Early Initiation of Antiretroviral Therapy Can Functionally Control Productive HIV-1 Infection in Humanized-BLT Mice

Abstract

Background: Recent reports showed that functional control of HIV-1 infection for a prolonged time is possible by early antiretroviral therapy (ART); however, its underlying mechanism needs to be studied with a suitable animal model. Recently, humanized-BLT (bone marrow, liver, and thymus) mouse (hu-BLT) was shown to be an excellent model for studying HIV-1 infection. We thus tested the feasibility of studying functional control of HIV-1 infection using hu-BLT mice.

Methods: Animals in 3 treatment groups (Rx-6h, Rx-24h, and Rx-48h) and untreated group were infected with HIV-1, followed by ART initiation at 6, 24, or 48 hours postinfection and continued daily for 2 weeks. Three weeks after stopping ART, CD8 T cells were depleted from all animals. Plasma viral load was monitored weekly using droplet digital polymerase chain reaction. Percentage of CD4 and CD8 T cells were measured by flow cytometry. In situ hybridization and droplet digital polymerase chain reaction were used to detect viral RNA (vRNA) and DNA.

Results: Although control animals had high viremia throughout the study, all Rx-6h animals had undetectable plasma viral load after ART cessation. After CD8 T-cell depletion, viremia increased and CD4 T cells decreased in all animals except the Rx-6h group. Viral DNA was detected in spleens of all animals and a few vRNA cells were detected by in situ hybridization in 1 of 3 Rx-6h animals.

Conclusions: Early ART did not act as prophylaxes but, rather, can control HIV-1 productive infection and prevented CD4 T-cell depletion in hu-BLT mice. This mouse model can be used to elucidate the mechanism for functional control of HIV-1.

Figure 1. Schematic of the experimental design

Thirteen adult hu-BLT mice were randomly divided into early treatment (Rx-6h, Rx-24h, Rx-48h, n=3 each) and control (n=4) groups. All animals were inoculated intraperitoneally (IP) with a mixture of two transmitted/founder HIV-1 viruses (2.5 × 10³ TCID₅₀ each). Daily ART was initiated at 6, 24 or 48 hours post-infection for each respective treatment groups. Three weeks after ART was stopped, CD8⁺ T-cells from all animals were depleted. The levels of CD8⁺ T-cells, CD4⁺ T-cells, PVL and vDNA in tissue were monitored as indicated.

Figure 2. Plasma viral load (PVL)

The identical control animals were included in all graphs for comparison purposes. Background cutoff value of 40 copies/ml was determined with plasma from non-infected hu-BLT mice. The 2 weeks on ART and CD8⁺ T-cells depletion period are shaded. (A) Rx-6h. (B) Rx-24h. (C) Rx-48h.

Figure 3. Flow cytometry

The identical control animals were included in all graphs for comparison purposes. (A) CD8⁺ T-cells percentage before and after CD8⁺ T-cells depletion. (B) CD4⁺ T-cells percentage of Rx-6h, (C) Rx-24h and (D) Rx-48h treatment groups over the study period.

Figure 4. HIV-1 DNA copy number in lymphatic tissues

Viral DNA was detected in the lymph node (LN) or Spleen (SP) of necropsied animals by ddPCR. Background cutoff value of 18 copies/10⁶ human cells was determined with non-infected hu-BLT mice spleen tissue. HM380 (Rx-24h) and 452 (Rx-48h) die before sacrifice, therefore their samples are unavailable. (A) HIV-1 DNA copies/10⁶ human cells for Control, Rx-6h, Rx-24h and Rx-48h group (B) Total number of human cells analyzed for HIV-1 DNA for each animal.

Figure 5. HIV-1 vRNA+ cells in the spleens of hu-BLT mice were detected using in situ hybridization

Spleen tissues were collected after >70 days p.i. and fixed in 4% paraformaldehyde. Clusters of green silver grains overlay HIV-1 vRNA+ cells after radioautography for 7 day exposure. (A) Representative image showed numerous HIV-1 vRNA+ cells in the control animal (HM370). (B) Representative image showed numerous HIV-1 vRNA+ cells in Rx-24h (HM430). (C) Representative image showed numerous HIV-1 vRNA+ cells in Rx-48h (HM383). (D and E) Undetectable vRNA+ cells in two animals of Rx-6h (HM323 and 353, respectively). (F) Isolated vRNA+ cell (arrow) in Rx-6h (HM344).