Background: 94% of HIV+ persons in the Canadian HIV and Aging Cohort Study (CHACS) are cytomegalovirus (CMV) co-infected. CMV infection drives the expansion of Natural Killer (NK) cells expressing the NKG2C activating receptor. HIV/CMV co-infection drive the expansion of NKG2C+ adaptive-like NK (adaptNK) cells to levels higher than that seen in CMV-mono-infected persons. However, these observations were made using samples from young individuals often aged <40 yrs. We questioned whether this was also the case for older CMV+ persons.

Methods: We studied 139 CMV+ individuals (n=89 ART treated HIV+; n=50 HIV-). Both HIV+ and HIV- groups were dichotomized by age (>40 vs <40 yrs). HIV+/- persons >40 yrs old were from the CHACS; those <40 yrs old were from the Montreal Primary Infection or the St Luc Injection Drug User Cohorts. Of those HIV+, 64 were >40 and 25 were <40 yrs old. Of those HIV-, 36 were >40 and 14 were <40 yrs old. Frozen and thawed PBMCs were stained for extracellular CD3, CD56 and NKG2C. We assessed the frequency (%) of NKG2C+ cells among CD3-CD56dim adapNK cells.

Results: As previously reported, young HIV+CMV+ subjects had a higher % of NKG2C+ adapNK cells than similarly aged CMV-mono-infected persons (median 50% versus 13.3%, respectively, p=0.02 Mann-Whitney test]. However, older HIV+CMV+ and CMV-mono-infected subjects had NKG2C+ adaptNK cell %s that were not significantly different (20% versus 14.2%, p=0.15 Mann-Whitney). The % of NKG2C+ adaptNK cells was negatively correlated with age (r= -0.23, p=0.04, Spearman correlation).

Conclusion: The higher % of NKG2C+ adaptNK cells in HIV+CMV+, versus CMV-mono-infected, persons is age-dependent; NKG2C% declines with age such that in those >40 yrs old their % is no longer significantly higher than that seen in CMV-mono-infected individuals. The mechanism underlying the decline in NKG2C+ adaptNK cell frequency requires further investigation.

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B-cell Activation Factor (BAFF) is a survival and differentiation factor involved in shaping the Marginal Zone (MZ) B-cell pool. We have shown that BAFF levels are augmented in HIV-1 infected progressors, as soon as the acute phase and up to 12 months post therapy. Excess BAFF is concomitant with chronic inflammation, autoimmunity manifestations and B-cell disorders, such as hyperglobulinemia and deregulation of MZ populations, shown to possess a Breg potential.
Excess BAFF has been linked to several chronic inflammatory conditions such as cardiovascular diseases (CVD) like atherosclerosis. Excess BAFF promotes endothelial dysfunction, a risk factor for atherosclerosis, and can be produced by adipocytes, creating a link between obesity and inflammation.
Long term HIV-1 infected individuals appear to develop premature comorbidities normally associated with aging, such as CVD and atherosclerosis. Our hypothesis is that excess BAFF and B-cell deregulations are involved in the premature development of CVD in long term HIV-1 infected individuals from the Canadian HIV and Aging Cohort Study (CHACS). To this end, we have found that HIV+ individuals possess higher levels of BAFF than HIV- individuals even after several years of ART therapy, with membrane BAFF levels being greater in CVD+ individuals. In HIV- individuals, BAFF levels correlate positively with Total Plaque Volume (TPV). Interestingly, levels of A Proliferation-Inducing Ligand (APRIL), an analog of BAFF, were lower in CVD+ individuals. Surprisingly, in HIV+ individuals, APRIL correlates negatively with TVP and with BAFF levels. This data suggests an unexpected atheroprotective role for APRIL. These results suggest that discrepancies in BAFF/APRIL levels could be associated with premature CVD development in long term HIV-1 infected individuals. Interestingly, MZ B-cells are involved in the immunosurveillance of atherosclerosis and palliate against its development in a mice model. BAFF-mediated deregulation of these populations could also contribute to CVD development in HIV+ individuals.

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MicroRNAs are small, non-coding RNAs that modulate gene expression. We set out to study the contribution of miRNAs in modulating HIV-1 infection of CD4+ T lymphocytes. To this end, using next generation RNAseq, we evaluated the expression profile of miRNAs and mRNAs in productively infected, virus-exposed bystander and uninfected lymphocytes. Data from our bioinformatics analyses of differentially expressed miRNAs and their potential mRNA targets reveal a remodeling of several key host pathways that are likely to modulate infection and pathogenesis. Concurrently, we focused our attention on miRNAs-103/107 that were recently shown to function as modulators of CCR5 in macrophages, and investigated their roles in lymphocytes. CD4+ EMT (effector-to-memory transitioning) T cells are a subpopulation of lymphocytes in which the potential for HIV-1 infection is briefly increased due to a transient elevation in CCR5 expression, a condition that promotes latent infection as EMT cells have a reduced ability to transcribe integrated proviral DNA. Our results suggest that miRNAs-103/107 have a role in this transient modulation of CCR5 expression as the progressive decrease in CCR5 coincides with an increase in miRNAs-103/107. In addition, nucleofection of miRNAs-103/107 mimics in CD4+ T cells decreased the level of CCR5 surface expression, while nucleofection of miRNA-103 antagomirs attenuated its decrease. Subsequently, using a double reporter virus (HIV-CRMZ), which allows for flow cytometry-based identification of productively and latently infected cells, we confirmed that CD4+ EMT T cells are more susceptible to latent infection. Experiments aimed at determining the impact of treatment with these miRNAs on the frequency of latent cells are currently underway. Given that the latent reservoir is the main obstacle to the eradication of HIV-1, these data bring new perspectives to counter the establishment of HIV-1 reservoirs.

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Background: We previously demonstrated that ATP-binding cassette (ABC) drug efflux transporters could contribute to low ARV intracellular concentrations in HIV-1 target tissues and cells. Furthermore, studies have reported that these transporters could be induced in activated and/or HIV-infected T-cells. The mammalian target of rapamycin (mTOR) signaling pathway is activated following HIV infection and T-cell activation. Therefore, we examined the regulation of ABC drug efflux transporters by mTOR, and their potential contribution to the inflammatory response following HIV-induced T-cell activation.

Methods: Human peripheral blood mononuclear cells (PBMCs) were exposed to HIV-1 envelope glycoprotein gp120IIIB, HIV pseudotype (pHIVNL4-3) and/or mTOR inhibitors. The expression of ABC transporters, T-cell activation marker CD69, mTOR and GFP+ pHIVNL4-3 was assessed in CD4+ T-cells by Flow cytometry. Proinflammatory cytokines (IL6, TNFα and INFγ) mRNA and protein levels were examined following exposure to pHIVNL4-3 and/or inhibitors of mTOR and ABC transporters by qPCR and ELISA analyses, respectively.

Results: Protein expression of ABC transporters (P-glycoprotein (P-gp), breast cancer resistance protein and multi-drug resistance associated protein-1 (MRP1)) was significantly increased in CD4+ T-cells exposed to gp120IIIB or pHIVNL4-3. Treatment with the selective mTOR pan-inhibitor OSI-027 reversed pHIVNL4-3-induced expression of these transporters in CD4+ T-cells. Inhibition of P-gp by verapamil or PSC833, or MRP1 by MK571, decreased concentrations of TNFα, INFγ and IL-6 in supernatants of PBMC exposed to pHIVNL4-3.

