optimal IR is a trait observed across the age spectrum, more common in females, and aligned with a specific immunocompetence-inflammation balance linked to favorable immunity-dependent health outcomes. IR metrics and mechanisms have utility both as biomarkers for measuring immune health and for improving health outcomes.

Purpose:TILRR is a modulator of genes in the NF-κB inflammation pathway. It regulates inflammation-responsive genes, the secretion of inflammatory mediators, and the migration of immune cells. Because inflammation drives the pathogenesis of many infectious and inflammatory diseases, it is important to know the expression of TILRR protein in tissues and cells. This study examined TILRR protein expression in healthy adult human and macaques' tissues and PBMCs (peripheral blood mononuclear cells).Methods and Results:Tissues (trachea, lungs, stomach, small intestine [ileum], cecum, colon, rectum, vagina, cervix, uterus, and penis) and PBMCs from humans and macaques were lysed in RIPA (radioimmunoprecipitation assay) lysis buffer. The TILRR protein was examined by fluorescent Western blot analysis. The relative fluorescence units (rfu) of TILRR protein expression were quantified by Image Studio software (LI-COR). The results showed that adult healthy female (n=1) rectal and cervicovaginal tissues expressed a higher level of TILRR protein than the other tissues (trachea, lungs, stomach, small intestine [ileum], cecum, colon, uterus, and penis) examined. Like humans, the lungs, colon, and rectal tissues of healthy adult female cynomolgus monkeys (Macaca fascicularis) (n=2) expressed the TILRR protein. In addition, PBMCs of healthy adult women (n=4), adult female cynomolgus monkeys (Macaca fascicularis) (n=4), and adult male and female rhesus monkeys (Macaca mulatta) (n=4) showed a similar expression level of TILRR protein (p= 0.2858). TILRR protein was not detected in most of the human cell lines examined, except in Jurkat cells.Conclusion:Our study for the first time showed that TILRR protein is expressed in healthy adult human and monkey tissues and PBMCs. The TILRR protein in these tissues and PBMCs may play a role in the inflammatory response of these tissues and cells in response to infectious pathogens.

Background: We analyzed the prevalence of pre-antiretroviral therapy (ART) drug resistance mutations (DRMs) in a Kenyan population. We also examined whether host HLA class I genes influence the development of pre-ART DRMs. Methods: The HIV-1 proviral DNAs were amplified from blood samples of 266 ART-naïve women from the Pumwani Sex Worker cohort of Nairobi, Kenya using a nested PCR method. The amplified HIV genomes were sequenced using next-generation sequencing technology. The prevalence of pre-ART DRMs was investigated. Correlation studies were performed between HLA class I alleles and HIV-1 DRMs. Results: Ninety-eight percent of participants had at least one DRM, while 38% had at least one WHO surveillance DRM. M184I was the most prevalent clinically important variant, seen in 37% of participants. The DRMs conferring resistance to one or more integrase strand transfer inhibitors were also found in up to 10% of participants. Eighteen potentially relevant (p < 0.05) positive correlations were found between HLA class 1 alleles and HIV drug-resistant variants. Conclusions: High levels of HIV drug resistance were found in all classes of antiretroviral drugs included in the current first-line ART regimens in Africa. The development of DRMs may be influenced by host HLA class I-restricted immunity.

