Background: During vaginal intercourse, women are twice as likely to be infected with HIV versus their male partners. Many factors drive this, including biological ones. Sex hormones, progesterone and estrogen interact closely with immune cells. The menstrual cycle or use of hormonal contraception could modify the immune response and, therefore, alter the risk of HIV acquisition. This study will explore immune changes during the menstrual cycle or when using the hormonal contraception depot-medroxyprogesterone acetate (DMPA) in female sex workers (FSWs).
Methods: FSWs from the Sex Worker Outreach Program in Nairobi, Kenya were enrolled in 4 groups: 1) women living with HIV (WLWH), 2) HIV uninfected, new to sex work (<3 years, NN) and not using hormonal contraception (no-HC), 3) HIV uninfected, >7 years in sex work HIV-exposed seronegative (HESN) and no-HC, and 4) HIV uninfected NN using DMPA contraception. Mucosal and blood samples were taken during the follicular and luteal phases of the menstrual cycle. Plasma Sex hormones and 22 cytokines/chemokines were assessed using Millipore bead array in plasma and CVL. T cells from the genital tract and blood and NK cells from the blood were phenotyped using flow cytometry. Data was analyzed using a mixed effects model controlling for age and menstrual cycle length.
Results: In HESN but not NN, we observed increases in the proportion of NK expressing the activation marker NKG2D during follicular phase. DMPA use was associated with higher CD4 (follicular:p=0.011, luteal:p=0.027) and lower CD8 (follicular:p=0.005, luteal:p=0.042) T cells compared to NN no HC. In WLWH, Tregs were lower during luteal phase (p=0.03).
Conclusion: We observed different immune profiles according to menstrual cycle phase, DMPA, HIV status, and sex work duration. This provides insights into the impact of sex hormones and hormonal contraception on HIV acquisition risk and transmission in the key population of sex workers.

Monkeypox virus (MPXV) infections in humans cause neurological disorders while studies of MPXV-infected animals indicate that the virus penetrates the brain. Pyroptosis is an inflammatory type of regulated cell death, resulting from plasma membrane rupture (PMR) due to oligomerization of cleaved gasdermins to cause membrane pore formation. Herein, we investigated the human neural cell tropism of MPXV compared to another orthopoxvirus, vaccinia virus (VACV), as well as its effects on immune responses and cell death. Astrocytes were most permissive to MPXV (and VACV) infections, followed by microglia and oligodendrocytes, with minimal infection of neurons based on plaque assays. Aberrant morphological changes were evident in MPXV-infected astrocytes that were accompanied with viral protein (I3) immunolabelling and detection of over 125 MPXV-encoded proteins in cell lysates by mass spectrometry. MPXV- and VACV-infected astrocytes showed increased expression of immune gene transcripts (IL12, IRF3, IL1B, TNFA, CASP1, and GSDMB). However, MPXV infection of astrocytes specifically induced proteolytic cleavage of gasdermin B (GSDMB) (50kDa), evident by the appearance of cleaved N-terminal-GSDMB (37 kDa) and C-terminal-GSDMB (18 kDa) fragments. GSDMB cleavage was associated with release of lactate dehydrogenase (LDH) and increased cellular nucleic acid staining, indicative of PMR. Pre-treatment with dimethyl fumarate reduced cleavage of GSDMB and associated PMR in MPXV-infected astrocytes. Human astrocytes support productive MPXV infection, resulting in inflammatory gene induction with accompanying GSDMB-mediated pyroptosis. These findings clarify the recently recognized neuropathogenic effects of MPXV in humans while also offering potential therapeutic options.

Background: Vaccine-elicited immune responses can be impaired among people living with HIV (PLWH). To examine this for COVID-19, we evaluated SARS-CoV-2 spike-specific T-cell responses after two, three and four COVID-19 vaccine doses, with or without breakthrough infection, in PLWH whose viremia was well-controlled on antiretroviral therapy.