Conclusion: To the best of our knowledge, we present novel data demonstrating that ABC drug efflux transporters are upregulated via mTOR signaling in CD4+ T-cells exposed to pHIVNL4-3. These transporters could limit ARV permeability in HIV target T-cells. Furthermore, these transporters could potentially contribute, in part, to HIV-associated proinflammatory cytokine secretion. This study provides a basis to further assess the role and regulation of ARV drug efflux transporters in T-cell activation and inflammatory response, in the context of HIV infection.

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Background: Drug efflux transporters and metabolic enzymes govern drug disposition and could render antiretroviral drug (ARV) intracellular concentrations suboptimal, thus facilitating HIV infection and HIV reservoir persistence in target cells, such as myeloid cells. In this study, we investigated the expression of these transporters and metabolic enzymes in monocyte subsets and monocyte-derived macrophages (MDMs) of people living with HIV (PLWH) receiving viral suppressive antiretroviral therapy (HIV+ART) and HIV-uninfected individuals (HIV-). Plasma and intracellular ARV concentrations were quantified in HIV+ART cells.

Methods: Classical, intermediate, and non-classical monocytes were identified based on their distinct CD14/CD16 expression. Total monocytes were isolated from peripheral blood mononuclear cells (PBMCs) by negative selection using magnetic beads and differentiated into MDMs by six days culture in the presence of macrophage colony-stimulating factor. mRNA and protein expression of drug transporters and metabolic enzymes were analyzed by qPCR and flow cytometry, respectively. Plasma and intracellular (PBMCs, monocytes, MDM) ARV concentrations were quantified by LC-MS/MS analysis.

Results: The mRNA and/or protein expression of relevant ARV transporters (ABCB1/P-gp, ABCG2/BCRP, ABCC1/MRP1, ABCC4/MRP4, SLC22A1/OCT1, and SLC29A2/ENT2) or metabolic enzymes (CYP2B6, CYP2D6, and UGT1A1), was detected in monocytes and matched MDMs. BCRP and MRP1 protein expression was higher in intermediate compared to classical monocytes of both HIV+ART and HIV- individuals. We obtained novel ARV intracellular concentration data in monocytes of PLWH, whereas concentrations in MDM were mostly undetectable. Plasma ARV concentrations were within previously reported therapeutic ranges.

Conclusion: Herein, we demonstrated the highest frequencies of ARV efflux transporters on intermediate monocytes, a subset expanded during HIV infection, expressing the highest levels of the HIV coreceptor CCR5. These transporters could potentially limit ARV intracellular concentrations and contribute to persistent HIV infection of these cells. These novel findings prompt future investigations on HIV reservoir persistence in tissue-resident myeloid cells in relationship with specific ART regimens.

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While the envelope glycoprotein gp120 is often viewed as the sole target on the surface of HIV virions for vaccines and therapeutics, numerous cellular proteins can also be incorporated into the viral envelope during viral egress (budding). While many cellular proteins have been documented in the HIV envelope, few have been well characterized for their impacts on HIV infection. Recently, the cellular protein P-Selectin Glycoprotein Ligand-1 (PSGL-1/CD162) was identified as a novel host restriction factor that is present in the HIV envelope. The CD162 protein was also shown to have inhibitory effects in other enveloped viruses, including influenza A, SARS-CoV-2 and murine leukemia virus. While the mechanisms by which CD162 reduces viral infection have been characterized, little is known on the mechanism behind incorporation of the protein into the HIV envelope. Similarly, no work has shown whether CD162 retains its biological function of binding to its receptor (P-selectin/CD62P) when present on virions. Here we show that CD162 is incorporated at a higher level than other previously described cellular proteins within the HIV envelope through both semiquantitative and quantitative techniques (antibody-based virus capture and flow virometry, respectively). Through the former we noted differential amounts of CD162 incorporated in viruses produced through transfection and infection. Using flow virometry we derive the most accurate estimates of the number of CD162 proteins on individual virus particles to date. Further, we show that CD162 incorporation employs a Gag-dependent mechanism, although other viral proteins may support optimal levels of incorporation. Finally, we demonstrate that CD162 on virions retains its ability to bind its cognate receptor (P-Selectin) which may have implications on how the virus is trafficked throughout the body, suggesting that the protein may play a more complex role in infection in vivo than what has currently been proposed.

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Background: While antiretroviral drugs effectively prevent human immunodeficiency virus (HIV) infection from progressing into serious disease, they are unable to eliminate the virus from the body. It may be possible to functionally cure HIV infection by modifying autologous hematopoietic stem cells ex vivo and retransplanting them into the patient. One modification strategy uses antiviral short hairpin (sh)RNAs which can be designed to target conserved HIV genomic sequences for degradation. Several anti-HIV-1 shRNAs have been identified that can be transduced into patient cells to make them resistant to HIV-1 replication. The choice of promoter from which shRNAs can be expressed can influence transcriptional efficiency, in turn impacting shRNA potency and potentially cytotoxicity. We aim to identify non-toxic combinations of shRNAs and promoters that will effectively inhibit HIV-1 replication.

Methods: We transduced cells with lentiviral vectors containing top shRNAs identified from three different screens. The best shRNAs were systematically expressed from one of the three human type 3 RNA Polymerase III promoters: 7SK, U6, and H1. Constructs were assessed for their ability to inhibit HIV replication using a reverse transcriptase assay and for their cytotoxicity with a competitive cell growth assay.

Results: Our data suggest that U6- and 7SK-promoted shRNAs delay viral replication. While some of these constructs also showed cytotoxicity, not all U6- and 7SK-promoted shRNAs demonstrated a negative impact. These findings suggest that the efficacy and toxicity of a shRNA depends on the promoter and on the shRNA sequence.

Conclusion: Our results give information on the best RNA-based molecules with the highest potential for clinical use and provide insights into the use of different RNA polymerase III promoters for shRNA gene therapy.

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Viral dissemination occurs early after infection targeting CD4 T cells and monocytes/macrophages. Monocytes derived from bone marrow and tissue resident macrophages (TRMs) derived from yolk sac, are short-lived and long-lived cells, respectively. HIV infects non-lymphoid tissues, such as liver and lung in which TRMs may represent viral reservoirs (VRs). Whereas we demonstrated that early antiretroviral therapy (ART) efficiently prevents infection of monocytes in the blood, spleen and intestine of SIV-treated rhesus macaques (RMs), little is known so far about the role of TRMs, and whether these cells may represent VRs in SIV-infected RMs.

Cells from liver and lung were isolated from RMs infected with SIVmac251. The phenotype of TRMs was analyzed by flow cytometry using specific antibodies including anti-CD14, anti-CD16, as well markers of TRMs such as CD44, CD59, CD35, CD117, CD206, MERKT, and LYVE. The levels of viral DNA and RNA were quantified by qPCR. `

Our results revealed that myeloid cells from the lungs and livers of SIV-infected RMs expressed mostly CD117, CD206 and LYVE markers. By performing a mechanical procedure, instead to use a cocktail of proteases, we preserved CD14 shedding that allowed to identify infiltrate cells. Thus, we also detected infiltrate monocytes (CD14+) that do express TRM markers in the infected tissues. Concomitantly, our data revealed that liver and lung of SIV-infected RMs both contain viral RNA and DNA.

To date we are assessing, which TRM subsets express viral DNA and RNA, and whether early ART prevents the infection of TRM in SIV-infected RMs. Understanding the nature of infected cells under ART is of crucial importance for developing strategies aim to eradicate HIV.

