Combined cART including Tenofovir Disoproxil, Emtricitabine, and Dolutegravir has potent therapeutic effects in HIV-1 infected humanized mice

Abstract

HIV-1 reservoirs persist in the presence of combined antiretroviral therapy (cART). However, cART has transformed HIV-1 infection into a chronic disease marked by control of HIV-1 viral load and mortality reduction. Major challenges remain, including viral resistance upon termination of cART and persistence and identification of tissue distribution of HIV-1 reservoirs. Thus, appropriate animal models that best mimic HIV-1 pathogenesis are important, and the current study complements our previously published validation of the CD34+ hematopoietic humanized mouse model for this purpose. Here we analyze viral suppression using the recently developed combination of antiretrovirals that include Tenofovir Disoproxil (TDF), Emtricitabine (FTC), and Dolutegravir (DTG), a choice based on recent clinical outcomes showing its improved antiretroviral potency, CD4+ T cell preservation, tolerability, and prevention of viral drug resistance compared to that of previous regimens. We used quantitative Airyscan-based super resolution confocal microscopy of selected mouse tissues. Our data allowed us to identify specific solid tissue reservoirs of human T cells expressing the HIV-1 core protein p24. In particular, lymph node, brain, spleen, and liver were visualized as reservoirs for residual infected cells. Marked reduction of viral replication was evident. Considering that detection and visualization of cryptic sites of HIV-1 infection in tissues are clearly crucial steps towards HIV-1 eradication, appropriate animal models with pseudo-human immune systems are needed. In fact, current studies with humans and non-human primates have limited sample availability at multiple stages of infection and cannot easily analyze the effects of differently administered combined antiretroviral treatments on multiple tissues. That is easier to manage when working with humanized mouse models, although we realize the limitations due to low human cell recovery and thus the number of cells available for thorough and comprehensive analyses. Nonetheless, our data further confirm that the CD34+ humanized mouse model is a potentially useful pre-clinical model to study and improve current anti-HIV-1 therapies.

Keywords: Antiretroviral therapies; HIV/AIDS pathogenesis; Hu-mouse models.

Fig. 1

Timeline of experimental steps. Newborn mice were irradiated and injected with human CD34+ hematopoietic stem cells. After 20 weeks, engrafted mice were infected with 10,000 TCID_50% HIV-1 BaL and a subset treated with cART 3 weeks post-infection. This subset of HIV-1 infected mice received oral cART containing Dolutegravir (DTG), Tenofovir Disoproxil (TDF), and Emtricitabine (FTC) for 17 weeks. After 20 weeks post-infection, mice were euthanized, and organs collected for qPCR and imaging experiments. Total DNA was quantified using qPCR in all four collected tissues, and infection sites mapped via p24 imaging in all four collected tissues using super resolution Airyscan imaging

Fig. 2

Confocal images of p24 in CD45+ lymphocytes or CD4+ T cells from hu-mouse tissues. Shown are a donor PBMC infected in vitro (A) and HIV(+) CD45+ (B), HIV(−) CD45+ (C), HIV(+) CD4+ (D), or HIV(−) CD4+ (E) lymphocytes or T cells in mouse liver, lymph node, and spleen from infected or uninfected hu-mice. Representative tissue sections were analysed by α-p24 mAb in red and human α-CD45 or -CD4 in green among the indicated groups of mice. White arrows indicate cells expressing p24 (A, B, and D). The bar size is 5 µm for PBMCs in panel A and 10 µm for tissue sample cells in panels B–E

Fig. 3

Peripheral blood viral loads. Mice were bled every few weeks and RNA isolated from plasma. RNA copies/mL were calculated from qRT-PCR as described in “Materials and methods”. Copies/mL are graphed on a log10 scale based on weeks post-infection. cART treatment was initiated at 3 weeks p.i. Each treatment group contained 3 mice. A color-coded relative geometric mean for each treatment group was added with HIV(+) in red, HIV(+) cART(+) in blue, and HIV(−) in green

Fig. 4

Viral DNA levels in tissues corresponding to visualized productive HIV-1 infection sites in Fig. 2. The first column in each panel represents tissue samples from infected mice; the second column represents tissues from HIV-1 infected, but cART treated mice; and the third column represents tissues from uninfected mice. The first panel is from LN, the second panel from liver, the third panel from brain, and the fourth panel from spleen tissues. The data include groups of 3 animals for the HIV-1 positive group, and 5 different animals for the HIV-1 positive, cART treated group and the HIV-1 negative control group. P values are 0.041, 0.001, < 0.001, and < 0.001 respectively, with the red lines indicating the average value of 3 or 5 mice per group. Copy numbers are graphed on a log₁₀ scale and reported as copies per 10⁶ human cells. *p < 0.05, **p < 0.01, ***p < 0.001