Methods: CD4+ and CD8+ T-cells reactive to overlapping peptides spanning the ancestral SARS-CoV-2 spike protein were quantified in 50 PLWH and 87 controls without HIV using an activation induced marker assay based on co-expression of CD137/4-1-BB and CD134/OX-40 (for CD4) or CD69 (for CD8). Antibody-mediated neutralization was quantified using a live SARS-CoV-2 infection assay. SARS-CoV-2 infection was assessed by seroconversion to nucleocapsid (N) antigen and self-reported PCR or rapid test results. All participants were COVID-naive until at least one month after their third vaccine dose. T-cell responses were further assessed in 21 PLWH who experienced their first SARS-CoV-2 infection after this time.

Results: CD4+ and CD8+ T-cell frequencies did not differ between PLWH and controls after the second vaccine dose. The third dose boosted T-cell frequencies in both groups (all p<0.0001), but CD8+ T-cell responses were modestly lower in PLWH at this time (p=0.02), a result that remained consistent in multivariable analyses (p=0.045). T-cell frequencies increased further in PLWH who subsequently experienced a breakthrough infection (all p<0.0001). CD8+ T-cell responses remained higher in these individuals even after a fourth vaccine dose (p=0.03), though CD4+ responses were similar to those of SARS-CoV-2-naive PLWH at this time. T-cell responses did not correlate with neutralizing antibody titers, nor predict breakthrough infection after multivariable correction. In multivariable analyses, only younger age associated with increased infection risk, consistent with higher infection rates among younger Canadians during the initial Omicron waves.

Conclusion: PLWH mount strong T-cell responses to COVID-19 mRNA vaccines and boosters, which may be enhanced following breakthrough infection.

An HIV/virus-like particle formulation (HLP) presented by MDDCs efficiently induces HIV latency reversal and release from CD4+ T cells harboring latent HIV in PLWH and receiving ART during early or chronic infection. For improved safety, HLP production was derived from near full-length (nfl) HIV-1 genome mutated to prevent genomic RNA packaging (HIV-1_nfl_ΔΨ_gRNA). However, in absence of gRNA, the HLP lost approximately 2-fold latency reversal related to an RNA adjuvant, TLR response in the CD4+ T cells. Thus, we explore two distinct ways to re-introduce an RNA adjuvant effect to HLP to enhance latency reversal, one involving the exogenous addition and the other preferential encapsidation of an optimized RNA adjuvant sequence into the HLP. For the former, the “Multi” RNA adjuvant containing 100x4 repeats of optimized RNA sequences for type 1 interferon and TNFα response was complexed into DOTAP vesicles. For the latter, the same optimized “Multi” RNA adjuvant sequence was placed downstream of the HIV-1 Ψ RNA packaging element with the HIV-1 Genome RNA Packaging Enhancer (GRPE) element to produce “Multi-Ψ-GRPE”. For optimal encapsidation of the “Multi-Ψ-GRPE” RNA adjuvant in the HLP and almost complete exclusion of remnant gRNA (HIV-1_nfl_ΔΨ_gRNA), a 4:1 plasmid ratio expressing the “Multi-Ψ-GRPE” versus the HIV-1_nfl_ΔΨ_gRNA was used. Testing immunostimulatory and latency reversal responses, both forms were added to PBMCs and to THP1-Dual™ cells to measure immunoinflammatory markers and TLR pathways through NFkB and IRF7 responses. We found that the exogenous form showed a greater response than the encapsidated form. However, in PBMCs, the encapsidated adjuvant had a targeted effect to HIV-specific CD4+ T cells, i.e. found to harbor the latent HIV majority. In contrast, the exogenous form might induce hyperimmune activation with the lack of specific cell targeting at the site of future intramuscular injections for treatment. Animal models are now being used to test this hypothesis.

Background
Autophagy and oxidative phosphorylation allow efficient anti-HIV T-cell responses, notably through IL-21 production by anti-HIV CD4 T-cells, which stimulates anti-HIV CD8 T-cells. Extracellular Acyl-CoA-Binding Protein (ACBP) inhibits autophagy, tricarboxylic acid (TCA) cycle and oxidative phosphorylation in mouse models. Herein, we assessed the levels of circulating ACBP and its influence on T-cell function in people living with HIV (PLWH) on antiretroviral therapy (ART).