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Control of RNA processing plays a central role in the expression and replication of HIV-1. From a single transcript, over 69 viral mRNAs are generated through the process of alternative RNA splicing. Disrupting the balance of HIV-1 RNA processing inhibits virus replication. Control is mediated in part through the action of host SR proteins whose activity, in turn, is regulated by multiple SR kinases (CLK1-4, SRPKs). Our studies demonstrate that SR kinases play disparate roles in modulating HIV-1 gene expression. Depletion of CLK1 enhanced HIV-1 gene expression, loss of CLK2 or SRPK1 suppressed it, while CLK3 depletion had a modest impact. Altered HIV-1 protein expression reflected changes in viral RNA accumulation. The opposing effects of CLK1 vs CLK2 depletion were due to action at distinct steps; loss of CLK1 increasing HIV-1 promoter function while depletion of CLK2 affected steps post-initiation. Loss of CLK1 also enhanced the response to several latency reversing agents, in part, by increasing the frequency of responding cells, consistent with a role in regulating provirus latency. To determine if modulation of SR kinase function by small molecules could be used to control HIV-1 replication, we screened the GSK library of kinase inhibitors and identified two compounds that suppress HIV-1 gene expression/replication with EC50~ 50 nM. The compounds resulted in dramatic suppression of HIV-1 proteins and viral RNA accumulation with minimal impact on cell viability. The compounds inhibited CLK1 and CLK2 but not CLK3 function and altered expression/activity of select SR proteins in cellulo. These findings demonstrate the unique roles individual SR kinases play in regulating HIV-1 gene expression and validate the targeting of these functions by small molecules for therapeutic benefit to enhance latency reversal, essential for “Kick-and-Kill” strategies, or to silence HIV protein expression for “Block-and-Lock” strategies.

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Human Immunodeficiency Virus (HIV) infection rates remain a global public health concern, and while the use of antiretroviral therapy (ART) has reduced perinatal mother to child transmission rates, there is a growing proportion of infants exposed to HIV in utero. In 2013, an estimated 22% of uninfected infants were born to HIV positive mothers in South Africa. HIV exposure during pregnancy has been linked to neurodevelopmental delay, increased infant mortality, and impaired immune response.
DNA methylation is an epigenetic regulator of gene expression, which changes over the course of a lifetime in response to environmental stimuli. This exploratory study aimed to determine whether HIV exposure during pregnancy could alter DNA methylation patterns of the infant. Umbilical cord blood was collected at birth from children enrolled in the Drakenstein Child Health Study, and DNA methylation was assessed using the Illumina 450K and EPIC arrays. An ANCOVA model accounting for natural killer cells, nucleated red blood cells, CD8 T cells, race, and child sex was run using RStudio. This analysis indicated that several differentially methylated sites between HIV-exposed uninfected (HEU) and HIV-unexposed uninfected infants. A similar model was used to assess DNA methylation of HEU infants whose mothers were on either two NRTIs + NNRTI, a single NRTI, or two NRTIs + PI, and identified differentially methylated loci which were not represented in the first analysis were identified, suggesting that HIV exposure and type of ART provide distinct methylation stimuli in the developing foetus.

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Despite the success of antiretroviral therapy implementation in pregnancy, fetal toxicity of antiretroviral drugs (ARVs) remains poorly understood, and existing literature often does not account for sex as a variable. Evidence of sex-linked neurotoxicity in ARV exposed children suggests sex-differential drug disposition, which may be explained by variable expression of membrane-associated ARV transporters of the placenta and fetal brain. This study evaluates the potential role of fetal drug transporters in modulating ARV penetration into the fetal brain.
Pregnant C57BL/6 mice were exposed to a clinically-relevant daily treatment of oral lamivudine + abacavir + atazanavir/ritonavir, or to a vehicle control. Tissues were collected on gestational day 18.5 and relative mRNA and protein expression of a panel of key ARV drug transporters was analyzed by qPCR and immunoblotting assays of fetal brain and placenta (n = 57 fetuses). In placenta, ARV exposure was associated with lower expression of Abcc1 (multi-drug resistance-associated protein 1) and Slc29a1 (Equilibrative nucleoside transporter 1). Additionally, greater expression of Abcc1 and Slc29a1 was observed in the placentae of female compared to male fetuses (p <0.05). Sex and treatment were not significantly associated with transporter expression in fetal brain. ARV penetration into maternal plasma, amniotic fluid and fetal brain was also quantified by LC-MS/MS (n = 56 fetuses). All four drugs were detected in amniotic fluid, while only abacavir and lamivudine were detected in the fetal brain at 12.6 ± 7.0 ng/mg and 434.6 ±125.5 ng/mg respectively.
This study provides novel evidence of sex-differential ARV transporter expression in the placenta, as well as characterization of ARV penetration into fetal brain and amniotic fluid. Due to the critical role that drug transporters play in modulating ARV exposure in the developing brain, these novel data contribute to a broader understanding of the influence of age and sex on antiretroviral drug neurotoxicity.

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Background:
Immune checkpoints are negative coreceptors that regulate immunity and are expressed during HIV and other cases of chronic activation. The immune checkpoint LAG3 is elevated on HIV-infected cells and being targeted by several dozen clinical trials for cancer treatment. Despite its promise, LAG3 is relatively understudied, with its mechanism remaining unknown.

Approach:
We have developed Jurkat cell lines expressing wild-type LAG3 and non-functional LAG3 lacking the cytoplasmic domain (CY) to study its mechanism and potential role in HIV-infection.

Results:
Upon activation with Staphylococcal enterotoxin E or D (SEE or SED) LAG3-negative or CY cell lines produce significantly more IL-2 than the cell lines expressing wild-type LAG3. Furthermore, activation of kinases involved in the TCR signaling pathway, including ERK (T202/T204), are also impaired in cells expressing wild-type LAG3 as compared with LAG3-negative cells.

Conclusion:
Our developed LAG3-expressing cell lines behave as expected. These cells can be used for further experiments characterizing the LAG3 mechanism and role in HIV-infection.

Relevance:
Blocking LAG3 has the potential to enhance anti-HIV immunity while helping to activate HIV transcription, thus potentially augmenting both the kick and kill aspects of a functional HIV cure.

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HIV-1 infection can be managed with antiretroviral therapies (ART), but these fail to eliminate the virus due to the establishment of latent infection in several cell types. Infected macrophages sustain viral replication and act as viral reservoirs even during ART (PMID: 28414330; 28811349). HIV-1 accumulates/buds into cytoplasmic virus-containing compartments (VCC) in macrophages, where it evades humoral immune responses (PMID: 22205742). HIV-1 assembly begins with the viral protein Gag translocation to the assembly site. Gag co-traffics with Late endosomes/Lysosomes (LEL), in HeLa cells, and repositioning of LEL to the cell periphery increased virus release (PMID: 17004321, 19286658). HIV-1 also maintains Gag/LEL peripheral positioning under various stress conditions (PMID: 28710431). Further studies demonstrated that the downregulation of LEL-related host proteins supresses HIV-1 release, suggesting that LELs direct Gag to membrane assembly sites (PMID: 22072966; 19451649). LEL proteins can be found in VCCs, so we propose LEL are essential for HIV-1 assembly at VCCs in macrophages (PMID: 12885763; 16087369). To answer this question, immunofluorescence was used to examine whether VCCs formed in the macrophage cell line THP-1 GagZip, following induction of HIV-1. We observed that Gag co-localized with the VCCs markers CD81 and CD9. ELISA assays demonstrated that VCCs formation correlated with decreased virus release, suggesting that HIV-1 accumulates within VCC-like structures in THP-1 GagZip macrophages. We studied LEL involvement in VCC phenomena using Rab7 as a marker, and observed that Rab7 colocalized with Gag within VCC-like structures, suggesting that HIV-1 repositions Rab7+ LEL to macrophage VCCs. Further investigation into the roles for VCCs aim to understand how to target VCCs for immune-mediated viral clearance and the design of new approaches to eradicate HIV-1.