BACKGROUND:TILRR (Toll-like Interleukin-1 Receptor Regulator) is a modulator of many genes in NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling. It promotes the production of inflammatory mediators and the migration of immune cells. Recently, we showed that TILRR protein circulates in human blood. Thus, it could influence systemic inflammation. Systemic and mucosal inflammations increase the susceptibility to HIV infection. In this study, we analyzed the TILRR protein levels of the archived plasma samples of women enrolled in the Pumwani cohort to determine whether the plasma TILRR protein levels before seroconversion are correlated with differential risk of HIV seroconversion.METHODS:TILRR protein of 941 archived HIV negative plasma samples from 390 women who were HIV negative at the cohort enrollment was quantified with an in-house developed multiplex bead array method. Proinflammatory cytokines/chemokines were measured using a 14-plex bead array method. Spearman rank correlation analysis was used to determine the correlation between plasma TILRR protein and proinflammatory cytokines/chemokines. Kaplan-Meier survival analysis was conducted to evaluate whether the median plasma TILRR protein levels correlate with increased risk of HIV seroconversion.FINDINGS:The level of plasma TILRR protein was positively correlated with plasma IL-1β (rho: 0.2593, p<0.0001), MCP-1 (rho: 0.2377, p<0.0001), and IL-17A (rho: 0.1225, p=0.0216). Women with median plasma TILRR protein levels ≥100 ng/ml seroconverted significantly faster than women with plasma TILRR protein levels <100 ng/ml (log-rank= 100.124, p<0.0001; relative risk= 3.72 and odds ratio= 15.29). Furthermore, the factors causing genital inflammation, such as STIs (sexually transmitted infections), vaginal discharge, and genital ulcers were not statistically significantly different among women with different median plasma TILRR protein levels.INTERPRETATION:The high plasma TILRR protein levels are highly correlated with several plasma proinflammatory cytokines/chemokines. High median plasma TILRR protein (≥100 ng/ml) strongly predicted an increased risk of HIV seroconversion. Reducing plasma TILRR protein levels may reduce the risk of HIV acquisition.FUNDING:The study was funded by an operating grant from the Canadian Institutes of Health Research (CIHR), operating grant-PA: CHVI Vaccine Discovery and Social Research (http://www.cihr-irsc.gc.ca/e/193.html), and National Microbiology Laboratory of Canada.

Background: We examined associations between NFκB1 polymorphisms and influenza A (H1N1) clinical outcomes in Canadian. Methods: A total of thirty-six Caucasian patients admitted to the intensive care unit (ICU) in hospitals in Canada were recruited during the 2009 H1N1 pandemic. Genomic DNA was extracted from the whole blood samples. The NFkB1 gene was targeted for genotyping using next-generation sequencing technology—Roche 454. Results: A total of 136 single nucleotide polymorphisms (SNPs) were discovered within the NFκB1 gene. Among them, 63 SNPs were significantly enriched in patients admitted in the ICU (p < 0.05) compared with the British Caucasian population in the 1000 Genomes study. These enriched SNPs are mainly intron variants, and only two are exon SNPs from the non-transcribing portion of the NFκB1 gene. Conclusions: Genetic variations in the NFκB1 gene could influence clinical outcomes of pandemic H1N1 infections. Our findings showed that sequence variations of the NFκB1 gene might influence patient response to influenza infection.

Background: TILRR (Toll-like Interleukin-1 Receptor Regulator) is a modulator of many genes in NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling. It promotes the production of inflammatory mediators and the migration of immune cells. Recently, we showed that TILRR protein circulates in human blood. Thus, it could modulate systemic inflammation. Systemic and mucosal inflammations increase the susceptibility to HIV infection. In this study, we analyzed the TILRR protein levels of the archived plasma samples of women enrolled in the Pumwani cohort to determine whether the plasma TILRR protein levels before seroconversion are correlated with HIV seroconversion status.Methods: TILRR protein of 941 archived HIV negative plasma samples from 390 women was quantified with an in-house developed multiplex bead array method. Proinflammatory cytokines/chemokines were measured using a 14-plex multiplex bead array method. Spearman rank correlation analysis was used to determine the correlation between plasma TILRR protein and proinflammatory cytokines/chemokines. Kaplan-Meier survival analysis was conducted to evaluate whether the median plasma TILRR protein levels influence HIV seroconversion.Findings: The level of plasma TILRR protein was positively correlated with plasma IL-1β ( p =1.0E-6), MCP-1 ( p =6.5E-6), and IL-17A ( p =0.0216). Women with median plasma TILRR protein levels ≥100 ng/ml seroconverted significantly faster than women with plasma TILRR protein levels <100 ng/ml (log-rank=100.124, p =1.4E-23; relative risk=3.71 and odds ratio=15.29). Furthermore, the factors causing genital inflammation, such as STIs (sexually transmitted infections), vaginal discharge, and genital ulcers were not significantly different among women with different median plasma TILRR protein levels.Interpretation: The high plasma TILRR protein levels are highly correlated with several plasma proinflammatory cytokines/chemokines. High median plasma TILRR protein (≥100 ng/ml) strongly predicted the risk of HIV seroconversion. Reducing plasma TILRR protein levels may reduce the risk of HIV acquisition.Funding Statement: The study was funded by an operating grant from the Canadian Institutes of Health Research (CIHR), operating grant-PA: CHVI Vaccine Discovery and Social Research (http://www.cihr-irsc.gc.ca/e/193.html)Declaration of Interests: The authors declare no competing interest.Ethics Approval Statement: This study was guided by the Helsinki Declaration on ethical principles for medical research involving human subjects. Ethics approval to conduct this study using human plasma samples was obtained from the University of Manitoba and the University of Nairobi/Kenyatta National Hospital Ethics Committee. All enrolled subjects gave written informed consent to participate in the study.