Methods
Plasma ACBP and cytokines were quantified by ELISA in 50 PLWH on ART (mean duration 14.7 years) and 30 uninfected controls with similar age. Metabolomic analyses were performed on serum samples by GC-MS (10 participants with high and low ACBP). In vitro, recombinant ACBP (recACBP) was added at concentrations up to 10µg/mL on PBMCs from PLWH on ART and controls. T-cell responses were assessed by flow cytometry.

Results
ACBP levels were higher in PLWH on ART compared to controls (median 127.5 vs 78.1 ng/mL, p=0.03), independently of age and sex.
In ART-treated PLWH, plasma ACBP levels were not associated with CD4 counts. However ACBP levels correlated positively with plasma levels of G-CSF, IFN-α2, IFN-γ, and IL-1β (r>0.3, p<0.05 for all), and inversely with plasma IL21 levels (r=-0.54, p<0.01).
PLWH with high plasma ACBP had higher serum levels of TCA intermediates glutamate (2-fold, p=0.02) and α-ketoglutarate (1.5-fold, p=0.09), respectively.
Addition of recACBP to PBMCs stimulated with anti-CD3 antibodies or HIV Gag, Nef and Env peptides, decreased IFN-γ, IL-2 and TNF-α production in CD4 and CD8 T-cells (p<0.03 for all). Upon anti-CD3 stimulation, IL-17A and IL-21 production were also decreased in CD4 T-cells while IL10 levels remained unaffected.

Conclusion
Higher plasma ACBP levels in ART-treated PLWH were associated with inflammation, unfit metabolism, and T-cell dysfunction. ACBP directly impedes polyclonal and anti-HIV T-cell functions, compelling the development of circulating ACBP inhibitors to improve anti-HIV T-cell responses in PLWH.

Introduction: Treatment of HIV using combination antiretroviral (ARV) therapy (ART) has been pivotal in reducing perinatal transmission. While most children who are HIV-exposed but uninfected (cHEU) remain in good health, they are at higher risk for neurodevelopmental impairments. We have developed mouse models of in-utero ARV exposure to understand the effects of different ARVs on the fetal development and subsequent neurodevelopmental and motor outcomes. Here, we compare the impact of in-utero exposure to protease inhibitor (PI)-based ART (ritonavir-boosted atazanavir, ATV/r), or integrase strand transfer inhibitor (INSTI)-based ART (dolutegravir, DTG) administered with either the dual NRTI backbone abacavir/lamivudine (ABC/3TC) or tenofovir/emtricitabine (TDF/FTC).
Methods: Plugged C57BL/6 female mice were randomly assigned to one of the five treatment arms, administered at therapeutic doses by oral gavage once daily throughout pregnancy. Treatment arms included: 1) control (water); 2) ABC/3TC+ATV/r, 3) TDF/FTC+ATV/r, 4) ABC/3TC+DTG, 5) TDF/FTC+DTG. Gross and fine motor (coordination and balance) of offspring (males and females) were assessed using the accelerating rotarod test. The striatum region (associated with locomotion) was extracted from the brain and used to study gene expression by real-time quantitative PCR.
Results: Female mice exposed in-utero to regimens containing TDF/FTC (either ATV/r or DTG) showed increased performance in the rotarod test suggesting hyperactivity and/or increased balance and coordination. Mice exposure to regimens containing ABC/3TC (either ATV/r or DTG) performed similar to controls. Gene expression analysis showed changes in neurotransmitter receptors regulating locomotion in the striatum. We found increased subunit (NR2A and NR2B) expression of NMDA glutamate receptors in TDF/FTC+ATV/r exposed mice.
Conclusion: Exposure to TDF/FTC, but not ABC/3TC during pregnancy in mice, regardless of whether the base drug was a PI or INSTI, revealed heightened locomotion and hyperactivity in a sex-dependent manner. These results imply that the choice of NRTI backbone may impact the neurological development of offspring.