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HIV-1 hijacks host protein function at multiple steps for its replicative advantage including host proteins involved in membrane trafficking, dynamics and fusion, directed transport, endocytosis, and autophagy. Despite their potential as possible targets for novel anti-viral therapies, only a few membrane trafficking proteins have been characterized to serve roles in HIV-1 replication. To elucidate their roles during HIV-1 replication, we performed a CRISPR-Cas9 screen of 140 membrane trafficking proteins. This led to the identification of host proteins whose knockout (KO) resulted in significant decreases in HIV-1 infectivity in CD4+ TZMbl reporter cells. Several of these host proteins had previously been characterized as intrinsic or related to autophagy- and clathrin-mediated endocytic pathways. As one example of a protein identified in this screen, PICALM, is known to regulate autophagy and is also a host dependency factor for other viruses such as herpes simplex virus (PMID: 31853228). To gain insight into how this and other membrane trafficking protein hits assist HIV-1 replication, we edited these genes to create stable SUP-T1 KO T-cell lines. After analyzing these KO cell lines, we found that the absence of certain host proteins, including PICALM, reduced viral entry by more than 2-fold. In addition, we found changes in autophagic flux resulting in altered intracellular trafficking and abundance of viral genomic RNA and HIV-1 Gag. This work will decipher the mechanisms by which membrane trafficking contributes to HIV-1 pathogenesis and will provide new targets with therapeutic potential.

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Background: Growth differentiation factor-15 (GDF15) is a transforming growth factor-β family member. Cellular stress leads to mitochondrial response releasing mitochondrial cytokines including GDF15, which in turn influence the whole-body energy homeostasis and inflammaging. In cardiovascular diseases, cancer and COVID19, elevated plasma GDF15 levels correlate with tissue stress response and are used as a biomarker for clinical outcome. Herein, we assessed whether plasma GDF15 levels were associated with clinical characteristics, inflammation and HIV reservoir size in people living with HIV (PLWH) taking antiretroviral therapy (ART).
Method: Blood was obtained from 55 ART-treated PLWH and 50 uninfected controls. GDF15, inflammation (IL1β, IL6, IL8, TNFα, IP10, CXCL13, sCD14), senescence (soluble urokinase plasminogen activator receptor [suPAR]) and gut permeability (LPS, REG3α and IFABP) markers were quantified in plasma by ELISA. HIV integrated DNA was quantified by nested-qPCR in sorted CD4 T-cells.
Results: PLWH were treated for a median of 14 years (viremia below 50 copies/mL), with a median age of 54 years. Plasma GDF15 levels were higher in PLWH than uninfected controls (p<0.01) and correlated with age and duration of infection. Type or class of ART had no influence on GDF15 levels in multivariate analyses. GDF15 levels were not associated with weight, BMI, inflammation, nor gut permeability markers. Conversely to other markers, GDF15 levels were strongly associated with integrated HIV DNA levels (r=0.59, p<0.01) independently of age, sex, and CD4 count. GDF15 levels were also associated with suPAR levels. In vitro stimulation of PBMC with inflammatory stimuli (LPS and/or PMA-Ionomycin) did not induce GDF15 expression.
Conclusion: In ART-treated PLWH, GDF15 levels were associated with suPAR, a marker of non-AIDS comorbidities, independently of other inflammatory and gut permeability markers. GDF-15 was also independently associated with HIV reservoir size. Further investigations are required to assess the role of GDF15 in HIV persistence and non-AIDS comorbidities.

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Background: Quantitative viral load assays hold the potential to advance COVID-19 control and prevention, but SARS-CoV-2 viral load tests are not yet widely available. SARS-CoV-2 molecular diagnostic tests, which typically employ real-time reverse transcriptase-polymerase chain reaction (RT-PCR), yield semi-quantitative results. Reverse transcriptase droplet digital PCR (RT-ddPCR) offers an attractive platform for SARS-CoV-2 RNA quantification.

Methods: Eight primer/probe sets developed for real-time RT-PCR-based SARS-CoV-2 diagnostic tests were evaluated for use in RT-ddPCR (Charité-Berlin E-Sarbeco; Pasteur Institute IP2, IP4; US-CDC-N1; Chinese CDC ORF, N; Hong Kong University ORF, N). Synthetic SARS-CoV-2 RNA standards of known copy number were used to determine assay analytical efficiency, precision and limits of quantification and detection. SARS-CoV-2 RNA viral loads and real-time RT-PCR cycle threshold (Ct) values (LightMix® 2019-nCoV real-time RT-PCR assay, E-gene target) were measured in a convenience panel of 48 COVID-19-positive diagnostic specimens.

Results: We identified three primer/probe sets, E-Sarbeco, IP2 and IP4, as the most efficient, precise and sensitive for RT-ddPCR-based SARS-CoV-2 RNA quantification. Analytical efficiency of the E-Sarbeco primer/probe set, for example, was ~83%, while assay precision (coefficient of variation) was ~2%. Lower limits of quantification and detection for this primer/probe set were 18.6 and 4.4 input SARS-CoV-2 RNA copies/reaction, respectively. SARS-CoV-2 RNA viral loads in COVID-19-positive diagnostic specimens spanned a 6.2log10 range, confirming substantial viral load variation in vivo. We calibrated SARS-CoV-2 E gene copy numbers against cycle threshold (Ct) values. The resulting log-linear relationship can be used to mathematically derive SARS-CoV-2 RNA copy numbers from Ct values.

Conclusion: Primer/probe sets for real-time RT-PCR-based COVID-19 diagnostic tests can be migrated to RT-ddPCR for SARS-CoV-2 RNA quantification. Mathematical inference of SARS-CoV-2 copy numbers from COVID-19 diagnostic test Ct values, possible via calibrations described above, will allow the wealth of existing diagnostic test data to be harnessed to answer foundational questions in SARS-CoV-2 biology.

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Between June 9 and August 31 2020, a long-term care facility in British Columbia experienced a prolonged COVID-19 outbreak. We describe our inter-laboratory effort to cross-validate whole-genome SARS-CoV-2 sequencing and bioinformatic methods and characterize outbreak viral dynamics.

Three independent laboratories sequenced available SARS-CoV-2 diagnostic specimens linked to the outbreak using Illumina MiSeq or Oxford Nanopore MinION platforms. Raw sequence data were processed using MiCall (MiSeq), BugSeq (MinION) or ARTIC (MinION) bioinformatic pipelines. For each specimen with sufficient material, at least two labs attempted sequencing with discrepancies resolved by a third. Consensus sequences representing the majority nucleotide at each position were compared for concordance, ignoring any positions with missing data. Single nucleotide polymorphisms were identified relative to a presumed founder virus.

Eighty-nine individuals were linked to the outbreak. Of the 65 specimens with Ct<30, 59 (90.7%) were successfully sequenced, while six sequences (25%) were sequenced from the remaining 24 specimens with Ct>30. In total, 65 (73.0%), 54 (60.7%) and 25 (28.1%) samples were sequenced in one, two or all three labs respectively. Pairwise nucleotide concordance between labs was >99.995%, with over 51.2% of sequence comparisons being identical. Non-identical sequences differed by a median of 1 [Q1-Q3: 1-2] nucleotide. Inter-lab concordance was generally higher for sequences collected on the same platform. Three samples collected early in the outbreak yielded identical sequences; this was presumed the outbreak founder virus. Subsequent samples over two months acquired up to five mutations relative to it, with 49 unique mutations observed across all outbreak sequences.

