A major barrier to the development of a cure for HIV-1 is the persistence of a reservoir of latently infected cells that is not eliminated by antiretroviral therapy (ART). Efforts are underway to test latency reversal agents that reactivate HIV-1 to expose latently infected cells to pharmaceutical and/or immunological elimination, the so-called shock and kill strategy. Many kill strategies incorporate aspects of the host immune response to destroy these cells. Cure studies would be greatly facilitated by a small animal model that could be infected with HIV-1, establish a reservoir of latently infected cells, undergo ART for sufficient time to suppress viremia, and then survive in a healthy state long enough to evaluate shock and kill approaches. Additionally, animals provide significantly greater sensitivity over in-vitro assays in the ability to detect remaining latent infection. In cases where the latent reservoir has been reduced but not eliminated, it could take significant time following treatment interruption for residual virus to emerge from latency, so long-lived models are essential.
BLT-humanized mice can be treated with ART to establish latent HIV-1 infections [1,2] and, importantly for cure studies, they also develop HIV-1-specific immune responses [3,4]. Another advantage of BLT-humanized mice is that up to 50 mice can be reconstituted with tissue from a single human donor, providing sufficient number of animals for statistically powerful studies. Although BLT-humanized mice are the best model for HIV-1 studies in most respects, they have a major drawback. Coincident with human reconstitution, BLT mice often develop graft versus host disease (GVHD) and begin to die around 22 weeks posthumanization . This time limitation represents a major obstacle in the use of BLT mice for cure studies . To avoid GVHD issues, humanized mice such as T-cell-only reconstituted mice, have been developed to study latency . However, such mice lack B-cell responses and human macrophages, which may be an important reservoir for HIV-1 .
Here, we investigate the utility of C57BL/6 Rag2−/−γc−/−CD47−/− triple knockout BLT (TKO-BLT) mice, which are resistant to the development of GVHD , to study long-term ART suppression of HIV-1. TKO-BLT mice develop high-levels of multilineage hematopoiesis with organized lymphoid tissues and have an intact complement system. They exhibit hallmarks of human HIV-1 infection including CD4+ T-cell depletion and immune activation. Infection is characterized by transient high-level viremia that is resolved to low levels, broad HIV-1-specific T-cell responses and low but detectable virus-specific immunoglobulin G (IgG) responses . Previously, TKO-BLT mice showed no signs of GVHD out to 29 weeks after humanization . The current study extends this to 45 weeks in mice generated from numerous human donors and includes 6 weeks of infection before initiating an 18-week regimen of oral ART to suppress HIV-1 followed by 4 weeks of therapy interruption. The results indicate that the TKO-BLT mouse is a practicable model in which to study HIV-1 cure strategies. Following the time to establish an HIV-1 reservoir and achieve virus suppression by ART, the mice survive at least another 5 months in a healthy state, sufficient time to rigorously test cure strategies. Given that the mice remained well reconstituted and healthy at the final time point, experiments may be extended even further.
Materials and methods
Anonymous tissue donations were obtained with informed written consent via Advanced Bioscience Resources. Research was conducted under the National Institutes of Health (NIH) office of human study participants research exemption #4980. Animal study proposals RML2014–032, RML2015–022, RML2015–081, and RML2016–030 were approved by the Rocky Mountain Laboratories Animal Care and Use Committee. Experiments were done in accordance with Public Health Service Office of Laboratory Animal Welfare regulations. Rocky Mountain Laboratories is fully accredited by Association for Assessment and Accreditation of Laboratory Animal Care.
Humanized triple knockout-BLT mice
C57BL/6 Rag2−/−γc−/−CD47−/− (TKO) mice were humanized using the BLT method as previously described [4,9]. Briefly, 6–10-week-old mice were irradiated and transplanted with 17–22-week gestation human thymus and liver followed by injection of autologous liver-derived CD34+ hematopoietic progenitor cells. Animals were housed under specific pathogen-free conditions.
