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Background: Despite successful antiretroviral therapy (ART), frequencies and immunological functions of memory CCR6 + Th17-polarised CD4 + T-cells are not fully restored in people with HIV (PWH). Moreover, long-lived Th17 cells contribute to HIV persistence under ART. However, the molecular mechanisms underlying these observations remain understudied., Methods: mRNA-sequencing was performed using Illumina technology on freshly FACS-sorted memory CCR6 + CD4 + T-cells from successfully ART-treated (ST), elite controllers (EC), and uninfected donors (HD). Gene expression validation was performed by RT-PCR, flow cytometry, and in vitro functional assays., Findings: Decreased Th17 cell frequencies in STs and ECs versus HDs coincided with reduced Th17-lineage cytokine production in vitro. Accordingly, the RORγt/RORC2 repressor NR1D1 was upregulated, while the RORγt/RORC2 inducer Semaphorin 4D was decreased in memory CCR6 + T-cells of STs and ECs versus HDs. The presence of HIV-DNA in memory CCR6 + T-cells of ST and EC corresponded with the downregulation of HIV restriction factors (SERINC3, KLF3, and RNF125) and HIV inhibitors (tetraspanins), along with increased expression of the HIV-dependency factor MRE11, indicative of higher susceptibility/permissiveness to HIV-1 infection. Furthermore, markers of DNA damage/modification were elevated in memory CCR6 + T-cells of STs and ECs versus HDs, in line with their increased activation (CD38/HLA-DR), senescence/exhaustion phenotype (CTLA-4/PD-1/CD57) and their decreased expression of proliferation marker Ki-67., Interpretation: These results reveal new molecular mechanisms of Th17 cell deficit in ST and EC PWH despite a successful control of HIV-1 replication. This knowledge points to potential therapeutic interventions to limit HIV-1 infection and restore frequencies, effector functions, and senescence/exhaustion in Th17 cells., Funding: This study was funded by the Canadian Institutes of Health Research (CIHR, operating grant MOP 142294, and the Canadian HIV Cure Enterprise [CanCURE 2.0] Team Grant HB2 164064), and in part, by the Réseau SIDA et maladies infectieuses du Fonds de recherche du Québec-Santé (FRQ-S)., Competing Interests: Declaration of interests We have no competing interest to declare., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Of the 37.9 million individuals infected with human immunodeficiency virus type 1 (HIV-1), approximately 50% exhibit HIV-associated neurocognitive disorders (HAND). We and others previously showed that HIV-1 viral RNAs, such as trans-activating response (TAR) RNA, are incorporated into extracellular vesicles (EVs) and elicit an inflammatory response in recipient naïve cells. Cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC), the primary cannabinoids present in cannabis, are effective in reducing inflammation. Studies show that cannabis use in people living with HIV-1 is associated with lower viral load, lower circulating CD16 + monocytes and high CD4 + T-cell counts, suggesting a potentially therapeutic application. Here, HIV-1 infected U1 monocytes and primary macrophages were used to assess the effects of CBD. Post-CBD treatment, EV concentrations were analyzed using nanoparticle tracking analysis. Changes in intracellular and EV-associated viral RNA were quantified using RT-qPCR, and changes in viral proteins, EV markers, and autophagy proteins were assessed by Western blot. Our data suggest that CBD significantly reduces the number of EVs released from infected cells and that this may be mediated by reducing viral transcription and autophagy activation. Therefore, CBD may exert a protective effect by alleviating the pathogenic effects of EVs in HIV-1 and CNS-related infections.

Despite the success of antiretroviral therapy (ART), people living with HIV continue to suffer from high burdens of respiratory infections, lung cancers and chronic lung disease at a higher rate than the general population. The lung mucosa, a previously neglected HIV reservoir site, is of particular importance in this phenomenon. Because ART does not eliminate the virus, residual levels of HIV that remain in deep tissues lead to chronic immune activation and pulmonary inflammatory pathologies. In turn, continuous pulmonary and systemic inflammation cause immune cell exhaustion and pulmonary immune dysregulation, creating a pro-inflammatory environment ideal for HIV reservoir persistence. Moreover, smoking, gut and lung dysbiosis and co-infections further fuel the vicious cycle of residual viral replication which, in turn, contributes to inflammation and immune cell proliferation, further maintaining the HIV reservoir. Herein, we discuss the recent evidence supporting the notion that the lungs serve as an HIV viral reservoir. We will explore how smoking, changes in the microbiome, and common co-infections seen in PLWH contribute to HIV persistence, pulmonary immune dysregulation, and high rates of infectious and non-infectious lung disease among these individuals., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Alexandrova, Costiniuk and Jenabian.)

