Background: Viral dissemination occurs early after infection targeting CD4 T cells and monocytes/macrophages. Despite the administration of antiretroviral therapy (ART), human and simian immunodeficiency virus (HIV/SIV) persists in treated individuals indicating viral reservoir seeding. Monocytes derived from bone marrow and tissue resident macrophages (TRMs) derived from yolk sac, are short-lived and long-lived cells, respectively. We recently demonstrated that early ART efficiently prevents infection of monocytes in SIV-infected rhesus macaques. Thus, our study focused on the role of TRMs in maintaining viral reservoirs.

Methods: Rhesus macaques were infected with SIVmac251 and treated at day 4 post-infection with a cocktail of antiretroviral drugs. Cells from liver and lung were mechanically isolated. The phenotype of TRMs was analyzed by flow cytometry using specific antibodies directed against the CD44, TIM-4, CD117, CD206, CD200R, CD64 and LYVE-1 molecules (previously defined in mice). The levels of viral DNA and RNA were quantified by qPCR. In situ hybridization was used to detect viral RNA. Furthermore, transcriptomic analysis was performed to assess gene profiles in both tissues.

Results: Our results revealed that CD117, CD206 and LYVE-1 are specific markers of TRM cells from liver and lung of SIV-infected rhesus macaques. In non-treated SIV-infected monkeys, higher levels of inflammatory and ISG transcripts were observed consistent with the detection of viral RNA and DNA in both tissues. The levels of viral RNA are positively correlated with viremia in the blood. Early ART prevented the establishment of viral dissemination in both tissues as well as the inflammation.

Conclusion: Herein, we analyzed the phenotypes of TRMs in the lung and liver of SIV-infected rhesus macaques. We demonstrated that early ART efficiently prevents viral seeding both in the liver and lung concomitantly by preventing inflammation. These results highlight the crucial importance of early treatment by decreasing anatomical viral reservoirs.

Background: HIV infection is associated with an increase in immunosuppressive FoxP3+ CD8 Tregs, which contribute to disease progression and immune dysfunction. However, the dynamics of CD8 Tregs in acute infection and following early ART initiation remain understudied.
Methods: Peripheral blood mononuclear cells (PBMCs) were collected from HIV-infected untreated individuals in acute (n=26) and chronic phases (n=10), ART-treated in early infection (n=10, median of ART initiation: 5,5 months post-infection), elite controllers (n=18), and HIV-uninfected controls (n=21). CD8 Tregs subsets were characterized by multiparameter flow cytometry.
Results: HIV infection was associated with an increase in total and effector memory CD8 Tregs in both acute and chronic infection, while early ART normalized only the frequencies of total CD8 Tregs but not their effector memory subset. CD8 Treg expression of CD31, a marker of recent Tregs migrating from the thymus, decreased overtime during infection, indicating the extra-thymic differentiation of CD8 Tregs, which was normalized by early ART. We also observed an overtime increase in CD8 Treg immune activation (HLADR+/CD38+), immune senescence (CD57+/CD28-), and PD-1 expression during both acute and chronic infection, while early ART initiation only restored PD-1+ CD8 Tregs. CD8 Tregs in untreated individuals expressed higher levels of immunosuppressive LAP(TGF-β1), which remained unchanged despite early ART. Additionally, the expression of gut homing markers CCR9 and Integrin-β7 by CD8 Tregs increased in both acute and chronic infections and remained higher than uninfected controls despite early ART. HIV elite controllers share most of the CD8 Treg characteristics in uninfected or early ART-treated individuals.
Conclusions: Although early ART initiation resulted in normalization of total CD8 Tregs frequencies, it was unable to reduce CD8 Treg gut homing potential nor LAP(TGF-β1) production, which in turn, may contribute to gut fibrosis, disease progression, and immune dysfunction despite early ART initiation.

Generating humanized mice with fully functional T cells currently relies on co-implantation of hematopoietic stem cells from fetal liver and autologous thymic tissue (BLT mice). However, access to such tissues has ethical and logistical challenges. Herein, we show that NOD/SCID/IL2rγnull mice humanized with cord blood-derived CD34+ cells and implanted in quadriceps with pediatric thymic tissues excised during cardiac surgeries (CCST mice) are an alternative to BLT mice. Our data reveal that T cells from CCST mice indeed originate from CD34+ progenitor cells; they proliferate efficiently in response to mitogenic stimulation ex vivo and are capable of rejecting allogeneic human leukemic cells in vivo. Despite having less T cells than BLT mice, CCST mice are just as susceptible to mucosal or intraperitoneal HIV-1 infection. Importantly, HIV-1-specific T-cell responses were significantly higher in CCST mice (median: 10.4% vs. 0.7%; p<0.0001 for CD8+ T cells and 3.9% vs. 0.7%; p<0.01 for CD4+ T cells, respectively p < 0.0001). As well, antiretroviral therapy (ART) robustly suppresses viremia and reduces the frequencies of cells carrying integrated HIV-1 DNA by up to 2 log in various tissues of CCST mice. Unsurprisingly, we observe a complete viral rebound in 67% of the animals by 2-4 weeks following ART interruption, suggesting the presence of HIV reservoirs. In conclusion, CCST mice represent an ethical and practical alternative to BLT mice, broadening the feasibility of utilizing humanized mice for research on HIV and other human diseases.