Inter-laboratory whole-genome SARS-CoV-2 sequence concordance was high despite sequencing/bioinformatics platform differences. Minor discrepancies nevertheless underscore the importance of laboratory cross-validation if sequencing is used to characterize emerging variants or to classify sequences as outbreak-related, as determination of genetic relatedness for SARS-CoV-2 can be influenced by as few as one nucleotide polymorphism.

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Currently, there are 38 million people living with human immunodeficiency virus (HIV-1) worldwide and there were 690,000 acquired immunodeficiency syndrome (AIDS)-related deaths in 2019 alone. The greatest cause of mortality in people living with HIV is infection with opportunistic pathogens such as tuberculosis (TB), which accounts for one third of AIDS-related deaths. In fact, HIV-positive individuals are 20 times more susceptible to TB infection. HIV and TB co-infection leads to significantly worsened outcomes in terms of both diseases. Humanized mouse (hu-mouse) models, which possess human immune cells for HIV to infect, have proven to be useful for HIV research. Our aim is to create hu-mouse models of HIV and HIV/TB co-infection to investigate disease progression, immune responses, therapeutics, prevention and vaccination. NOD-Rag1null-IL2rgnull (NRG) mice are highly immunocompromised mice that are traditionally used to generate hu-mouse models. We are also developing NRG mice that are transgenic for human HLA-DR4 and HLA-A2 (DRAG-A2). Previous studies reveal that humanized DRAG-A2 mice develop significantly higher T cell reconstitution compared to NRG mice and are able to undergo immunoglobulin isotype-switching to produce human antibodies such as IgG. NRG and DRAG-A2 mice were humanized with hematopoietic stem cells obtained from human umbilical cord blood. 52% of NRG mice were successfully reconstituted with human T cells, B cells and monocytes to greater than 10% of total circulating leukocytes, whereas 100% of DRAG-A2 mice were successfully engrafted, as determined by flow cytometry. In a proof-of-concept experiment, female humanized NRG mice were infected intravaginally with NL4.3-Bal-HIV-1 and assessed for HIV load via vaginal wash and blood plasma qRT-PCR. Humanized NRG mice were also successfully infected intranasally with TB as demonstrated by flow cytometry and histology. Preliminary DRAG-A2 experiments are ongoing. In future experiments, hu-mice will be co-infected with HIV and TB to enable us to investigate co-infection.

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Introduction: Having a licensed HIV self-test in Canada provides new opportunities to increase the access, uptake and frequency of HIV testing and more effectively reach the undiagnosed especially in priority populations. The objectives of this study were to (1) evaluate the INSTI HIV Self-Test (HIV-ST) performance compared with laboratory reference testing, (2) document if intended users can perform the steps to use the HIV-ST device, and (3) document if intended users can successfully interpret contrived positive, negative, and invalid results. Study was submitted to Health Canada for review for license purposes.

Methods: The study used a cross-sectional design and recruited consenting adults from four community sites across Ontario, Québec, and Manitoba between August 2019 and March 2020. The results of the observed HIV-ST were compared with results of the Abbott Architect HIV Ag/Ab Combo test.

Results: Primary efficacy analysis on 678 completed HIV-ST revealed a positive percent agreement of 100% and a negative percent agreement of 99.5% with the comparator method. A total of 6 previously undiagnosed participants were identified through study testing and linked to care and treatment. Of the 708 participants who took part in the usability study, 92.4% successfully performed the steps determined to be “critical” for successful completion of the test, 96.7% found the instructions easy to follow, and 95% indicated that they would use the test again. Of the 404 participants who interpreted the strong positive, weak positive, negative and invalid contrived results, successful interpretation ranged from 90.6% (for weak positive) to 99.3% (for negative).

Conclusions: This study showed high levels of accuracy, usability and acceptance by participants, leading to license by Health Canada of the INSTI HIV Self-Test in November 2020. A REACH 3.0 national implementation science project will provide 60,000 free HIV-ST to participants across Canada starting in February 2021.

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Background: Despite antiretroviral therapy (ART), HIV continues to persist in anatomical and cellular sanctuaries. HIV infects the brain and replicates in microglia and trafficking macrophages. We previously demonstrated the limited impact of ART on persistence of virus in brains of patients with undetectable plasma viral load. This study investigates the efficacy of ART in brain using primary human microglial cultures and brain tissues from SIV-infected nonhuman primates and an expanded cohort of HIV-infected humans in the absence or presence of ART.
Methods: To determine the EC50 levels of antiretroviral drugs, HIV-infected microglia and lymphocytes were treated with dolutegravir, ritonavir, emtricitabine, raltegravir, and tenofovir. Total DNA and RNA were extracted from post-mortem brains of SIV-infected Rhesus macaques (n=19) and HIV-infected patients (n=15) receiving ART and quantified by ddPCR.
Results: All ART drugs displayed lower EC50 values in lymphocytes than in microglia except tenofovir, which showed two-fold greater activity. SIV RNA, total and integrated DNA was detected in brain regions among animals receiving suppressive, interrupted and no ART comprised of the same drugs examined in the in vitro studies. Similarly, HIV RNA, total and integrated DNA were detected in brain tissues of all HIV-infected patients regardless of ART duration and plasma viral load. Analysis of host antiviral immune responses showed these genes were increased in the brains of all HIV-infected persons compared to uninfected control brains. Moreover, SIV and HIV antigens were detected in brain, largely in microglia/macrophage cells regardless of treatment regimen. This might be in part due to higher expression of efflux genes in microglia compared to lymphocytes.
Conclusions: Contemporary ART is relatively less effective in HIV-infected microglia, which might contribute to HIV persistence in the brain that is seemingly independent of blood viral load. New pharmacological approaches are required to enhance ART efficacy in the brain.

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T-follicular helper cells (TFH) express high levels of B-cell Lymphoma 6 (BCL6) transcription factor, and help B-cells throughout development and activation. During HIV infection TFH are productively and latently infected at a higher frequency than other CD4 T-cell subsets. These characteristics mark TFH as a substantial barrier to HIV cure. It is established that the well conserved accessory protein viral protein R (Vpr) induces a G2/M cell cycle arrest in cycling cells but its function during HIV infection remains elusive. In an HIV-1 Vpr BioID analysis, we identified the Polycomb complex component BCL6 co-repressor (BCOR) as a potential target of Vpr. The BCL6/BCOR complex is well characterized to silence genes based on CPG rich regions. Using a Vpr inducible HeLa cell line, we show that Wild-Type (WT), and cell cycle arrest-defective Vpr mutants deplete endogenous BCOR but not a mutant defective in recruiting the Cul4a-DDB1-DCAF1 (DCAF1com) E3 ligase ubiquitin complex. Using this system, we knocked down DCAF1 by siRNA and confirmed that it is essential for Vpr-dependent BCOR depletion. These results imply that DCAF1com recruitment but not G2/M cell cycle arrest is required for BCOR depletion. We further show that Vpr-dependent BCOR degradation requires a functional proteasome. Importantly, we also show that Vpr is essential for BCOR degradation during HIV infection, and that BCOR is highly expressed in latently but not productively infected cells. Knockdown of BCOR in latently infected cells is sufficient to reactivate HIV-1. Finally, CHIP-qPCR analysis showed that BCOR occupies the HIV-LTR in latently infected cells. Our results raise the possibility that Vpr drives BCOR degradation via DCAF1com to evade transcriptional silencing of provirus, potentially increasing productive infection of TFH.