People living with HIV have high burdens of chronic lung disease, lung cancers, and pulmonary infections despite antiretroviral therapy (ART). The rates of tobacco smoking by people living with HIV vastly exceed that of the general population. Furthermore, we showed that HIV can persist within the lung mucosa despite long-term ART. As CD8 T cell cytotoxicity is pivotal for controlling viral infections and eliminating defective cells, we explored the phenotypic and functional features of pulmonary versus peripheral blood CD8 T cells in ART-treated HIV + and uninfected controls. Bronchoalveolar lavage fluid and matched blood were obtained from asymptomatic ART-treated HIV + smokers ( n = 11) and nonsmokers ( n = 15) and uninfected smokers ( n = 7) and nonsmokers ( n = 10). CD8 T cell subsets and phenotypes were assessed by flow cytometry. Perforin/granzyme B content, degranulation (CD107a expression), and cytotoxicity against autologous Gag peptide-pulsed CD4 T cells (Annexin V + ) following in vitro stimulation were assessed. In all groups, pulmonary CD8 T cells were enriched in effector memory subsets compared with blood and displayed higher levels of activation (HLA-DR + ) and exhaustion (PD1 + ) markers. Significant reductions in proportions of senescent pulmonary CD28 - CD57 + CD8 T cells were observed only in HIV + smokers. Pulmonary CD8 T cells showed lower perforin expression ex vivo compared with blood CD8 T cells, with reduced granzyme B expression only in HIV + nonsmokers. Bronchoalveolar lavage CD8 T cells showed significantly less in vitro degranulation and CD4 killing capacity than blood CD8 T cells. Therefore, pulmonary mucosal CD8 T cells are more differentiated, activated, and exhausted, with reduced killing capacity in vitro than blood CD8 T cells, potentially contributing to a suboptimal anti-HIV immune response within the lungs., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Sub-Saharan Africa has the vast majority (∼90%) of new pediatric acquired immunodeficiency syndrome cases worldwide. Biologically monitoring HIV-infected pediatric populations remains challenging. The differential interest of human immunodeficiency virus (HIV)-1 RNA loads and CD4 T-cell counts is debated for the treatment of pediatric acquired immunodeficiency syndrome patients.Long-term antiretroviral treatment (ART) outcomes regarding immunological and virological surrogate markers were longitudinally evaluated between 2009 and 2014 (over 57 months) in 245 perinatally HIV-1-infected children and adolescents born from HIV-infected mothers, treated at inclusion for at least 6 months by the World Health Organization-recommended ART in Bangui, Central African Republic.Patients were monitored over time biologically for CD4 T-cell counts, HIV-1 RNA loads, and drug resistance mutation genotyping.Children lost to follow-up totaled 6%. Four categories of immunovirological responses to ART were observed. At baseline, therapeutic success with sustained immunological and virological responses was observed in 80 (32.6%) children; immunological and virologic nonresponses occurred in 32 (13.0%) children; finally, the majority (133; 54.2%) of the remaining children showed discordant immunovirological responses. Among them, 33 (13.4%) children showed rapid virological responses to ART with an undetectable viral load, whereas immunological responses remained absent after 6 months of treatment and increased progressively over time in most of the cases, suggesting slow immunorestoration. Notably, nearly half of the children (40.8% at baseline and 48.2% at follow-up) harbored discordant immunovirological responses with a paradoxically high CD4 T-cell count and HIV-1 RNA load, which are always associated with high levels of drug resistance mutations. The latter category showed a significant increase over time, with a growth rate of 1.23% per year of follow-up.Our STROBE-compliant study demonstrates the high heterogeneity of biological responses under ART in children with frequent passage from 1 category to another over time. Close biological evaluation with access to routine plasma HIV-1 RNA load monitoring is crucial for adapting the complex outcomes of ART in HIV-infected children born from infected mothers.

Background: Regenerating islet-derived protein 3α (REG3α) is an antimicrobial peptide secreted by intestinal Paneth cells. Circulating REG3α has been identified as a gut damage marker in inflammatory bowel diseases. People living with human immunodeficiency virus (PWH) on antiretroviral therapy (ART) present with an abnormal intestinal landscape leading to microbial translocation, persistent inflammation, and development of non-AIDS comorbidities. Herein, we assessed REG3α as a marker of gut damage in PWH., Methods: Plasma from 169 adult PWH, including 30 elite controllers (ECs), and 30 human immunodeficiency virus (HIV)-uninfected controls were assessed. REG3α plasma levels were compared with HIV disease progression, epithelial gut damage, microbial translocation, and immune activation markers., Results: Cross-sectionally, REG3α levels were elevated in untreated and ART-treated PWH compared with controls. ECs also had elevated REG3α levels compared to controls. Longitudinally, REG3α levels increased in PWH without ART and decreased in those who initiated ART. REG3α levels were inversely associated with CD4 T-cell count and CD4:CD8 ratio, while positively correlated with HIV viral load in untreated participants, and with fungal product translocation and inflammatory markers in all PWH., Conclusions: Plasma REG3α levels were elevated in PWH, including ECs. The gut inflammatory marker REG3α may be used to evaluate therapeutic interventions and predict non-AIDS comorbidity risks in PWH., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

HIV's ability to persist during suppressive antiretroviral therapy is the main barrier to cure. Immune-privileged tissues, such as the testes, may constitute distinctive sites of HIV persistence, but this has been challenging to study in humans. We analyzed the proviral burden and genetics in the blood and testes of 10 individuals on suppressive therapy who underwent elective gender-affirming surgery. HIV DNA levels in matched blood and testes were quantified by quantitative PCR, and subgenomic proviral sequences ( nef region) were characterized from single templates. HIV diversity, compartmentalization, and immune escape burden were assessed using genetic and phylogenetic approaches. Diverse proviruses were recovered from the blood (396 sequences; 354 nef -intact sequences) and testes (326 sequences; 309 nef -intact sequences) of all participants. Notably, the frequency of identical HIV sequences varied markedly between and within individuals. Nevertheless, proviral loads, within-host unique HIV sequence diversity, and the immune escape burden correlated positively between blood and testes. When all intact nef sequences were evaluated, 60% of participants exhibited significant blood-testis genetic compartmentalization, but none did so when the evaluation was restricted to unique sequences per site, suggesting that compartmentalization, when present, is attributable to the clonal expansion of HIV-infected cells. Our observations confirm the testes as a site of HIV persistence and suggest that individuals with larger and more diverse blood reservoirs will have larger and more diverse testis reservoirs. Furthermore, while the testis microenvironment may not be sufficiently unique to facilitate the seeding of unique viral populations therein, differential clonal expansion dynamics may be at play, which may complicate HIV eradication. IMPORTANCE Two key questions in HIV reservoir biology are whether immune-privileged tissues, such as the testes, harbor distinctive proviral populations during suppressive therapy and, if so, by what mechanism. While our results indicated that blood-testis HIV genetic compartmentalization was reasonably common (60%), it was always attributable to differential frequencies of identical HIV sequences between sites. No blood-tissue data set retained evidence of compartmentalization when only unique HIV sequences per site were considered; moreover, HIV immune escape mutation burdens were highly concordant between sites. We conclude that the principal mechanism by which blood and testis reservoirs differ is not via seeding of divergent HIV sequences therein but, rather, via differential clonal expansion of latently infected cells. Thus, while viral diversity and escape-related barriers to HIV eradication are of a broadly similar magnitude across the blood and testes, clonal expansion represents a challenge. The results support individualized analysis of within-host reservoir diversity to inform curative approaches., (Copyright © 2019 American Society for Microbiology.)