A rare subset of people living with HIV (PLWH), known as elite controllers (EC), control their viral load (VL) below detectable levels whereas viremic controllers (VC) maintain VLs at <3000 copies of HIV RNA/ml of plasma spontaneously, without antiretroviral treatment. The underlying immunological mechanisms behind this phenomenon are under investigation. HIV gp120/Env-specific antibody dependent (AD) cellular cytotoxicity (ADCC) was identified as a correlate of protection against HIV infection in the RV144 vaccine trial. If AD functions also play a role in controlling infection, then ECs and VCs would be expected to have more potent AD functions compared to PLWH who are untreated (UTP) or treated progressors (TP). We generated a novel HIV-infected target cell line that expressed Env in a closed conformation (siCEM cells) to 1) quantify Env-specific antibodies in plasma from EC, VC, UTP and TP and 2) to measure their AD functional competence. We tested plasma antibodies for four AD functions: activation of monocytes to induce phagocytosis (ADCP) and trogocytosis (ADCT), NK cell-mediated ADCC and activation of the complement cascade (ADCD). Anti-Env-specific antibodies from EC, VC and UTP demonstrated similar levels of AD functions that were higher than those in TP. ECs differed from all the other groups by having a more strongly correlated, polyfunctional anti-Env-specific AD response. The distribution of IgG subclasses and glycosylation patterns of anti-gp120-specific antibodies was measured. Multivariate dimensionality-reduction tools could not distinguish the four subject groups based on these parameters. Normalization of AD responses by anti-Env antibody concentration eliminated between-group differences in AD functions suggesting that quantity, and not the quality, of these antibodies drove the AD function potency. Reservoir size was measured by integrated HIV-DNA PCR. ADCC function was higher in controllers (EC+VC) who maintained an undetectable reservoir size than controllers with a detectable reservoir size.

Background: Understanding the mechanisms that are responsible for natural immune T-cell protection against HIV-1 in elite controllers (EC) is key to achieve successful cure strategies. This is truer considering that a second case of EC recently showed a complete treatment-free remission of HIV-1 infection (Esperanza patient). Here, our aim was to show that autophagy, a lysosomal degradative system, drove superior anti-HIV-1 responses in EC by providing cells with diverse cellular energy stores.

Methods: We assessed autophagy in anti-HIV-1 CD8 and CD4 T-cells from EC and age matched patients under antiretroviral therapies (ART). We used complementary methods including electron microscopy and flow cytometry to measure numbers of autophagic vesicles along with expression of autophagic markers. We inhibited autophagy in EC by Beclin-1 gene silencing or with lysosomal blockers and assessed mitochondrial metabolism. Our Seahorse device allowed us to assess not only mitochondrial energy production, but also to what extent the latter was fueled by the fatty acid oxidation (FAO) and glutaminolysis. Finally, we investigated polyfunctional anti-HIV-1 T-cell responses in EC with or without lysosomal and specific metabolic inhibitors (etomoxir and BPTES, respectively for FAO and glutaminolysis).

Results: We confirmed highly active AMPK-dependent autophagy in anti-HIV-1 T-cells in EC when compared to ART. Autophagy in EC provided lipid- and protein-based energy substrates, respectively in CD8 and CD4 T-cells, which fueled mitochondrial energy production. We validated that autophagy-related FAO and glutaminolysis in EC respectively ensured proper anti-HIV-1 CD8 and CD4 responses. Finally, anti-HIV-1 T-cell responses in ART could be reinforced with the AMPK inducer AICAR.

Conclusions: Our data revealed that autophagy and metabolic pathways are working together in EC to fuel energy production and drive optimal T-cell immunity against HIV-1. The fact that we could reinforce anti-HIV-1 responses in ART by targeting those pathways has to be considered for the next cure developments.

Background
Current antiretroviral drugs block different steps of the viral replication cycle but not the transcription, a process under the control of host-cell transcription factors. In previous studies, we demonstrated that the transcriptional signature associated with HIV permissiveness in Th17 cells includes the circadian clock components/regulators BMAL1 and REV-ERBs. Of note, REV-ERBs act as transcriptional repressors of BMAL1 (a transcriptional activator binding to E-boxes in the HIV promoter) and RORC2 (the master regulator of Th17 polarization). Thus, we hypothesized that REV-ERBs regulate both BMAL1-mediated HIV replication and RORC2-mediated effector functions in Th17 cells.

