Provirus reactivation is impaired in HIV-1 infected individuals on treatment with dasatinib and antiretroviral therapy

Abstract

The latent viral reservoir formed by HIV-1, mainly in CD4 + T cells, is responsible for the failure of antiretroviral therapy (ART) to achieve a complete elimination of the virus in infected individuals. We previously determined that CD4 + T cells from individuals with chronic myeloid leukemia (CML) on treatment with dasatinib are resistant to HIV-1 infection ex vivo. The main mechanism for this antiviral effect is the preservation of SAMHD1 activity. In this study, we aimed to evaluate the impact of dasatinib on the viral reservoir of HIV-infected individuals with CML who were on simultaneous treatment with ART and dasatinib. Due to the low estimated incidence of HIV-1 infection and CML (1:65,000), three male individuals were recruited in Spain and Germany. These individuals had been on treatment with standard ART and dasatinib for median 1.3 years (IQR 1.3-5.3 years). Reservoir size and composition in PBMCs from these individuals was analyzed in comparison with HIV-infected individuals on triple ART regimen and undetectable viremia. The frequency of latently infected cells was reduced more than 5-fold in these individuals. The reactivation of proviruses from these cells was reduced more than 4-fold and, upon activation, SAMHD1 phosphorylation was reduced 40-fold. Plasma levels of the homeostatic cytokine IL-7 and CD4 effector subpopulations TEM and TEMRA in peripheral blood were also reduced. Therefore, treatment of HIV-infected individuals with dasatinib as adjuvant of ART could disturb the reservoir reactivation and reseeding, which might have a beneficial impact to reduce its size.

Keywords: CD4+ T cells; Dasatinib; HIV functional cure; HIV reservoir; Proviral reactivation; SAMHD1.

Fig. 1.. Treatment with ART and dasatinib reduced the frequency of latently infected cells.

(A) Quantification by dPCR of proviral DNA per million of PBMCs from HIV-infected individuals on treatment with ART (participants 33, 34 and 35) and dasatinib in comparison with individuals only on ART. (B) Followup of the previously indicated participants, highlighting the sample represented in the previous bar graph for participants 33, 34 and 35 (black triangle) or individuals on treatment only with ART (black circle). Each dot corresponds to one sample and lines represent mean ± standard error of the mean (SEM). Statistical significance was calculated using Mann–Whitney U test. *p < 0.05.

Fig. 2.. Treatment with dasatinib interfered efficiently with TCR-mediated reactivation of HIV-1 provirus.

The synthesis of HIV-1 core antigens (A) and the phosphorylation of SAMHD1 (B) were quantified by flow cytometry in CD4+ T cells isolated from PBMCs of HIV-infected individuals only on ART activated for 7 days with antiCD3/CD28/IL-2 in the presence (+) or absence (−) of dasatinib in the culture medium. The synthesis of HIV-1 core antigens (C) and the phosphorylation of SAMHD1 (D) were also quantified by flow cytometry in CD4+ T cells isolated from PBMCs of HIV-infected individuals on treatment only with ART or with ART and dasatinib (Participants 33, 34 and 35), activated for 7 days with antiCD3/CD28/IL-2. Each dot corresponds to one sample and lines represent mean ± SEM. Open symbols stand for undetected values. Statistical significance was calculated using Mann–Whitney U test. ** p < 0.01.

Fig. 3.. Treatment with dasatinib did not change significantly the proportion of intact and defective HIV-1 proviruses in PBMCs from individuals 33 and 34.

The proportion of intact proviruses (A), hypermutated/3’-deleted (B) and 5’-deleted (C) proviruses per million of cells was evaluated by IPDA in PBMCs from individuals 33 and 34 in comparison with HIV-infected individuals treated only with ART. Each dot corresponds to one sample and lines represent mean ± SEM. Open symbols stand for undetected values. Statistical significance was calculated using Mann–Whitney U test.

Fig. 4.. Treatment with dasatinib modified the distribution of CD4+ T cell subpopulations in HIV-infected individuals.

The proportion of CD3+ (A) and CD4+ (B) cells in PBMCs from HIV-infected individuals treated only with ART or treated with ART and dasatinib (individuals 33, 34 and 35) was compared to individuals HIV negative affected with CML on treatment only with dasatinib and with healthy donors. (C) The distribution of CD4+ T cell subpopulations was determined by flow cytometry in PBMCs isolated from these three groups of individuals after staining with antibodies against CCR7 and CD45RA. Each dot corresponds to one sample and lines represent mean ± SEM. Statistical significance was calculated using One-way ANOVA. *p < 0.05; **p < 0.01.

Fig. 5.. Treatment with dasatinib changed the levels of homeostatic cytokines in plasma of HIV-infected individuals.

Plasma levels of IL-7 (A), IL-21 (B), IL-15 (C) and IL-2 (D) from HIV-infected individuals on ART and dasatinib (Participants 33, 34 and 35) or only with ART were analyzed by Luminex assay and compared to plasma levels of HIV negative individuals with CML on treatment with dasatinib and healthy donors. Each dot corresponds to one sample and lines represent mean ± SEM. Statistical significance was calculated using Mann–Whitney U test.