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Background: Interleukin -1α and IL-1β are pro-inflammatory cytokines induced by M. tuberculosis (Mtb) infection that drive resistance to infection. Murine studies reveal IL-1α, not IL-1β, is the main mediator of these protective innate responses. Mtb also induces production of interferon α/β, which in mouse studies negatively regulate IL-1α and IL-1β potentially driving progression from latent infection to active tuberculosis (TB). The IL-1s inhibit IFN α/β levels through a negative feedback mechanism. However, the effects of individual IFN subtypes (IFN-α and IFN-β) on IL-1α and IL-1β in clinical phenotypes of Mtb infection in humans have not been defined.
Hypothesis: Stimulation of peripheral blood mononuclear cells (PBMCs) with IFN-α or IFN-β suppresses IL-1A mRNA expression more than the IL-1B gene in Mtb infection.
Methods: PBMCs from 11 healthy controls (HC), 12 tuberculin skin test (TST) positive, IFN-γ-release assay (IGRA) negative individuals [TST+IGRA-] (TST), 19 IGRA-positive individuals (LTBI); and 19 active TB patients (ATB) were stimulated with either IFN-α or IFN-β then assessed for expression of IL-1A and IL-1B mRNA using qPCR.
Results: In unstimulated samples, higher expression of IL-1A and IL-1B mRNA were observed in LTBI and ATB relative to healthy controls; the expression of these genes was lower in LTBI compared to ATB. Stimulation with type I IFNs downregulated the expression of IL-1A and IL-1B in all the clinical phenotypes, with IL-1A mRNA suppressed more than IL-1B. Higher suppression of the genes was observed in LTBI compared to ATB; this suppressive effect was greater with IFN-β than with IFN-α.
Conclusion: Our study supports the concept that IFN-α and IFN-β suppress IL-1A and IL-1B expression in Mtb infection. Our findings also demonstrate differences in the effects of IFN-α- and IFN-β on the expression of IL-1A and IL-1B that may contribute to TB disease progression.

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A better understanding of factors that contribute to HIV latency and reactivation is needed to improve clinical strategies to eliminate viral reservoirs. HIV Nef promotes the formation of exosomes that contribute to autocrine and paracrine cell signaling. Exosomes containing Nef and cellular ADAM17 can trigger CD4 T cell activation, leading to secretion of TNF-α and HIV reactivation in cell culture; however, Nef’s role in modulating latency remains understudied.

To examine whether exosomes produced by naturally arising Nef alleles differ in their ability to mediate viral reactivation from latency, we treated a latent HIV-infected human CEM T cell clone harbouring a single integrated provirus lacking Nef (C-Lat-Nef-KO) with exosomes produced using wild-type Nef (SF2), a mutant Nef (AXXA) that is unable to activate ADAM17, and two Nef isolates representing major pandemic HIV subtypes B and C. HIV reactivation (Gag-p24 expression) was quantified by flow cytometry.

Consistent with prior reports, we observed significantly higher viral reactivation (~ 5 fold) in cells treated with exosomes produced using wild type Nef compared to those treated with exosomes produced using mutant Nef. Interestingly, the natural Nef isolates differed in their abilities to mediate viral reactivation from latency (p<0.001), with the subtype B clone exhibiting higher activity (~2 fold higher Gag24 expressing cells) compared to the subtype C clone.

Our results indicate that Nef’s ability to mediate viral reactivation from latency may differ among natural arising Nef alleles. A larger, more diverse panel of Nef clones will be examined to validate our observations and possibly illuminate Nef polymorphisms that are crucial for this function. This work highlights the role of Nef in modulating viral reactivation from latency, which might lead to novel approaches to enhance HIV eradication strategies.

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Viremic controllers are underrepresented in HIV reservoir dynamics studies. We combined single-genome sequencing (SGS), proviral quantification, phylogenetics and mathematical modeling to: 1) reconstruct within-host pre-ART HIV evolutionary histories, 2) measure proviral burden, age and diversity on-ART, and 3) estimate proviral half-lives, in 4 viremic controllers.
Three participants broadly maintained pVL <~2000, while one eventually lost control before initiating ART. We performed HIV nef RNA SGS on a median of 12 longitudinal pre-ART plasma samples/participant and HIV nef DNA SGS on proviruses sampled a median 1.9 years following ART. Reservoir size on-ART was estimated using the Intact Proviral DNA Assay. Proviral sequence ages were inferred using a phylogenetic approach that leverages within-host pre-ART HIV evolutionary rates. We then applied a published mathematical model of reservoir dynamics to infer host-specific proviral half-lives.
We collected 356 unique plasma HIV RNA sequences (range 52-173/participant) and 206 intact unique proviral sequences (12-118/participant). All within-host phylogenies exhibited molecular clock signal pre-ART (range 1.16×10-5–5.35×10-5 substitutions/base/day). Pre-ART pVL areas under the curve correlated strongly with longitudinal pre-ART plasma HIV sequence diversity, total on-ART proviral burden and overall on-ART proviral diversity (all Spearman's ρ=1; p=0.08). Intact proviral percentage ranged from 10-94%, where the latter was observed in the individual who lost control prior to ART. For two participants, inferred proviral integration dates ranged from shortly following infection to cART initiation. For the other two, including the participant who lost control, proviruses dated well into chronic infection. For three of four participants, the best-fit proviral half-life estimates were <~1 year, suggesting rapid proviral turnover pre-ART. The fourth participant's proviral pool was consistent with negligible decay following deposition.
Despite viremic control, significant within-host pre-ART HIV evolution nevertheless gave rise to diverse within-host proviral pools with varying intact genome burden. HIV eradication strategies must overcome within- and between-host proviral diversity.

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Background: The foreskin is a primary site of HIV-1 acquisition in heterosexual males; however, a lack of relevant in vitro models that recapitulate important epithelial barrier functions has limited our understanding of susceptibility at this site. We hypothesized an organotypic in vitro foreskin model would better mimic the foreskin tissue compared to current models.
Methods: Organotypic foreskins were generated from primary foreskin fibroblasts and keratinocytes isolated from adult men undergoing elective circumcision, and matured at the air-liquid interface over 5 weeks. Organotypic foreskins were compared to explanted foreskin tissue maintained in culture for seven days (explants) and tissues cryopreserved immediately after surgery (representative of the in vivo state we hope to recapitulate). Organotypic cultures, explants, and cryopreserved foreskin tissue were assessed for tissue architecture by immunofluorescent microscopy and for epithelial permeability using a fluorescent tracer dye applied for 6-24 hours (dextran).
Results: Organotypic foreskins displayed a stratified, keratinized epithelium with localization of keratin (Filaggrin) to terminally differentiated keratinocytes on the apical aspect and robust expression of cell junction proteins (E-cadherin) (Figure 1). Organotypic foreskins successfully excluded the tracer dye while explants did not.
Impact: Organotypic in vitro foreskins mimic in vivo tissue structure and epithelial barrier function while conventional explants do not. Development of this model will facilitate discerning the mechanism(s) by which circumcision reduces HIV-1 susceptibility, while permitting future testing of microbicides on the penile microbiome to examine its relation to susceptibility.