FREM1 (Fras-related extracellular matrix 1) and its splice variant TILRR (Toll-like interleukin-1 receptor regulator) have been identified as integral components of innate immune systems. The potential involvement of FREM1 in HIV-1 (human immunodeficiency virus 1) acquisition was suggested by a genome-wide SNP (single nucleotide polymorphism) analysis of HIV-1 resistant and susceptible sex workers enrolled in the Pumwani sex worker cohort (PSWC) in Nairobi, Kenya. The studies showed that the minor allele of a FREM1 SNP rs1552896 is highly enriched in the HIV-1 resistant female sex workers. Subsequent studies showed that FREM1 mRNA is highly expressed in tissues relevant to mucosal HIV-1 infection, including cervical epithelial tissues, and TILRR is a major modulator of many genes in the NF-κB signal transduction pathway. In this article, we review the role of FREM1 and TILRR in modulating inflammatory responses and inflammation, and how their influence on inflammatory responses of cervicovaginal tissue could enhance the risk of vaginal HIV-1 acquisition.

PURPOSE:TILRR (Toll-like interleukin-1 receptor regulator), a variant of FREM1 (Fras-related extracellular matrix 1), is a modulator of many genes in NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling and inflammatory responses. It enhanced the expression of multiple genes in the NF-κB signaling pathway and promoted the production of multiple pro-inflammatory cytokines/chemokines. TILRR is an extracellular matrix protein and expressed in cells and tissues, and has never been considered to exist in the blood. The study aimed to identify circulating TILRR protein in human plasma as a biomarker of systemic inflammation.METHODS AND RESULTS:We developed a multiplex bead array method (Bio-Plex) using 4 monoclonal antibodies targeting different protein domains of FREM1/TILRR to investigate whether TILRR can be detected in blood plasma. The results of the multiplex bead array method were validated by Western blot analysis of affinity-purified TILRR from patient plasma samples. We subsequently analyzed 640 plasma samples from women enrolled in the Pumwani Sex Worker cohort (PSWC) (Nairobi, Kenya). Our study showed that TILRR exists in all patient plasma samples, but its quantities vary greatly among the patients, ranging from 2.38 ng/mL to 5196.79 ng/mL. The plasma TILRR below 2.38 ng/mL can only be detected by affinity purification and Western blot analysis.CONCLUSION:Our in-house developed multiplex bead array method can successfully quantify TILRR protein in plasma samples. Because TILRR is an important modulator of many inflammation-responsive genes, it may be an inflammation biomarker in blood and play a role in modulating systemic inflammation.

