Amplification of Replication Competent HIV-1 by Adoptive Transfer of Human Cells From Infected Humanized Mice

Abstract

Detection of latent human immunodeficiency virus type 1 (HIV-1) in "putative" infectious reservoirs is required for determining treatment efficiency and for viral elimination strategies. Such tests require induction of replication competent provirus and quantitative testing of viral load for validation. Recently, humanized mice were employed in the development of such tests by employing a murine viral outgrowth assay (mVOA). Here blood cells were recovered from virus infected antiretroviral therapy suppressed patients. These cells were adoptively transferred to uninfected humanized mice where replication competent virus was recovered. Prior reports supported the notion that an mVOA assay provides greater sensitivity than cell culture-based quantitative VOA tests for detection of latent virus. In the current study, the mVOA assays was adapted using donor human hematopoietic stem cells-reconstituted mice to affirm research into HIV-1 elimination. We simulated an antiretroviral therapy (ART)-treated virus-infected human by maintaining the infected humanized mice under suppressive treatment. This was operative prior to human cell adoptive transfers. Replication-competent HIV-1 was easily detected in recipient animals from donors with undetectable virus in plasma. Moreover, when the assay was used to investigate viral presence in tissue reservoirs, quantitative endpoints were determined in "putative" viral reservoirs not possible in human sample analyses. We conclude that adoptive transfer of cells between humanized mice is a sensitive and specific assay system for detection of replication competent latent HIV-1.

Keywords: HIV-1 infection; adoptive transfers; humanized mice; lymphoid tissue; viral latency; viral outgrowth assays; viral reservoirs.

Figure 1

Descriptors of donor humanized mice. (A) Flow cytometric gating strategy for human cell reconstitution in NSG-humanized mice. In brief, human cells (hCD45+) were gated from total lymphocytes and single cells and subsequently grouped into human T lymphocytes (hCD3+) and B lymphocytes (hCD19+). Human CD3+ cells were separated into human CD4+ (hCD4+) and CD8+ (hCD8+) T lymphocytes. Detailed immune and viral information of individual animal (n = 18) was presented as scatter dots covering (B) peripheral human CD45+, CD3+, CD4+, CD8+, and CD19+ cell levels, (C) CD4+/CD8+ T cell ratio, (D) plasma viral load (pVL), (E) tissue HIV-1 DNA, and (F) tissue HIV-1 RNA distributed across spleen, BM, gut, lung, brain, liver, and kidney. Data are expressed as mean ± SEM. Orange dots in (D) indicated that pVL was below the detection limit (DL) of 400 HIV-1 RNA copies/ml as shown by the dotted line. Orange dots in (E,F) shows tissue viral levels of animals with undetected pVL. The DL is below 10 HIV-1 copies/10⁶ human CD45+ cells as measured using semi-nested real time qPCR. The dotted line corresponded to the DL. (G) Spearman's rank correlation tests were conducted between humanized mice pVL and respective spleen DNA (black), spleen RNA (red), BM DNA (blue), and BM RNA (green). The Spearman correlation coefficient (r) and p-value for each analysis were displayed.

Figure 2

Murine HIV-1 outgrowth assay. A mixture of mouse and human cells were isolated from donor humanized mouse spleen or BM and immediately intraperitoneally injected into naïve humanized mice as described in the scheme. Recipient animals were maintained for 1 month before sacrifice and evaluated for HIV-1 recovery.

Figure 3

Descriptors of recipient humanized mice. Peripheral human cell levels (hCD45+, hCD3+, hCD4+, hCD8+, and hCD19+ cells) before (blue) and after (red) adoptive transfer were depicted for (A) total recipient (TR) humanized mice, and subgroups (C) spleen recipient (SR) and (D) bone marrow recipient (BR) humanized mice. (B) Scatter plots represented CD4+/CD8+ T cell ratio of individual TR, SR, and BR before (blue) and after (red) adoptive transfer. (E) Plasma viral load (pVL) of individual recipient humanized mouse at sacrifice was displayed for TR (black), SR (blue), and BR (red). Orange dots represented humanized mice receiving engraftment from donors with undetected plasma VL. The detection limit is <400 HIV-1 RNA copies/ml. A Spearman's rank correlation test was conducted to investigate the association (F) between recipient humanized mouse plasma VL and respective peripheral CD4+ T cell loss and (G) between recipient humanized mouse plasma VL and respective end-point peripheral CD4+/CD8+ T cell ratio. The Spearman correlation coefficient (r) and p-value for each analysis were displayed. In (A–E), data are expressed as mean ± SEM and considered *, *** statistically significant, at p < 0.05 and p < 0.001.

Figure 4

Adoptive transfer of donor humanized mice with undetected or close-to-detection limit viral load. (A) The experimental scheme. In brief, donor humanized mice were first infected with HIV-1_ADA for 2 weeks. Animals were then maintained under LASER ART which contained a combination of nanoformulated long-acting named NMCAB, NMABC, NM3TC, and NRPV for 3 months until pVL reached undetectable level or close to detection limit of 400 HIV-1 RNA copies/ml. Donor humanized mouse splenocytes and PBMCs were extracted and separately engrafted to respective naïve humanized mice. The recipient animals were maintained for 9 weeks to monitor HIV-1 recovery. The dynamic changes of individual donor humanized mouse were recorded for (B) pVL, (C) peripheral human CD45+, and (D) peripheral human CD4+ T cells. (E) Tissue HIV-1 DNA and (F) tissue HIV-1 RNA in individual donor humanized mice was evaluated across spleen, BM, gut, lung, brain, liver, and kidney, using semi-nested real time qPCR. The dotted line in (B) indicated the detection limit (DL) of 400 HIV-1 RNA copies/ml. The dotted line in (E,F) showed the DL below 10 HIV-1 copies/10⁶ human CD45+ cells.

Figure 5

Recipient humanized mice engrafted from donor humanized mice under effective treatment. A total of 5 naïve humanized mice received adoptive transfer among which three (M344, M467, and M473) received splenocytes engraftment, and two (M329 and M358) received PBMCs engraftment. (A) Peripheral VL of individual spleen and PBMCs recipients after adoptive transfer, respectively. (B) Peripheral CD4+ T cell levels of individual spleen and PBMCs recipients after adoptive transfer. (C,E) tissue HIV-1 DNA and (D,F) tissue HIV-1 RNA in individual donor humanized mice was evaluated across spleen, gut, lung, brain, liver, and kidney, using semi-nested real time qPCR. M344 (red) received spleen transfer from donor M352. M473 and M358 (blue) received spleen and PBMCs engraftment from M375, respectively. M467 and M329 (green) received spleen and PBMCs engraftment from M387, respectively. The dotted line in (A) indicates the detection limit (DL) of 400 HIV-1 RNA copies/ml. The dotted line in (C–F) showed the DL below 10 HIV-1 copies/10⁶ human CD45+ cells.