Interleukin-18 (IL-18) is a pleiotropic cytokine of the IL-1 family with multiple context dependent functions. We and others have shown that HIV infection is accompanied by increased circulating levels of IL-18 along with decreased levels of its antagonist, Interleukin-18 Binding Protein (IL-18BP). The infection is also accompanied by intestinal inflammation and decreased intestinal integrity as measured by intestinal permeability, regeneration and repair. However, little is known concerning the relation between high level of IL-18 associated with the viral infection and intestinal permeability. Here we demonstrate that HIV treatment increases production of IL-18 and decreases that of IL-18BP production in human intestinal epithelial cell (IEC) lines. IL-18 causes apoptosis of the IEC by activating caspase-1 and caspase-3. It induces epithelial barrier hyperpermeability by decreasing and disrupting both tight and adherens junction proteins, occludin, claudin 2 and beta-catenin. Disorganization of F-actin was also observed in the IEC that were exposed to the cytokine. Moreover IL-18 decreases transepithelial electrical resistance (TEER) in Caco-2 and increases permeability in HT29 monolayers. The cells' treatment with IL-18 causes an increase in the expression of phosphorylated myosin II regulatory light-chain (p-MLC) and myosin light-chain kinase (MLCK), and a decrease in phosphorylated Signal Transducer and Activator of Transcription (p-STAT)-5. This increase in p-MLC is suppressed by a Rho-kinase (ROCK)-specific inhibitor. Interestingly, the levels of the cytokine correlate with those of LPS in the circulation in three different categories of HIV infected patients (HAART-naïve and HAART-treated HIV-infected individuals, and Elite controls) as well as in healthy controls. Collectively, these results suggest that the HIV-induced IL-18 plays a role in increased intestinal permeability and microbial translocation observed in HIV-infected individuals.

A large cohort of 220 HIV-1-infected children (median [range] age: 12 [4-17] years) was cared and followed up in the Central African Republic, including 198 in 1st-line and 22 in 2nd-line antiretroviral regimens. Patients were monitored clinically and biologically for HIV-1 RNA load and drug resistance mutations (DRMs) genotyping. A total of 87 (40%) study children were virological responders and 133 (60%) nonresponders. In children with detectable viral load, the majority (129; 97%) represented a virological failure. In children receiving 1st-line regimens in virological failure for whom genotypic resistance test was available, 45% displayed viruses harboring at least 1 DRM to NNRTI or NRTI, and 26% showed at least 1 major DRM to NNRTI or NRTI; more than half of children in 1st-line regimens were resistant to 1st-generation NNRTI and 24% of the children in 1st-line regimens had a major DRMs to PI. Virological failure and selection of DRMs were both associated with poor adherence. These observations demonstrate high rate of virological failure after 3 to 5 years of 1st-line or 2nd-line antiretroviral treatment, which is generally associated with DRMs and therapeutic failure. Overall, more than half (55%) of children receiving 1st-line antiretroviral treatment for a median of 3.4 years showed virological failure and antiretroviral-resistance and thus eligible to 2nd-line treatment. Furthermore, two-third (64%) of children under 2nd-line therapy were eligible to 3rd-line regimen. Taken together, these observations point the necessity to monitor antiretroviral-treated children by plasma HIV-1 RNA load to diagnose as early as possible the therapeutic failure and operate switch to a new therapeutic line.

IL-18 is a pro-inflammatory cytokine belonging to the IL-1 family and is produced in the body from macrophages, epithelial and dendritic cells, keratinocytes, adrenal cortex etc. The cytokine is produced as an inactive precursor that is cleaved inside cells into its mature form by activated caspase 1, which exists as an inactive precursor in human cells and requires assembly of an inflammasomes for its activation. We show here for the first time that human platelets contain transcripts for the IL-18 gene. They synthesize the cytokine de novo, process and release it upon activation. The activation also results in the assembly of an inflammasome and activation of caspase-1. Platelets also contain the IL-18 antagonist, the IL-18-Binding Protein (IL-18BP); however, it is not synthesized in them de novo, is present in pre-made form and is released irrespective of platelet activation. IL-18 and IL-18BP co-localize to α granules inside platelets and are secreted out with different kinetics. Platelet activation contributes to plasma concentrations in healthy individuals, as their plasma samples contain abundant IL-18, while their platelet-poor plasma samples contain very little amounts of the cytokine. The plasma and PPP samples from these donors, however, contain comparable amounts of IL-18BP. Unlike healthy individuals, the platelet-poor plasma from HIV-infected individuals contains significant amounts of IL-18. Our findings have important implications for viral infections and other human diseases that are accompanied by platelet activation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

An imbalance between IL-18 and its antagonist, IL-18 Binding Protein, occurs in the circulation of HIV-infected individuals. We show here for the first time that HIV-infected Long Term Non-Progressors (LTNPs) do not develop this imbalance, and maintain normal levels of IL-18BP in the circulation. Their circulating levels of the antagonist correlate negatively with viral loads and show a positive trend with CD4+ T cells counts. The maintenance of normal production of IL-18BP may contribute, at least in part, to the ability of LTNPs to delay AIDS progression., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Th17 cells are permissive to HIV-1 infection and their depletion from the gut of infected individuals leads to microbial translocation, a major cause for non-AIDS co-morbidities. Most recent evidence supports the contribution of long-lived Th17 cells to HIV persistence during antiretroviral therapy (ART). However, the identity of long-lived Th17 cells remains unknown., Results: Here, we performed an in-depth transcriptional and functional characterization of four distinct Th17 subsets and investigated their contribution to HIV reservoir persistence during ART. In addition to the previously characterized CCR6(+)CCR4(+) (Th17) and CCR6(+)CXCR3(+) (Th1Th17) subsets, we reveal the existence of two novel CCR6(+) subsets, lacking (double negative, CCR6(+)DN) or co-expressing CXCR3 and CCR4 (double positive, CCR6(+)DP). The four subsets shared multiple Th17-polarization markers, a fraction of cells proliferated in response to C. albicans, and exhibited lineage commitment and plasticity when cultured under Th17 and Th1 conditions, respectively. Of note, fractions of CCR6(+)DN and Th17 demonstrated stable Th17-lineage commitment under Th1-polarization conditions. Among the four subsets, CCR6(+)DN expressed a unique transcriptional signature indicative of early Th17 development (IL-17F, STAT3), lymph-node homing (CCR7, CD62L), follicular help (CXCR5, BCL6, ASCL2), and self-renewal (LEFI, MYC, TERC). Cross sectional and longitudinal studies demonstrated that CCR6(+)DN cells were the most predominant CCR6(+) subset in the blood before and after ART initiation; high frequencies of these cells were similarly observed in inguinal lymph nodes of individuals receiving long-term ART. Importantly, replication competent HIV was isolated from CCR6(+)DN of ART-treated individuals., Conclusions: Together, these results provide new insights into the functional heterogeneity of Th17-polarized CCR6(+)CD4(+) T-cells and support the major contribution of CCR6(+)DN cells to HIV persistence during ART.