Methods
To test this hypothesis, we used the REV-ERB agonists reported to be efficient in decreasing Th17-mediated autoimmune pathology in mice. A viral outgrowth assay (VOA) was performed with memory CD4+ T cells of ART-treated PLWH activated via CD3/CD28 in the presence/absence of the REV-ERB agonists. Lentiviral vectors were used to over express BMAL1 in primary CD4+ T cells. Cytokines and HIV-p24 levels were measured by ELISA. HIV-DNA integration was quantified by PCR.

Results
The REV-ERB agonists potently inhibited HIV replication in vitro and viral outgrowth in VOA. The antiviral effects coincided with decreased IL-17A and IFN-γ production. We also observed a decreased of RORC2 mRNA that we identified as a novel cell-specific target for HIV-1 therapy in Th17 cells. Single-round infection with a VSV-G-pseudotyped HIV showed decreased HIV-p24 expression/production but no differences in HIV-DNA integration in presence of REV-ERB agonists, indicative of an inhibitory effect post-integration, likely during transcription. Finally, we confirmed that BMAL1 overexpression increases HIV replication.

Conclusion
These results provide a strong rationale for further evaluating the possibility to therapeutically target REV-ERBs as a way to limit BMAL1-dependent HIV transcription and subsequently diminish chronic immune activation and non-AIDS co-morbidities during ART.

Background: HIV persists through the proliferation of latently infected CD4+ T cells. Although most proviruses persist in a latent form, a small number of infected cells produce virions. Immunosuppressants given to people undergoing kidney transplants to prevent rejection block several aspects of the immune response including proliferation and activation of T cells. We hypothesized that immunosuppressive therapy could reduce both the proliferation of reservoir cells and markers associated with residual production of HIV.

Methodology: Seven HIV-infected participants on suppressive ART who underwent kidney transplantation and initiated immunosuppressive therapy were enrolled. Longitudinal blood samples were collected before and after the transplant (6-7 samples per participant, over 2 years). The effects of immunosuppressants on T cell activation and plasma HIV-envelope antibody responses (a surrogate of virion production) were assessed by flow cytometry and ELISA, respectively. HIV DNA and RNA were quantified by qPCR in isolated CD4+ T cells.

Results: As expected, we observed a significant decrease in the expression levels of HLA-DR and Ki67 in CD4+ T cells after transplantation, which was accompanied by an increase in the frequency of central memory cells and a sharp decrease in the frequency of regulatory T cells. Antibody levels to the HIV envelope also decreased following immunosuppressive therapy initiation. Overall, there was a modest decrease in HIV DNA and RNA levels after kidney transplantation (median fold change = 0.9 and 0.6 respectively), however this decrease was not sustained.

Conclusion: Immunosuppressive therapy decreases the proliferation and activation of CD4+ T cells which is concomitant to a modest and transient decrease in HIV reservoir markers in most participants. Our results suggest that current immunosuppressive drugs are insufficient to profoundly and durably affect the HIV reservoir and suggest that the homeostatic forces that maintain the pool of infected cells during suppressive ART will be difficult to counteract.

Background: Humoral responses to COVID-19 vaccines, particularly third doses, remain incompletely characterized in people living with HIV (PLWH).

Methods: We measured antibodies against the SARS-CoV-2 spike protein receptor-binding domain (RBD), ACE2 displacement and viral neutralization activities one month following the first COVID-19 vaccine dose, one, three and six months following the second dose, and one month after the third/booster dose in 99 adult PLWH and 152 controls. All PLWH were receiving suppressive ART, with median CD4+ T-cell counts of 715 (Q1-Q3 545-943) cells/mm3.

Results: After adjustment for sociodemographic, health and vaccine-related variables, HIV was associated with significantly lower anti-RBD antibody concentrations and ACE2 displacement activity, but not lower viral neutralization, after one vaccine dose. One and three months after the second dose however, HIV was no longer significantly associated with the magnitude of any humoral response after adjustment for these factors. Rather, older age, a higher burden of chronic health conditions, and having received two ChAdOx1 doses were associated with lower responses in both PLWH and controls. There was no significant correlation between the most recent or nadir CD4+ T-cell count and vaccine responses after two doses. Six months after the second dose, antibody concentrations had declined by an average of 0.7 log10 from those measured one month after the second dose. In both PLWH and controls, the booster dose increased antibody concentrations to an average of 0.4 log10 higher than peak responses after the second dose.

Conclusions: Early data indicate that PLWH with well-controlled viral loads on ART and CD4+ T-cell counts in a healthy range have robust humoral responses to second and third COVID-19 vaccine doses, similar to those of HIV-uninfected controls. Factors such as older age, co-morbidities, initial vaccine regimen and the rise of SARS-CoV-2 variants will influence when PLWH will benefit from booster doses.