The HIV-1 accessory protein, Nef, plays a central role in HIV virulence and pathogenesis by impairing T cell activation and maturation, subverting apoptosis, and modulating major histocompatibility complex class I (MHC-I) expression. Following infection and upon interacting with Src family tyrosine kinases (SFK), Nef initiates and misdirects a signaling cascade that leads to MHC-I cell surface down-regulation and sequestration within the trans-Golgi network. Additionally, this also reduces the presentation of MHC-I-restricted HIV-derived epitopes to limit immune recognition and represents a major impediment towards the development of HIV cure strategies. By performing an in silico docking screen, we identified a small molecule inhibitor (H3-1) of the Nef-SFK interaction. Preliminary studies in HIV-infected primary human CD4+ T cells demonstrated that H3-1 addition to ex vivo culture counteracted Nef-dependent MHC-I down-regulation in the absence of cytotoxicity. When testing this Nef-SFK inhibitor in a transgenic mouse model of a HIV/AIDS-like phenotype, H3-1 lead to enhanced cell surface presentation of both total MHC-I and MHC-I presenting a model epitope; despite being rapidly cleared in vivo after oral and intraperitoneal administration as assessed using mass spectrometry. Given the proteolytically-labile peptidic nature of H3-1, we used organic synthesis to generate a peptidomimetic panel of H3-1 analogues predicted to have improved biostability. As expected, ex vivo plasma stability was improved in these analogues and will be further characterized for their in vivo plasma stability in transgenic mice. Using a luciferase-based readout of protein-protein interaction, we have identified two analogues that significantly impair the intracellular interaction of Nef with Hck, a prominent SFK, in live cells for upwards of 24 hours without measured cytotoxicity. The synthesis and characterization of biostable H3-1 analogues that combat Nef-mediated MHC-I down-regulation is an important step towards evaluating the role of Nef-SFK inhibitors as adjuvants within a HIV/AIDS functional cure.

Introduction.
Over 1 million pregnant people living with HIV receive antiretroviral therapy (ART) annually. Integrase strand transfer inhibitors (INSTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) are commonly used. While the benefits of ART outweigh the risks, antiretrovirals are associated with adverse birth outcomes and metabolic complications. The Na+-independent System L-amino acid transporters (LAT1,2,4) transfer essential amino acids (mainly leucine) across the placenta and are crucial for proper fetal development. Although lower System L transport activity is noted in pregnancies complicated by fetal growth restriction, these transporters have not been investigated in the context of HIV/ART exposure. We explored the effects of antiretrovirals on the expression and activity of System L transporters using a human placental cell line.

Methods.
BeWo cells were induced to syncytialize with 1µM dibutyryl-cAMP for 24h followed by 24h exposure to INSTIs (dolutegravir, raltegravir, bictegravir, cabotegravir) or NNRTIs (efavirenz) at clinically relevant concentrations. Cytoplasmic and membrane expression of LAT1, LAT2 and LAT4 were quantified by Western blot, normalized to α-tubulin or E-cadherin respectively. System L transport activity was measured by tritium-labelled leucine uptake assays. Data were compared using Student’s t-test.

Results.
LAT1 and LAT2 proteins were prominently expressed in the cell membrane while LAT4 was cytoplasmic. LAT1 and LAT2 membrane and LAT4 cytoplasmic protein levels were lower in the bictegravir-treated (C50, Cmax) group compared to control. LAT1 and LAT2 membrane protein levels were lower in the dolutegravir-treated (Cmax) and efavirenz-treated (Cmax) groups compared to control (n=3). Lower leucine uptake was observed in bictegravir-treated cells, correlating with lower levels of System L isoforms.

Conclusions.
Our data suggest that exposure to bictegravir is associated with lower expression and activity of System L isoforms in BeWo cells. Further work is required to understand the impact of INSTI-based ART on placental System L-amino acid transfer and potential impacts on the fetus.