IL-18 is a pleiotropic and multifunctional cytokine that belongs to the IL-1 family. It is produced as a biologically inactive precursor, which is cleaved into its active mature form mainly by caspase-1. The caspase becomes active from its inactive precursor (procaspase-1) upon assembly of an inflammasome. Because of IL-18's potential pro-inflammatory and tissue destructive effects, its biological activities are tightly controlled in the body by its naturally occurring antagonist called IL-18BP. The antagonist is produced in the body both constitutively and in response to an increased production of IL-18 as a negative feedback mechanism. Under physiological conditions, most of IL-18 in the circulation is bound with IL-18BP and is inactive. However, an imbalance in the production of IL-18 and its antagonist (an increase in the production of IL-18 with a decrease, no increase or an insufficient increase in the production of IL-18BP) has been described in many chronic inflammatory diseases in humans. The imbalance results in an increase in the concentrations of free IL-18 (unbound with its antagonist) resulting in increased biological activities of the cytokine that contribute towards pathogenesis of the disease. In this article, we provide an overview of the current biology of IL-18 and its antagonist, discuss how the imbalance occurs in HIV infections and how it contributes towards development of AIDS and other non-AIDS-associated clinical conditions occurring in HIV-infected individuals undergoing combination anti-retroviral therapy (cART). Finally, we discuss challenges facing immunotherapeutic strategies aimed at restoring balance between IL-18 and its antagonist in these patients., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

HIV-1 infection leads to a depletion of CD4 T-cells associated with a persistent immune inflammation and changes in cellular metabolism. Most effort of managing HIV infection with combination of antiretroviral therapies (ART) has been focused on CD4 T-cell recovery, while control of persistent immune inflammation and metabolism were relatively underappreciated in the past. Recent discoveries on the interplay between innate immunity, inflammation (especially the inflammasome) and metabolic changes in the context of cancer and autoimmunity provide an emerging field for chronic viral infections including HIV-1. In a previous review, we described the deregulated metabolism contributing to immune dysfunctions such as alteration of memory T-cell responses, mucosal protection, and dendritic cell-related antigen presentation. Here, we summarize the latest knowledge on the detrimental influence of long-lasting inflammation and inflammasome activation induced by HIV-1, gut dysbiosis, and bacterial translocation, on metabolism during the course of viral infection. We also report on the inability of ART to fully counteract inflammation, resulting in partial metabolic improvement and leading to an insufficient decrease in the risk of non-AIDS events. Further advances in our understanding of the relationship between inflammation, altered metabolism, and long-term ART is warranted. Additionally, there is a critical need for developing new strategies to regulate the pro-inflammatory signals to enhance cellular metabolism and immune functions in order to improve the quality of life of individuals living with HIV-1., (Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.)

Background: The HIV-1 infection is characterized by profound CD4(+) T cell destruction and a marked Th17 dysfunction at the mucosal level. Viral suppressive antiretroviral therapy restores Th1 but not Th17 cells. Although several key HIV dependency factors (HDF) were identified in the past years via genome-wide siRNA screens in cell lines, molecular determinants of HIV permissiveness in primary Th17 cells remain to be elucidated., Results: In an effort to orient Th17-targeted reconstitution strategies, we investigated molecular mechanisms of HIV permissiveness in Th17 cells. Genome-wide transcriptional profiling in memory CD4(+) T-cell subsets enriched in cells exhibiting Th17 (CCR4(+)CCR6(+)), Th1 (CXCR3(+)CCR6(-)), Th2 (CCR4(+)CCR6(-)), and Th1Th17 (CXCR3(+)CCR6(+)) features revealed remarkable transcriptional differences between Th17 and Th1 subsets. The HIV-DNA integration was superior in Th17 versus Th1 upon exposure to both wild-type and VSV-G-pseudotyped HIV; this indicates that post-entry mechanisms contribute to viral replication in Th17. Transcripts significantly enriched in Th17 versus Th1 were previously associated with the regulation of TCR signaling (ZAP-70, Lck, and CD96) and Th17 polarization (RORγt, ARNTL, PTPN13, and RUNX1). A meta-analysis using the NCBI HIV Interaction Database revealed a set of Th17-specific HIV dependency factors (HDFs): PARG, PAK2, KLF2, ITGB7, PTEN, ATG16L1, Alix/AIP1/PDCD6IP, LGALS3, JAK1, TRIM8, MALT1, FOXO3, ARNTL/BMAL1, ABCB1/MDR1, TNFSF13B/BAFF, and CDKN1B. Functional studies demonstrated an increased ability of Th17 versus Th1 cells to respond to TCR triggering in terms of NF-κB nuclear translocation/DNA-binding activity and proliferation. Finally, RNA interference studies identified MAP3K4 and PTPN13 as two novel Th17-specific HDFs., Conclusions: The transcriptional program of Th17 cells includes molecules regulating HIV replication at multiple post-entry steps that may represent potential targets for novel therapies aimed at protecting Th17 cells from infection and subsequent depletion in HIV-infected subjects.

Background: Because of factors only partly understood, the generalized elevated immune activation and inflammation characterizing HIV-1-infected patients are corrected incompletely with antiretroviral therapy (ART). Extracellular vesicles (EVs) including exosomes and microvesicles released by several cell types may contribute to immune activation and dysfunction. EV size, abundance, and content appear to differ according to infection phase, disease progression, and ART., Methods: We examined whether the size of EVs and the abundance of exosomes in plasma are associated with cell and tissue activation as well as with viral production. Acetylcholinesterase-bearing (AChE+) exosomes in plasma were quantified using an AChE assay. EV size was analyzed using dynamic light scattering. Proteins and microRNAs present in EVs were detected by Western blot and real-time polymerase chain reaction, respectively., Results: Exosomes were found more abundant in the plasma of ART-naive patients. EV size was larger in ART-naive than in ART-suppressed patients, elite controllers, or healthy control subjects. Both exosome abundance and EV sizes were inversely correlated with CD4/CD8 T-cell ratio and neutrophil, platelet, and CD4 T-cell counts and positively correlated with CD8 T-cell counts. A negative correlation was found between CD4 T-cell nadir and exosome abundance, but not EV size. Levels of miR-155 and miR-223 but not miR-92 were strongly correlated negatively with EV abundance and size in ART-naive patients., Conclusions: Monitoring of circulating EVs and EV-borne microRNA is possible and may provide new insight into HIV-1 pathogenesis, disease progression, and the associated inflammatory state, as well as the efficacy of ART and the treatments intended to reduce immune activation.

