Humanized NOD/SCID/IL2rγnull (hu-NSG) Mouse Model for HIV Replication and Latency Studies

Abstract

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in high demand. With a close genetic and behavioral resemblance to humans, small animals such as the mouse are good candidates for human disease models, through which human-like symptoms and responses can be recapitulated. Further, the mouse genetic background can be altered to accommodate diverse demands. The NOD/SCID/IL2rγ^null (NSG) mouse is one of the most widely used immunocompromised mouse strains; it allows engraftment with human hematopoietic stem cells and/or human tissues and the subsequent development of a functional human immune system. This is a critical milestone in understanding the prognosis and pathophysiology of human-specific diseases such as HIV/AIDS and aiding the search for a cure. Herein, we report a detailed protocol for generating a humanized NSG mouse model (hu-NSG) by hematopoietic stem cell transplantation into a radiation-conditioned neonatal NSG mouse. The hu-NSG mouse model shows multi-lineage development of transplanted human stem cells and susceptibility to HIV-1 viral infection. It also recapitulates key biological characteristics in response to combinatorial antiretroviral therapy (cART).

Figure 1.. Syringe/needle setup used for intrahepatic injection.

The custom-made Hamilton 280508 syringe/needle setup includes a 30-gauge, 51-mm-long needle with a beveled edge and an attached 50-μL glass syringe. Maximum injection volume in this procedure is 25 μL.

Figure 2.. Flow cytometry data represent successful engraftment and the developments of lymphoid and myeloid cells in the peripheral blood.

Successfully prepared peripheral blood samples should have discrete population separations, and a well-engrafted hu-NSG mouse should have T-cell, B-cell and monocyte positive populations presented in the peripheral blood. A CD34:CD8 chart is recommended for ratio calculation. a) Successful engraftment showed more than 25% CD45+ human leukocytes in the peripheral blood; discrete population of b) B-cells, c) monocytes, d) T-cells among human CD45+ leukocyte; e) CD4+ helper T-cells and f) CD8+ cytotoxic T-cells are well separated, and g) yields a ratio between 1.5 to 2.5 in this uninfected hu-NSG.

Figure 3.. CD4+ cell count changes throughout the course of viral infection, cART and cART withdraw.

As infection progresses, the CD44:CD8 ratio decreases from 1.5–2.5 to >1.0, CD4:CD8 ratio has been served as a clinical parameter in evaluating HIV prognosis as well as treatment efficacies. a) Representative flow data indicating the change in CD4:CD8 ratios during the experimental course; b) Comparison trend chart indicating the effectiveness of cART, which can be identified as restored percentage of CD4+ T cells. Detection of CD4+ T cell count by flow cytometry. N: number of tested mice = 6; Error bars: means ± SEM. * p <0.05, ** p <0.01, ***p <0.001, **** p <0.0001, ns: no significant different. Two-way ANOVA analysis is employed. Figure is reprinted with permission.

Figure 4.. Changes of serum viral RNA copy numbers throughout the course of viral infection, cART and cART withdraw

Detection of plasma viremia in the HIV-infected hu-NSG mice by qRT-PCR. The shaded area indicates the time period during which the mice received cART (from Day 28 to Day 70 as shown). The limit of detection (indicated by the dashed line) of the PCR assay is (~110–160 RNA copies/mL) in 50 to 80 μL of plasma obtained through the tail vein. Star (*) indicates viral RNA not detected in cART treated animals at Day 56. Serum viral RNA copy number analyzed from peripheral blood samples serves as direct evidence concerning the degree of viral infection. It should be in the agreement with the CD4 flow analysis. N: number of tested mice = 6; Error bars: means ± SEM. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001, ns: no significant different. Two-way ANOVA analysis is employed. Figure is reprinted with permission.