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Different interferon-alpha (IFN-α) subtypes have been shown to elicit distinct control of different viral infections. Along with its direct antiviral effects, IFN-α also strongly modulates host innate and adaptive immune responses. Our previous work showed that IFN-α subtypes differentially control HIV-1 infection and mediate distinct effects on immune function. Clinical use of the IFN-α2 subtype has not been highly effective in reducing viral or proviral HIV-1 and high levels of endogenous IFN-α2 has been associated with CD8+ T cell hyperactivation and dysfunction in HIV-1 patients. Our prior study with the IFN-α14 subtype suggested that some IFN-α subtypes may be beneficial in HIV-1 infection. Using HIV-1-infected TKO-BLT humanized mice, we demonstrated that after 3 weeks of treatment IFN-α14 significantly reduced markers of CD8+ T cell-related dysfunction such as hyperactivation, exhaustion and apoptosis and, unlike ART, these low levels were maintained even after the treatment was withdrawn. IFN-α14 treated mice also maintained a more naïve CD8+ T cell profile as opposed to the development of the larger effector memory subset observed in HIV-1 infected and IFN-α2 treated mice. Although IFN-α14 reduced the activation profile and proliferative capacity of CD8+T cells, it did not change their ability to secrete cytokines or degranulate upon stimulation ex vivo. Also, IFN-α14 treatment did not reduce the CD4+ T cell count supporting the hypothesis that IFN-α14 treatment does not exacerbate disease progression and may have therapeutic potential to alleviate CD8+ T cell dysfunction during HIV-1 infection.

My Project was funded by SHRF (Saskatchewan Health Research Foundation)

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Background: The only confirmed cases of an HIV-1 cure have been two individuals who received hematopoietic stem cell transplants (HSCT) from donors with natural resistance to HIV-1. To translate this into a treatment option for HIV infection, several clinical trials have been initiated to insert antiviral genes into an infected person's cells during an autologous HSCT. Combinations of safe and effective genes need to be used so that HIV-1 replication is completely inhibited, and no resistant virus emerges. Although several antiviral genes have been identified, there are limited data on how they compare to one another both within and between classes.

Methods: Genes expressing anti-HIV-1 ribozymes, aptamer/decoy RNAs, short hairpin (sh) RNAs and U1 interference (U1i) RNAs were directly compared for efficacy and safety in HEK293T and SupT1 cells. Potent shRNA candidates were also compared when expressed from different promoters (U6, 7SK and H1).

Results: shRNAs and U1i RNAs were much more effective at inhibiting HIV-1 replication compared to other RNA classes. For U1i RNAs, those that enhance HIV-1 RNA splicing were more effective compared to those that inhibit polyadenylation. For the most effective U1i RNA, we were able to increase its specificity by lengthening its recognition domain. For shRNAs, the 7SK and U6 promoters resulted in higher levels and potency compared to the H1 promoter, but their use resulted in cell growth defects. These defects could be mitigated in some cases by altering the shRNA loop.

Conclusions: So far, data from clinical trials has shown that HSCT with antiviral genes is safe but that better antiviral genes are needed. By directly comparing some of the top candidates from the literature we have identified the genes that will have the greatest chance of success in curing HIV infection in the clinic and we have started evaluating different gene combinations.

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Nuclear pore complexes (NPCs), made up of nucleoporins (Nups), are the main channels for the transportation of macromolecules and molecular complexes between cytoplasm and nucleus. Previously it has been shown that Nups such as Nup153 and RANBP2 directly interact with the HIV-1 capsid protein (CA), modulating nuclear entry and DNA integration into the host genome. Cell-autonomous innate immune mechanisms might be activated upon HIV-1 infection, leading to the secretion of type I interferons (IFN-I) that induce antiviral responses through the expression of many antiviral restriction factors (effectors). One of the restriction factors, Mx2, uses NPCs for its targeting of HIV-1 CA. We postulate that NPCs are involved in the innate response to HIV-1 infection; specifically, we hypothesize that HIV-1 and IFN-I modulate the composition of NPCs. Label-free quantitative mass spectrometry (MS) was performed on nuclear membrane extracts from monocytic cells infected with HIV-1 vectors with and without IFN-β treatment. Using this approach, we quantified 33 different Nups and 4813 other proteins. Around 7.52 % proteins showed significant variation upon infection and/or IFN-β treatment including one Nup, Translocated Promoter Region (TPR) and the NPC associated protein vimentin. We also exploited the generated MS data to quantify the HIV-1 proteins present at the nuclear envelope at the early stages of infection. Interestingly, CA and other Gag proteins were more abundant at the nuclear envelope following IFN-β treatment. To investigate the possibility that Mx2 is the restriction factor responsible for sequestering HIV-1 at the nuclear membrane upon IFN-I treatment, we generated Mx2-knockdown cells. MS and immunofluorescence microscopy experiments underway will provide clarification on a possible role for Mx2 in this phenotype. This project constitutes the first MS-based characterization of nuclear pores in the early stages of HIV-1 and may lead to the identification of novel cellular targets for HIV-1-inhibiting drugs.

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Background: Unwanted pregnancies and sexually transmitted infections (STIs) are major health concerns of women worldwide. These concerns have prompted efforts to develop Multipurpose Prevention Technologies (MPTs), which simultaneously provide contraception and prevent STIs, including HIV. LL-37, the only human cathelicidin and an effective spermicide on human sperm, has broad antimicrobial activity including in vitro activity against HIV. 17BIPHE2 is a mimic of a truncated LL-37 peptide, engineered to contain 5 unnatural residues, thus limiting its protease degradation by vaginal fluid. Hence, this AMP represents a promising MPT agent. We, therefore, hypothesize that 17BIPHE2 will be a potent inhibitor of HIV infection.

Methods: PMA-stimulated ACH-2 cells, a chronically HIV-infected T cell line, were incubated with LL-37 or 17BIPHE2, and HIV replication was evaluated by p24 concentration in the supernatant via ELISA. Alternatively, HIV was incubated with 17BIPHE2 prior to infection of target cells. Infection was quantified by luciferase activity in an HIV reporter TZM-bl cell line or by p24 ELISA in activated CD4+ T cells.
Results: 17BIPHE2 inhibited HIV replication in stimulated ACH-2 cells in a dose dependent manner, while this was not observed with LL-37. Pre-incubation with 17BIPHE2 decreased the ability of HIV to infect TZM-bl cells in a dose-dependent manner across multiple titers of HIV. Preliminary results demonstrated that when HIV was incubated with 17BIPHE2 before infecting CD4+ T cells, HIV infection decreased with increasing amounts of 17BIPHE2.

Conclusion: Initial results show that 17BIPHE2 can reduce the ability of HIV to infect relevant target cells. The mechanisms of this activity and the specific stages of HIV replication where 17BIPHE2 exerts anti-HIV activity remain to be established. This project provides the groundwork to study 17BIPHE2 in other cells/tissues of the female reproductive tract and eventually in in vivo models of HIV infection.