Background: Despite the benefit of maintaining inactive Nucleotide/side reverse transcriptase inhibitors (NRTIs) in salvage regimens, they are associated with increased toxicity and treatment costs. Current evidence suggests that NRTI-sparing regimens in patients failing ART are non-inferior to NRTI-including regimens. This study aimed to evaluate the impact of removing at least one inactive NRTI on virologic, safety, and financial outcomes., Methods: Drug-resistant, virologically suppressed patients with CD4 >250 cells/ml on a stable regimen of four or more antiretrovirals (ARVs) were enrolled in a 48-week prospective, open-label pilot trial. One inactive NRTI was removed at baseline. Patients taking over five ARVs had a second inactive NRTI removed at 24 weeks. Viral load, CD4 count, and adverse events were assessed at baseline, 24, and 48 weeks., Results: Thirty-one male patients participated. Twenty-nine (94%) patients had lamivudine (3TC) or emtricitabine (FTC) removed and four patients had an additional NRTI removed. One patient was excluded at week 26 for discontinuing an active NRTI. All patients maintained undetectable viral loads at weeks 24 (100%) and 48 [PP = 100%; Intent-to-treat (ITT) = 97%]. At 48 weeks, patients had a median gain of 20 CD4 (IQR: - 50, +133; mean +39) compared to baseline. Three patients exhibited Grade III bilirubin elevation (two Grade II and one Grade III at baseline), which returned to baseline levels. No serious adverse events were observed. Removal of one or two ARVs equated to a mean annual savings of $3319 CDN (11%) and $8630 CDN (24%), respectively., Conclusion: Removing inactive NRTIs in patients with a controlled viral load appears to be safe, maintains virological suppression, and reduces treatment costs.

Background: Depletion of mucosal Th17 cells during HIV/SIV infections is a major cause for microbial translocation, chronic immune activation, and disease progression. Mechanisms contributing to Th17 deficit are not fully elucidated. Here we investigated alterations in the Th17 polarization potential of naive-like CD4(+) T-cells, depletion of Th17-commited subsets during HIV pathogenesis, and Th17 restoration in response to antiretroviral therapy (ART)., Results: Peripheral blood CD4(+) T-cells expressing a naive-like phenotype (CD45RA(+)CCR7(+)) from chronically HIV-infected subjects receiving ART (CI on ART; median CD4 counts 592 cells/μl; viral load: <50 HIV-RNA copies/ml; time since infection: 156 months) compared to uninfected controls (HIV-) were impaired in their survival and Th17 polarization potential in vitro. In HIV- controls, IL-17A-producing cells mainly originated from naive-like T-cells with a regulatory phenotype (nTregs: CD25(high)CD127(-)FoxP3(+)) and from CD25(+)CD127(+)FoxP3(-) cells (DP, double positive). Th17-polarized conventional naive CD4(+) T-cells (nT: CD25(-)CD127(+)FoxP3(-)) also produced IL17A, but at lower frequency compared to nTregs and DP. In CI on ART subjects, the frequency/counts of nTreg and DP were significantly diminished compared to HIV- controls, and this paucity was further associated with decreased proportions of memory T-cells producing IL-17A and expressing Th17 markers (CCR6(+)CD26(+)CD161(+), mTh17). nTregs and DP compared to nT cells harbored superior levels of integrated/non-integrated HIV-DNA in CI on ART subjects, suggesting that permissiveness to integrative/abortive infection contributes to impaired survival and Th17 polarization of lineage-committed cells. A cross-sectional study in CI on ART subjects revealed that nTregs, DP and mTh17 counts were negatively correlated with the time post-infection ART was initiated and positively correlated with nadir CD4 counts. Finally, a longitudinal analysis in a HIV primary infection cohort demonstrated a tendency for increased nTreg, DP, and mTh17 counts with ART initiation during the first year of infection., Conclusions: These results support a model in which the paucity of phenotypically naive nTregs and DP cells, caused by integrative/abortive HIV infection and/or other mechanisms, contributes to Th17 deficiency in HIV-infected subjects. Early ART initiation, treatment intensification with integrase inhibitors, and/or other alternative interventions aimed at preserving/restoring the pool of cells prone to acquire Th17 functions may significantly improve mucosal immunity in HIV-infected subjects.

Upon discontinuation of highly active antiretroviral therapy (HAART), human immunodeficiency virus (HIV)-infected individuals experience a brisk rebound in blood plasma viremia due to the exodus of HIV from various body reservoirs. Assessment of HIV dynamics during HAART and following treatment discontinuation is essential to better understand HIV persistence. Here we will first provide a brief overview of the molecular mechanisms involved in HIV reservoir formation and persistence. After a summary of HAART-mediated HIV decay within peripheral blood, we discuss findings from clinical studies examining the effects of HAART initiation and interruption on HIV reservoir dynamics in major anatomical compartments, including lymph nodes and spleen, gut associated lymphoid tissue, reproductive organs, the central nervous system, and the lungs. Features contributing to these reservoirs as distinct compartments, including anatomical features, the presence of drug transporters, and the effect of co-infection, are also discussed.

Background: Approximately 30% of HIV-1-infected patients receiving antiretroviral therapy who achieve virologic control have unsatisfactory immune reconstitution, with CD4+ T-cell counts persistently below 350 cells/μL. These patients are at elevated risk for clinical progression to AIDS and non-AIDS events. CD4+ T-cell depletion following infection and persistent immune activation can partially explain this low CD4+ T-cell recovery. Recent data suggest a link between the tryptophan oxidation pathway, immune activation and HIV disease progression based on overstimulation of the tryptophan oxidation pathway by HIV antigens and by interferon-gamma. This overstimulation reduces levels of circulating tryptophan, resulting in inflammation which has been implicated in the development of neurocognitive dysfunction. Niacin (vitamin B3) is able to control the excess tryptophan oxidation, correcting tryptophan depletion, and therefore represents an interesting strategy to improve CD4 recovery.We aim to design a crossover proof-of-concept study to assess supplementation with an extended-release form of niacin (Niaspan FCT™) in combination with antiretroviral therapy, compared to antiretroviral therapy alone, on T-cell immune activation as defined by changes in the percentage of CD8+ CD38+ HLA-DR+ T-cells., Methods/design: This randomized, open-label, interventional crossover study with an immediate versus deferred use of Niaspan FCT for 24 weeks will assess its ability to reduce immune activation and thus increase CD4 recovery in 20 HIV-infected individuals with suboptimal immune responses despite sustained virologic suppression. A substudy evaluating neurocognitive function will also be conducted., Discussion: This randomized trial will provide an opportunity to evaluate the potential benefit of oral extended-release niacin, a drug that can indirectly increase tryptophan, to reduce immune activation and in turn increase CD4+ T-cell recovery. The study will also allow for the evaluation of the impact of Niaspan FCT on neurocognitive function in HIV-infected individuals with suboptimal immune responses despite sustained virologic suppression., Trial Registration: This study was registered with ClinicalTrials.gov on 17 December 2013 (registration number: NCT02018965).

