Summary CD32 has raised conflicting results as a putative marker of the HIV-1 reservoir. We measured CD32 expression in tissues from viremic and virally suppressed humanized mice treated relatively early or late after HIV-1 infection with combined antiretroviral therapy. CD32 was expressed in a small fraction of the memory CD4 + T-cell subsets from different tissues in viremic and aviremic mice, regardless of treatment initiation time. CD32 + memory CD4 + T cells were enriched in cell-associated (CA) HIV-1 DNA but not in CA HIV-1 RNA as compared to the CD32 − CD4 + fraction. Using multidimensional reduction analysis, several memory CD4 + CD32 + T-cell clusters were identified expressing HLA-DR, TIGIT, or PD-1. Importantly, although tissue-resident CD32 + CD4 + memory cells were enriched with translation-competent reservoirs, most of it was detected in memory CD32 - CD4 + T cells. Our findings support that CD32 labels highly activated/exhausted memory CD4 + T-cell subsets that contain only a small proportion of the translation-competent reservoir.

HIV-1 infection results in the activation of inflammasome that may facilitate viral spread and establishment of viral reservoirs. We evaluated the effects of the caspase-1 inhibitor VX-765 on HIV-1 infection in humanized NSG mice engrafted with human CD34+ hematopoietic stem cells. Expression of caspase-1, NLRP3, and IL-1β was increased in lymph nodes and bone marrow between day 1 and 3 after HIV-1 infection (mean fold change (FC) of 2.08, 3.23, and 6.05, p<0.001, respectively). IFI16 and AIM2 expression peaked at day 24 and coincides with increased IL-18 levels (6.89 vs 83.19 pg/ml, p=0.004), increased viral load and CD4+ T cells loss in blood (p<0.005 and p<0.0001, for the spleen respectively). Treatment with VX-765 significantly reduced TNF-α at day 11 (0.47 vs 2.2 pg/ml, p=0.045), IL-18 at day 22 (7.8 vs 23.2 pg/ml, p=0.04), CD4+ T cells (44.3% vs 36,7%, p=0.01), viral load (4.26 vs 4.89 log 10 copies/ml, p=0.027), and total HIV-1 DNA in the spleen (1 054 vs 2 889 copies /106 cells, p=0.029). We demonstrated that targeting inflammasome activation early after infection may represent a therapeutic strategy towards HIV cure to prevent CD4+ T cell depletion and reduce immune activation, viral load, and the HIV-1 reservoir formation.

HIV-1 persists in viral reservoirs of latently infected CD4+ T cells containing integrated replication-competent viral DNA. Combined Antiretroviral Therapy (cART) does not eradicate HIV-1 reservoirs and treatment interruption will ultimately lead to viral load rebound. HIV-1 infection dramatically reduces the proportion of functional NK cell subsets and increases the expression of the checkpoint inhibitors NKG2A and KIR2DL. In this regard, we developed novel recombinant molecules combining multimers of the IL-15/IL-15Rα complex with the single-chain fragment variables (scFvs) of NKG2A or KIR2DL, and named them as Natural killer activating Multimeric immunotherapeutic compleXes (NaMiX). NaMiX significantly improved the cytotoxic activity of NK cells against HIV-1 positive ACH-2 cells and resistant Raji cancer cells by increasing their degranulation capacity, release of granzyme B, perforin and IFN-γ expression. Targeting the NKG2A receptor had a stronger effect compared to the targeting of the KIR2DL receptor due to its higher expression on NK cells. In a viral inhibition assay using CD4+ T cells from HIV-1 positive patients under cART, NaMiX initially increased viral replication which was subsequently inhibited by stimulated NK cells. In humanized NSG tg-huIL-15 mice showing functional NK cells, we observed enhanced activation, degranulation and killing by NK cells from the spleen of mice treated with anti-NKG2A NaMiX compared to the cells of control mice previously infected with HIV-1 and treated with cART. Although NaMiX did not delay viral load rebound after treatment interruption in a first attempt, it tend to decrease total HIV-1 DNA in the lungs of the mice. Blocking the inhibitory receptor NKG2A in combination with targeted multimers of IL-15 on NK cells could therefore be a promising immunotherapeutic strategy towards HIV-1 functional cure.

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Background: HIV-1 infection results in the activation of inflammasome involving NLRP3, IFI16, caspase-1 and release of IL-1β and IL-18. Early inflammasome activation may facilitate viral spread and establishment of the viral reservoir. We evaluated the effect of the caspase-1 inhibitor VX-765 on virological and immunological parameters after HIV-1 infection in humanized mice. Methods: NSG mice were engrafted with human CD34+ hematopoietic stem cells and were infected with HIV-1 JRCSF. 15 mice were first sacrificed serially to investigate kinetics of the HIV-1 related inflammasome activation. Infected mice (n=24) were then treated with VX-765 or vehicle from day 1 post infection for 21 days. Blood and organs were collected at different time points, and analysed for inflammasome genes expression, cytokines levels, viral load, CD4 cell count, and total HIV-1 DNA. Results: Expression of caspase-1, NLRP3 and IL1-β was increased in lymph nodes and bone marrow on day 1 and 3 post infection (mean fold change (FC) of 2.08, 3.23, and 6.05, p< 0.001 respectively between day 1 and 3). IFI16 expression peaked at day 24 in lymph node and bone marrow (FC 1.49 and 1.64, p<0.05) and coincides with increased IL-18 levels in plasma (6.89 vs. 83.19 pg/ml, p=0.004). AIM2 and IFI16 expression correlated with increased viral load in tissues (p<0.005 for the spleen) and loss of CD4+ T cells percentage in blood (p<0.0001 for the spleen). Treatment with VX-765 significantly reduced TNF-α at day 11 (0.47 vs. 2.2 pg/ml, p=0.045), IL-18 at day 22 (7.8 vs 23.2 pg/ml, p=0.04), CD4 + T cells (44.3% vs 36,7%, p=0.01) and the CD4/CD8 ratio (0.92 vs 0.67, p=0.005) in plasma. Importantly, viral load (4.26 vs. 4.89 log 10 copies/ml, p=0.027) and total HIV-1 DNA (1 054 vs. 2 889 copies /106 cells, p=0.029) were decreased in VX-765-treated mice as compared to vehicle-treated mice. Discussion: we report here an early inflammasome activation before detectable viral dissemination in humanized mice. We demonstrated that targeting inflammasome activation early after HIV-1 infection may represent a potential therapeutic strategy to prevent CD4+ T cell depletion as well as to reduce immune activation, viral load and the HIV-1 reservoir formation.