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During HIV-1 infection, Th17-cells are highly susceptible to infection and depleted from mucosal sites, resulting in mucosal barrier integrity alterations, microbial translocation, systemic inflammation, and disease progression. Additionally, HIV-infected Th17-cells can be long-lived and harbor viral reservoirs(VR) in people living with HIV(PLWH) receiving antiretroviral therapy(ART). Thus, Th17-cells are key players in HIV pathogenesis and VR persistence. Here, we evaluated the role of RORC2, the master regulator of Th17-cell differentiation, on HIV replication and outgrowth.
Memory CD4+T-cells were isolated from PBMCs of HIV-uninfected individuals(HIV-), ART-treated(ART+PLWH) and untreated(ART-PLWH) PLWH by negative selection using magnetic beads. Subsequently, cells expressing the Th17 markers RORC2 and CCR6 were isolated by FACS. The NL4.3BAL and THRO-HIV-1 strains were used for infections in vitro. A viral outgrowth assay(VOA) was performed with cells from ART+PLWH and ART-PLWH. HIV replication/outgrowth were evaluated by FACS and ELISA. HIV integration was evaluated by nested real-time PCR. RORC2 silencing in Jurkat cells and primary T-cells was performed. RORC2 overexpression was performed in 293T and Jurkat cells infected with VSV-G-pseudotyped HIV-1LAI env-GFP(HIV-1GFP). For Chromatin immune precipitation(ChIP) experiments, Jurkat cells transduced with a retroviral vector expressing RORC2-myc were infected with HIV-1GFP. Real-time PCR signal for HIV-LTR-NRRE-1 and HIV-CS-Pol was subsequently evaluated.
The inhibition of RORC2 by tool small molecule decreased HIV replication in CD4+T-cells in vitro, as well as viral outgrowth from T-cells of ART+PLWH and ART-PLWH. Consistently, RORC2 expression was higher within HIV-p24+ compared to total T-cells in ART-PLWH. Moreover, CCR6+RORC2+ compared to CCR6-RORC2- T cells of ART+PLWH were enriched in proviral DNA ex vivo. Furthermore, RORC2 silencing inhibited HIV-1 infection, specifically in CCR6+T-cells, whereas RORC2 overexpression led to enhanced viral replication. Finally, RORC2 promoted viral gene expression and ChIP revealed that RORC2 binds to the HIV-1 promoter.
Altogether, these results point to RORC2 as a new Th17-specific target for HIV-1 therapy.

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HIV broadly neutralizing antibodies (bnAbs) represent tools to guide prophylactic strategies. Most bnAbs have been cloned as IgG, and their protective ability at mucosal surfaces is typically evaluated in that format. However, superior protection by dimeric IgA (dIgA) against HIV mucosal challenge has been reported in a few studies. Mucosal IgA has also been suggested as a correlate of protection in HIV highly exposed seronegative individuals. While past evaluation of HIV-specific dIgA includes antibodies to the gp120 V3 loop and CD4-binding site, here we report on a first step to evaluate the HIV-neutralizing activity and protective ability of bnAb PGT128 in a dIgA format. PGT128 binds a conserved patch of oligomannose-type glycans on HIV Env that is of interest to vaccine design. Recombinant PGT128 dIgA2, chosen for evaluation first because of the higher proportion of IgA2 in colonic and vaginal external secretions in people, was expressed in 293F cells and purified by affinity chromatography. In an ELISA inhibition assay, PGT128 dIgA2 incubation with an HIV-1 SOSIP trimer in solution resulted in ~5-fold greater inhibition of antigen capture onto ELISA plate wells coated with PGT128 IgG compared to incubation with PGT128 IgG, which suggest that the greater valency of PGT128 dIgA2 allows it to bind more avidly. When assayed in a pseudovirus-based neutralization assay at equimolarity, PGT128 dIgA2 exhibited 2- to 3-fold greater potency than the IgG, suggesting that dIgA2 valency increases binding avidity for Env on the surface of virions. Studies are now underway to assess dIgA1. Nevertheless, our results so far provide impetus for probing whether at least the dIgA2 form of PGT128, alone or in combination with IgG, might offer better protection against mucosal HIV challenge than the IgG form alone. Such insight would be informative to ongoing efforts to elicit oligomannose-specific bnAbs like PGT128.

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Background: HIV infection leads to the expansion of immunosuppressive regulatory T-cells (Tregs), contributing to immune dysfunction, mucosal fibrosis, and disease progression. Antiretroviral treatment (ART) upon HIV exposure improves CD4 count and decreases immune activation and reservoir size. However, the dynamics of Tregs following early ART initiation remain understudied.
Methods: Peripheral blood mononuclear cells (PBMCs) were collected from 123 individuals consisting of HIV-infected untreated in acute and chronic phases, ART-treated in early infection (median of ART initiation: 6.7 months post-infection), elite controllers (EC), immunological controllers (IC), and HIV-uninfected controls. Tregs subsets were characterized by multiparameter flow cytometry. The methylation status of six regulatory regions of the foxp3 gene was assessed using MiSeq sequencing technology.
Results: HIV infection decreased the CD4/CD8 ratio and increased T-cell activation (CD38+HLA-DR+) and immunosenescence (CD28-CD57+) phenotypes, which were all improved after early ART initiation. Total Treg frequency increased over time during HIV infection, which was normalized in early ART recipients. Tregs in untreated individuals expressed higher levels of activation and immunosuppressive markers (CTLA4, CD39 and LAP(TGF-β1)), which remained unchanged following early ART. Expression of gut migration markers (CCR6, CCR9, Integrin-β7) by Tregs was elevated during HIV infection and suppressed after ART initiation. Importantly, gut-homing Tregs expressing LAP(TGF-β1) and CD39 remained higher despite early treatment. Additionally, the increase in LAP(TGF-β1)+ Tregs and extra-thymic Helios-FoxP3+ Tregs overtime during HIV infection were consistent with higher demethylation of conserved non-coding sequence (CNS)-1 in the foxp3 gene, which is induced by TGF-β1. Notably, LAP(TGF-β1)-expressing Tregs in EC and IC were significantly higher than uninfected subjects, while the markers of Treg activation, migration, and function remained similar in these individuals.
Conclusions: Early ART initiation was unable to control the levels of immunosuppressive Treg subsets and their gut migration potential, which could ultimately contribute to gut tissue fibrosis and disease progression.

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Antiretroviral therapy (ART) suppresses HIV infection to undetectable levels. However, upon withdrawal of ART, viremia rebounds rapidly to pre-treatment levels, indicating the presence of latently-infected cells during ART. Understanding the mechanisms that induce/maintain latent cells is critical to achieving HIV cure. Naïve and memory CD4+ T cells recirculate through secondary lymphoid organs, where they continuously contact dendritic cells and are exposed to the homeostatic cytokine IL-7. Signals generated from the T-cell receptor and IL-7R induce homeostatic proliferation and maintain memory T cell survival at steady-state. In this study, we tested our hypothesis that similar mechanisms maintain latent T cells under ART. To address this, we generated a full-length, R5-tropic dual-fluorescent HIV reporter that encodes two fluorescent markers: a Nef-E2Crimson fusion protein under the control of the HIV LTR, and the EF1a-HTLV1 fusion promoter driving the expression of ZsGreen1 (HIVNef-CRMZY). Infection of primary CD4+ T cells with this reporter allowed us to visually identify productively-infected (E2Crimson+ZsGreen1+) and latently-infected (E2Crimson-ZsGreen1+) cells. Functional Nef expression was confirmed by significant CD4 downregulation in productively-infected cells. Using both intracellular p24 staining and cell surface CD4 receptor expression as a sensitive marker for viral protein production, we further define a truly latent T cell population as E2Crimson-ZsGreen1+CD4high p24neg, which we followed longitudinally for up to 15 days in culture. We show that while IL-7 alone increased the proportion of latently-infected T cell population in vitro, co-culture with syngeneic monocyte-derived dendritic cells greatly expanded latent T cell populations. Our data demonstrate that latently-infected T cells may exploit/utilize physiological signals within lymphoid tissues to achieve long-term survival.
Significance and Conclusion: Investigations into the signals (tonic or antigen-derived) that help maintain the HIV reservoir in memory T cells may uncover a possible immunological approach to reducing the size of the HIV reservoir.

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