Human immunodeficiency virus type 1 (HIV-1) infection is characterized by chronic immune activation and suppressed T-lymphocyte functions. Here we report that CD73, both a coactivator molecule of T cells and an immunosuppressive ecto-enzyme through adenosine production, is only weakly expressed by CD8+ T cells of HIV-infected patients and only partially restored after successful antiviral treatment. CD73 expression on CD8+ T cells correlates inversely with cell activation both ex vivo and in vitro. However, CD8+ T cells from HIV controllers (HICs), which spontaneously control HIV replication, express CD73 strongly, despite residual immune activation. Finally, we demonstrate that CD73 is involved in the HIV-specific CD8+ T-cell expansion. Thus, we show that CD73 is central to the functionality of HIV-specific CD8+ T cells and that the preservation of HIV-specific CD73+ CD8+ T cells is a characteristic of HICs. These observations reveal a novel mechanism involved in the control of viral replication.

Failure of antiretroviral therapy to eradicate HIV, even in individuals who suppress the virus to undetectable levels, is a consequence of persistent infection in latently infected cells and within anatomical reservoirs. Support for the notion that the lungs are distinct anatomical reservoirs of HIV comes from a spectrum of studies that have documented different levels of HIV within the lungs compared with the peripheral blood. Different HIV variants have also been found within these two compartments, including variants with distinct antiretroviral resistance mutations. Given that macrophages are long-lived cellular reservoirs of HIV because of their resistance to apoptosis, HIV can persist for prolonged periods within alveolar macrophages that are abundant within the lungs. Furthermore, the large number of cells in close proximity within the lungs provides fertile grounds for cell-to-cell spread of HIV. Distinct immunological pressures in the lungs compared with the peripheral blood likely account for differences in HIV levels within these two compartments in addition to the finding of different variants within these regions. Furthermore, coinfections and tobacco may serve as local stimuli to induce further HIV replication within the lungs. Herein, we review the evidence supporting the notion that lungs are important reservoirs of HIV infection, and we discuss various factors influencing HIV burden within these reservoirs., (Copyright © 2013 John Wiley & Sons, Ltd.)

Background: We previously demonstrated that primary Th1Th17 cells are highly permissive to HIV-1, whereas Th1 cells are relatively resistant. Molecular mechanisms underlying these differences remain unknown., Results: Exposure to replication competent and single-round VSV-G pseudotyped HIV strains provide evidence that superior HIV replication in Th1Th17 vs. Th1 cells was regulated by mechanisms located at entry and post-entry levels. Genome-wide transcriptional profiling identified transcripts upregulated (n = 264) and downregulated (n = 235) in Th1Th17 vs. Th1 cells (p-value < 0.05; fold change cut-off 1.3). Gene Set Enrichment Analysis revealed pathways enriched in Th1Th17 (nuclear receptors, trafficking, p38/MAPK, NF-κB, p53/Ras, IL-23) vs. Th1 cells (proteasome, interferon α/β). Differentially expressed genes were classified into biological categories using Gene Ontology. Th1Th17 cells expressed typical Th17 markers (IL-17A/F, IL-22, CCL20, RORC, IL-26, IL-23R, CCR6) and transcripts functionally linked to regulating cell trafficking (CEACAM1, MCAM), activation (CD28, CD40LG, TNFSF13B, TNFSF25, PTPN13, MAP3K4, LTB, CTSH), transcription (PPARγ, RUNX1, ATF5, ARNTL), apoptosis (FASLG), and HIV infection (CXCR6, FURIN). Differential expression of CXCR6, PPARγ, ARNTL, PTPN13, MAP3K4, CTSH, SERPINB6, PTK2, and ISG20 was validated by RT-PCR, flow cytometry and/or confocal microscopy. The nuclear receptor PPARγ was preferentially expressed by Th1Th17 cells. PPARγ RNA interference significantly increased HIV replication at levels post-entry and prior HIV-DNA integration. Finally, the activation of PPARγ pathway via the agonist Rosiglitazone induced the nuclear translocation of PPARγ and a robust inhibition of viral replication., Conclusions: Thus, transcriptional profiling in Th1Th17 vs. Th1 cells demonstrated that HIV permissiveness is associated with a superior state of cellular activation and limited antiviral properties and identified PPARγ as an intrinsic negative regulator of viral replication. Therefore, triggering PPARγ pathway via non-toxic agonists may contribute to limiting covert HIV replication and disease progression during antiretroviral treatment.