After over three decades of research, an effective anti-HIV vaccine remains elusive. Unconventional and novel vaccine strategies are needed. Here, we report that a vaccine focusing the immune response on the sequences surrounding the 12 viral protease cleavage sites (PCSs) provides greater than 80% protection of Mauritian cynomolgus macaques (MCMs) against repeated intravaginal SIVmac251 challenges. The PCS-specific T cell responses are correlated with vaccine efficacy. The PCS vaccine does not induce immune activation and inflammation known to be associated with increased susceptibility to HIV infection. Machine learning analyses revealed that the immune environment generated by the PCS vaccine predicts vaccine efficacy. Our study demonstrates for the first time that a novel vaccine which targets viral maturation, but lacks full Env and Gag proteins as immunogens, can prevent intravaginal infection in a highly stringent NHP/SIV challenge model. Targeting HIV maturation thus offers a novel approach to developing an effective HIV vaccine. One Sentence Summary The anti-PCS T cell responses and the immune environment induced by the novel PCS vaccine are key correlates of vaccine efficacy

After over 3 decades of research, an effective anti-HIV vaccine remains elusive. The recently halted HVTN702 clinical trial not only further stresses the challenge to develop an effective HIV vaccine but also emphasizes that unconventional and novel vaccine strategies are urgently needed. Here, we report that a vaccine focusing the immune response on the sequences surrounding the 12 viral protease cleavage sites (PCSs) provided greater than 80% protection to Mauritian cynomolgus macaques against repeated intravaginal SIVmac251 challenges. The PCS-specific T cell responses correlated with vaccine efficacy. The PCS vaccine did not induce immune activation or inflammation known to be associated with increased susceptibility to HIV infection. Machine learning analyses revealed that the immune microenvironment generated by the PCS vaccine was predictive of vaccine efficacy. Our study demonstrates, for the first time to our knowledge, that a vaccine which targets only viral maturation, but lacks full-length Env and Gag immunogens, can prevent intravaginal infection in a stringent macaque/SIV challenge model. Targeting HIV maturation thus offers a potentially novel approach to developing an effective HIV vaccine.

Studies have shown that vaccine vectors and route of immunization can differentially activate different arms of the immune system. However, the effects of different HIV vaccine immunogens on mucosal inflammation have not yet been studied. Because mucosal sites are the primary route of HIV infection, we evaluated the cervico-vaginal inflammatory cytokine and chemokine levels of Mauritian cynomolgus macaques following immunization and boost using two different SIV vaccine immunogens. The PCS vaccine delivers 12 20-amino acid peptides overlapping the 12 protease cleavage sites, and the Gag/Env vaccine delivers the full Gag and full Env proteins of simian immunodeficiency virus. We showed that the PCS vaccine prime and boosts induced short-lived, lower level increases of a few pro-inflammatory/chemotactic cytokines. In the PCS-vaccine group only the levels of MCP-1 were significantly increased above the baseline (P = 0.0078, Week 6; P = 0.0078, Week 17; P = 0.0234; Week 51) following multiple boosts. In contrast, immunizations with the Gag/Env vaccine persistently increased the levels of multiple cytokines/chemokines. In the Gag/Env group, higher than baseline levels were consistently observed for IL-8 (P = 0.0078, Week 16; P = 0.0078, Week 17; P = 0.0156, Week 52), IL-1β (P = 0.0234, Week 16; P = 0.0156, Week 17; P = 0.0156, Week 52), and MIP-1α (P = 0.0313, Week 16; P = 0.0156, Week 17; P = 0.0313, Week 52). Over time, repeated boosts altered the relative levels of these cytokines between the Gag/Env and PCS vaccine group. 18 weeks after final boost with a higher dosage, IP-10 levels (P = 0.0313) in the Gag/Env group remained higher than baseline. Thus, the influence of vaccine immunogens on mucosal inflammation needs to be considered when developing and evaluating candidate HIV vaccines.