Background: We evaluated whether B cell-derived immune defenses of the gastro-intestinal tract are activated to produce HIV-specific antibodies in children continuously exposed to HIV via breast-feeding., Methods: Couples of HIV-1-infected mothers (n = 14) and their breastfed non HIV-infected (n = 8) and HIV-infected (n = 6) babies, and healthy HIV-negative mothers and breastfed babies (n = 10) as controls, were prospectively included at the Complexe Pédiatrique of Bangui, Central African Republic. Immunoglobulins (IgA, IgG and IgM) and anti-gp160 antibodies from mother's milk and stools of breastfed children were quantified by ELISA. Immunoaffinity purified anti-gp160 antibodies were characterized functionally regarding their capacity to reduce attachment and/or infection of R5- and X4- tropic HIV-1 strains on human colorectal epithelial HT29 cells line or monocyte-derived-macrophages (MDM)., Results: The levels of total IgA and IgG were increased in milk of HIV-infected mothers and stools of HIV-exposed children, indicating the activation of B cell-derived mucosal immunity. Breast milk samples as well as stool samples from HIV-negative and HIV-infected babies exposed to HIV by breast-feeding, contained high levels of HIV-specific antibodies, mainly IgG antibodies, less frequently IgA antibodies, and rarely IgM antibodies. Relative ratios of excretion by reference to lactoferrin calculated for HIV-specific IgA, IgG and IgM in stools of HIV-exposed children were largely superior to 1, indicating active production of HIV-specific antibodies by the intestinal mucosa. Antibodies to gp160 purified from pooled stools of HIV-exposed breastfed children inhibited the attachment of HIV-1NDK on HT29 cells by 63% and on MDM by 77%, and the attachment of HIV-1JRCSF on MDM by 40%; and the infection of MDM by HIV-1JRCSF by 93%., Conclusions: The intestinal mucosa of children exposed to HIV by breast-feeding produces HIV-specific antibodies harbouring in vitro major functional properties against HIV. These observations lay the conceptual basis for the design of a prophylactic vaccine against HIV in exposed children.

Human urine has been poorly investigated with regard to infection with human immunodeficiency virus (HIV). Here, we have studied the anti-infective functional properties of human urine against HIV. The effect of fresh urine pools on CCR5- and CXCR4-tropic HIV-1 was evaluated by using four in vitro mucosal models: reduction of infectivity of urine-treated HIV-1 particles, HIV-1 attachment to immature monocyte-derived dendritic cells (iMDDC), transfer of HIV-1 particles from iMDDC to autologous CD4 T cells, and HIV-1 transcytosis through epithelial cells. Human urine partially disrupted both CCR5- and CXCR4-tropic HIV-1 particles, moderately decreased the adsorption of HIV-1 on dendritic cells, and partially decreased the transfer of HIV-1 particles from dendritic cells to autologous T cells. These findings demonstrate partial inactivation of HIV infectivity and suggest that voiding urine after coitus could play a potential role in reducing the risk of HIV infection by both mechanically flushing out and neutralizing the infectivity of HIV-1 particles present in the genital tract.

Background: Evaluation of the dynamics of enfuvirtide (ENF) resistance mutations after ENF withdrawal in patients with virological failure under salvage therapy may be helpful to optimize the management of ENF in human immunodeficiency virus (HIV)-infected patients., Methods: Seven patients with a failing ENF-containing regimen, initiated for at least 3 months (median 6.4 months, range 3-14), were included and followed up prospectively at the time of virological failure. Genotypic analysis of the gp41 region by bulk sequencing and clonal analysis was performed in plasma and/or peripheral blood mononuclear cells to detect ENF resistance mutations., Results: Genotypic profiles of ENF-resistant variants at ENF discontinuation were as follows: V38A in 3 patients, V38A+N42T+N43D in 1 patient, N43D in 2 patients, and N43K in 1 patient. Clonal analysis showed that maintaining ENF treatment after virological failure has an impact on both (1) the number of resistance profiles detected, and (2) the time of persistence of ENF-resistant variants. ENF-resistant variants were archived in HIV DNA in 5/7 patients. At 1 month after ENF withdrawal, no significant increase in HIV-1 viral load was observed., Conclusion: The persistence of ENF-resistant variants was found to be correlated to exposure time to failing drug. ENF withdrawal should be considered in patients with virological failure to preserve the possible efficacy of ENF recycling or upcoming entry inhibitors.

We showed herein the potent virucidal effect of soap and water solutions against both CCR5-tropic and CXCR4-tropic cell-free HIV-1 strains, and cytotoxicity for HIV-1-infected lymphocytes during short incubation durations, ranging from 30 seconds to 2 minutes. These observations indicate a rapid inhibitory effect of soap and water on viral infectivity.

Objectives: To block the different mechanisms of HIV mucosal transmission, it is likely that use of several microbicide molecules will lead to the best protection against HIV transmission. Indeed, the combination of microbicides with complementary mechanisms of action is expected to increase the antiviral potency of the formulation., Methods: The gp120-interacting plant lectin HHA ('Hippeastrum hybrid agglutinin'), the non-nucleoside reverse transcriptase inhibitor KRV2110 and the fusion inhibitor enfuvirtide (T20) were combined in 12 drug associations by using the Ray combination design method. Their activity against HIV-1(BaL) was assessed by the lymphocyte infectivity reduction assay and by the single-cycle BaL pseudovirus (PV) assay. In addition, their cell tolerance was evaluated for HEC-1 and HeLa epithelial cell lines, both originating from genital tissue., Results: All evaluated combinations showed synergistic activity in both lymphocyte infectivity reduction and single-cycle BaL PV assays. The combination HHA + KRV2110 resulted in the highest cell viability, whereas the combinations including T20 exhibited a dose-dependent decrease in cell viability, demonstrating the differential tolerance of epithelial cell lines to the combinations., Conclusions: These observations provide a rational basis for in vitro testing of microbicide candidate molecule combinations, including anti-HIV-1 and cytotoxic cellular assays.

Objective: To characterize the functional properties of natural autoantibodies capable of preventing in vitro infection by HIV-1, present in normal human serum (NHS), and denoted as IgG-reactive antibodies., Methods: IgG-reactive antibodies were affinity purified both from normal human serum (NHS) and from a GammaBind G Sepharose Flowthrough (GBF) fraction of NHS by affinity chromatography on IgG coupled to CNBr-activated Sepharose (IgG-Sepharose)., Results: The GBF fraction was shown, by Capture ELISA relative to isotype-matched standards, to contain in addition to IgM and IgA isotypes, a low but constant level of IgG isotype. About 15% of the GBF fraction's IgG, compared to only about 0.3% of the NHS IgG, was affinity purified on IgG-Sepharose. On IgG subclass analysis, in contrast to the characteristic dominance of IgG1 in pooled NHS, the IgG-reactive antibodies obtained from NHS and from the GBF fraction each showed a dominance of IgG2. Western blot analysis confirmed the abundance of IgG2, a major IgG subclass reactive against carbohydrate antigens, and showed the presence of IgG2 dimers. The IgG-reactive antibodies separated from the GBF fraction were able to neutralize HIV-1(BaL) strain with approaching 100% and 80% effectiveness at 2 microg/ml and 0.6 microg/ml, respectively, as well as the primary isolates HIV-1(NDK) (X4-tropic isolate) and HIV-1(JR-CSF) (R5-tropic isolate) with an IC50 between 0.4 microg/ml and 1.8 microg/ml for two different preparations., Conclusion: These findings further support our previous proposal for IgG-reactive antibody preparations to be used in the treatment of HIV-1 infected individuals.