TILRR has been identified as an important modulator of inflammatory responses. It is associated with NF-κB activation, and inflammation. Our previous study showed that TILRR significantly increased the expression of many innate immune responsive genes and increased the production of several pro-inflammatory cytokines/chemokines by cervical epithelial cells. In this study, we evaluated the effect of TILRR-induced pro-inflammatory cytokines/chemokines on the migration of immune cells. The effect of culture supernatants of TILRR-overexpressed cervical epithelial cells on the migration of THP-1 monocytes and MOLT-4 T-lymphocytes was evaluated using Transwell assay and a novel microfluidic device. We showed that the culture supernatants of TILRR-overexpressed HeLa cells attracted significantly more THP-1 cells (11-40%, p = 0.0004-0.0373) and MOLT-4 cells (14-17%, p = 0.0010-0.0225) than that of controls. The microfluidic device-recorded image analysis showed that significantly higher amount with longer mean cell migration distance of THP-1 (p < 0.0001-0.0180) and MOLT-4 (p < 0.0001-0.0025) cells was observed toward the supernatants of TILRR-overexpressed cervical epithelial cells compared to that of the controls. Thus, the cytokines/chemokines secreted by the TILRR-overexpressed cervical epithelial cells attracted immune cells, such as monocytes and T cells, and may potentially influence immune cell infiltration in tissues.

TILRR (Toll-like interleukin-1 receptor regulator), a transcript variant of FREM1, is a novel regulatory component, which stimulates innate immune responses through binding to IL-1R1 (Interleukin-1 receptor, type 1) and TLR (Toll-like receptor) complex. However, it is not known whether TILRR expression influences other genes in the NFκB signal transduction and pro-inflammatory responses. Our previous study identified FREM1 as a novel candidate gene in HIV-1 resistance/susceptibility in the Pumwani Sex worker cohort. In this study, we investigated the effect of TILRR overexpression on expression of genes in the NFκB signaling pathway in vitro. The effect of TILRR on mRNA expression of 84 genes related to NFκB signal transduction pathway was investigated by qRT-PCR. Overexpression of TILRR on pro-inflammatory cytokine/chemokine(s) secretion in cell culture supernatants was analyzed using Bioplex multiplex bead assay. We found that TILRR overexpression significantly influenced expression of many genes in HeLa and VK2/E6E7 cells. Several cytokine/chemokine(s), including IL-6, IL-8 (CXCL8), IP-10, MCP-1, MIP-1β, and RANTES (CCL5) were significantly increased in the cell culture supernatants following TILRR overexpression. Although how TILRR influences the expression of these genes needs to be further studied, we are the first to show the influence of TILRR on many genes in the NFκB inflammatory pathways. The NFκB inflammatory response pathways are extremely important in microbial infection and pathogenesis, including HIV-1 transmission. Further study of the role of TILRR may identify the novel intervention targets and strategies against HIV infection.

Within the Pumwani sex worker cohort, a subgroup remains seronegative, despite frequent exposure to HIV-1; some of them seroconverted several years later. This study attempts to identify viral variations in 5'LTR-leader sequences (5'LTR-LS) that might contribute to the late seroconversion. The 5'LTR-LS contains sites essential for replication and genome packaging, viz, primer binding site (PBS), major splice donor (SD), and major packaging signal (PS). The 5'LTR-LS of 20 late seroconverters (LSC) and 122 early seroconverters (EC) were amplified, cloned, and sequenced. HelixTree 6.4.3 was employed to classify HIV subtypes and sequence variants based on seroconversion status. We find that HIV-1 subtypes A1.UG and D.UG were overrepresented in the viruses infecting the LSC (P < 0.0001). Specific variants of PBS (Pc < 0.0001), SD1 (Pc < 0.0001), and PS (Pc < 0.0001) were present only in the viral population from EC or LSC. Combinations of PBS [PBS-2 (Pc < 0.0001) and PBS-3 (Pc < 0.0001)] variants with specific SD sequences were only seen in LSC or EC. Combinations of A1.KE or D with specific PBS and SD variants were only present in LSC or EC (Pc < 0.0001). Furthermore, PBS variants only present in LSC co-clustered with PBS references utilizing tRNAArg; whereas, the PBS variants identified only in EC co-clustered with PBS references using tRNALys3 and its variants. This is the first report that specific PBS, SD1, and PS sequence variants within 5'LTR-LS are associated with HIV-1 seroconversion, and it could aid designing effective anti-HIV strategies.