Heterosexual contact is the primary mode of human immunodeficiency virus (HIV) type 1 (HIV-1) transmission worldwide. The chemokine receptor CCR5 is the major coreceptor that is associated with the mucosal transmission of R5-tropic HIV-1 during sexual intercourse. The CCR5 molecule is thus a target for antibody-based therapeutic strategies aimed at blocking HIV-1 entry into cells. We have previously demonstrated that polyreactive natural antibodies (NAbs) from therapeutic preparations of immunoglobulin G and from human breast milk contain NAbs directed against CCR5. Such antibodies inhibit the infection of human macrophages and T lymphocytes by R5-tropic isolates of HIV in vitro. In the present study, we demonstrate that human immunoglobulins from the cervicovaginal secretions of HIV-seronegative or HIV-seropositive women contain NAbs directed against the HIV-1 coreceptor CCR5. Natural affinity-purified anti-CCR5 antibodies bound to CCR5 expressed on macrophages and dendritic cells and further inhibited the infection of macrophages and dendritic cells with primary and laboratory-adapted R5-tropic HIV but not with X4-tropic HIV. Natural anti-CCR5 antibodies moderately inhibited R5-tropic HIV transfer from monocyte-derived dendritic cells to autologous T cells. Our results suggest that mucosal anti-CCR5 antibodies from healthy immunocompetent donors may hamper the penetration of HIV and may be suitable for use in the development of novel passive immunotherapy regimens in specific clinical settings of HIV infection.

Background: Plant lectins such as Galanthus nivalis agglutinin (GNA) and Hippeastrum hybrid agglutinin (HHA) are natural proteins able to link mannose residues, and therefore inhibit HIV-target cell interactions. Plant lectins are candidate for microbicide development., Objective: To evaluate the activity against HIV of the mannose-specific plant lectins HHA and GNA at the cellular membrane level of epithelial cells and monocyte-derived dendritic cells (MDDC), two potential target cells of HIV at the genital mucosal level., Methods: The inhibitory effects of HHA and GNA were evaluated on HIV adsorption to genital epithelial HEC-1A cell line, on HIV transcytosis throughout a monolayer of polarized epithelial HEC-1A cells, on HIV adsorption to MDDC and on transfer of HIV from MDDC to autologous T lymphocytes., Results: HHA faintly inhibited attachment to HEC-1A cells of the R5-tropic HIV-1Ba-L strain, in a dose-dependent manner, whereas GNA moderately inhibited HIV adsorption in the same context, but only at high drug doses. Only HHA, but not GNA, inhibited HIV-1JR-CSF transcytosis in a dose-dependent manner. By confocal microscopy, HHA, but not GNA, was adsorbed at the epithelial cell surface, suggesting that HHA interacts specifically with receptors mediating HIV-1 transcytosis. Both plant lectins partially inhibited HIV attachment to MDDC. HHA inhibited more efficiently the transfer of HIV from MDDC to T cell, than GNA. Both HHA and GNA lacked toxicity below 200 microg/ml irrespective the cellular system used and do not disturb the monolayer integrity of epithelial cells., Conclusion: These observations demonstrate higher inhibitory activities of the lectin plant HHA by comparison to GNA, on HIV adsorption to HEC-1A cell line, HIV transcytosis through HEC-1A cell line monolayer, HIV adsorption to MDDC and HIV transfer from MDDC to T cells, highlighting the potential interest of HHA as effective microbicide against HIV.

Currently, there is no cure for human immunodeficiency virus type 1 (HIV-1) infection. However, combined antiretroviral therapy (cART) aids in viral latency and prevents the progression of HIV-1 infection into acquired immunodeficiency syndrome (AIDS). cART has extended many lives, but people living with HIV-1 (PLWH) face lifelong ailments such as HIV-associated neurocognitive disorders (HAND) that range from asymptomatic HAND to HIV-1-associated dementia. HAND has been attributed to chronic inflammation and low-level infection within the central nervous system (CNS) caused by proinflammatory cytokines and viral products. These molecules are shuttled into the CNS within extracellular vesicles (EVs), lipid bound nanoparticles, and are released from cells as a form of intercellular communication. This study investigates the impact of cannabidiol (CBD), as a promising and potential therapeutic for HAND patients, and a similar synthetic molecule, HU308, on the EVs released from HIV-1-infected myeloid cells as well as HIV-1-infected 3D neurospheres. The data shows that both CBD and HU308 decrease non-coding and coding viral RNA (TAR and env) as well as proinflammatory cytokines as IL-1β and TNF-α mRNA. This decrease in viral RNA occurs in in vitro differentiated primary macrophages, in EVs released from HIV-1-infected cells monocytes, and infected neurospheres. Furthermore, a 3D neurosphere model shows an overall decrease in proinflammatory mRNA with HU308. Finally, using a humanized mouse model of HIV-1 infection, plasma viral RNA was shown to significantly decrease with HU308 alone and was most effective in combination with cART, even when compared to the typical cART treatment. Overall, CBD or HU308 may be a viable option to decrease EV release and associated cytokines which would dampen the virus spread and may be used in effective treatment of HAND in combination with cART. [ABSTRACT FROM AUTHOR]

Introduction: Herpes Simplex Virus-2 (HSV-2) is known to be a potent co-factor of Human Immunodeficiency type 1 virus (HIV-1) heterosexual transmission. We were interested in assessing the distribution of HIV-1 and HSV-2 epidemics at the national level in Chad. Methodology: In 2007, a population-based anonymous serosurvey for HIV-1 and HSV-2 infections, using dried blood spots, was conducted. The study included 548 adults living in 15 regions of Chad. After specimen elution, serological testing for HIV and HSV-2 infections was performed. Results: Countrywide, the HIV-1 and HSV-2 seroprevalences were 11.1% and 15.7%, respectively. A positive correlation was observed with the highest HIV-1 prevalence seen in regions of the highest HSV-2 prevalence, especially in two conflict-affected eastern provinces of Darfur. Conclusion: Urgent public health interventions are needed in regions of Chad where high HSV-2 prevalence may be increasing the risk of HIV propagation. [ABSTRACT FROM AUTHOR]