HIV-1 challenge and antiretroviral therapy
HIV-1 stocks (strain JR-CSF) were prepared as previously described . Intraperitoneal infections were one time, 100-μl injections of 10 000 tissue culture infectious units (TCIU). Liquid formulations of tenofovir disoproxil fumarate (TDF), emtricitabine (FTC) and dolutegravir were provided by Gilead Sciences (Foster City, California, USA) and administered daily by subcutaneous injection. Tablets of TDF, FTC, and raltegravir (RAL; Merck & Co., Kenilworth, New Jersey, USA) were crushed and formulated with TestDiet 5B1Q feed as previously described . Briefly, powdered TDF, FTC, and RAL were incorporated into TestDiet 5B1Q irradiated feed pellets at a final concentration of 720 mg/kg TDF, 520 mg/kg FTC, and 4800 mg/kg RAL.
Isolation of plasma and human leukocytes
Blood was collected in ethylenediaminetetraacetic acid (EDTA) and centrifuged to obtain plasma. Blood leukocytes were purified using red blood cell Lysis Buffer (BioLegend, San Diego, California, USA). Splenocytes and thymic organoid cells were obtained by passage through 100-μm filters and bone marrow was flushed from femurs and tibias prior to lysis with (NH4Cl 0.15 M, KHCO3 10-mm, EDTA 0.1 mol/l). CD4+ splenocytes used for proviral quantification were preenriched using positive selection (Miltenyi, San Diego, California, USA).
Leukocytes were stained using CD3-V450, CD4-allophycocyanin (APC), CD45-V500, CD123-phycoerythrin, and lin-1 (CD3, CD14, CD16, CD19, CD20, CD56)-fluorescein isothiocyanate (BD Biosciences, San Jose, California, USA); CD11c-APC and human leukocyte antigen - antigen-D related (HLA-DR) Alexa Fluor 700 (Biolegend); CD8-APC-eFluor 780, CD19 PerCP-Cy5.5 and CD45 eFluor450 (eBioscience, San Diego, California, USA). Samples were run on an LSRII (BD Biosciences) and analyzed using FlowJo 9.9 (TreeStar Inc., Ashland, Oregon, USA).
Quantification of HIV-1 antigenemia, viral RNA, and proviral DNA
Plasma HIV-1 p24 levels were determined by ELISA (Advanced Bioscience Laboratories, Rockville, Maryland, USA). Plasma and cell-associated viral RNA (ca-vRNA) was measured by using either the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, v2.0 (Roche, Pleasanton, California, USA) or Abbott RealTime HIV-1 assay on the m2000 system (Abbott, Des Plaines, Illinois, USA). Genomic DNA was isolated with the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Determination of proviral DNA was performed by quantitative real-time PCR as detailed previously  using lysates from 5 × 106 CD4+-enriched splenocytes.
Statistical calculations were performed using GraphPad Prism (GraphPad Software, La Jolla, California, USA).
Triple knockout-BLT mice sustain long-term human engraftment and HIV-1 infection
To assess the durability of human engraftment in TKO-BLT mice, blood was analyzed at 12, 35, and 45 weeks posttransplant (wpt). Increasing frequencies of human leukocytes (hCD45+) were observed over the 45-week period (Fig. 1a top) but the absolute number of human leukocytes remained stable between 12 and 45 wpt peaking at 35 wpt (1.5 × 106/ml; Fig. 1a bottom). This suggested that the human compartment remained stable and the residual mouse myeloid compartment was gradually lost. Over time, total T cells (CD3+) increased with slightly higher proportions and numbers of CD4+ cells and lower CD8+ cells (Fig. 1a top). The loss of B cells (Fig. 1a top) between 12 and 35 wpt is characteristic of TKO-BLT mice  and is likely because of the earlier release of B cells into the periphery compared with T cells, which first migrate and mature within the human thymic organoid. However, the overall reduction in B-cell numbers (Fig. 1a bottom) suggests that the murine mileu is insufficient for the maintenance of a robust B-cell compartment, which is a concern in BLT-humanized mice [12,13]. It is interesting but unclear why B-cell numbers slightly rebounded between 35 and 45 wpt. At study termination (45 wpt), spleens were harvested and analyzed. Cell frequencies (Fig. 1b, top) and absolute numbers (Fig. 1b, bottom) for individual mice were variable, but all spleens contained at least 10% human leukocytes and ∼4 × 106 human cells/spleen. CD4+ to CD8+ T cells ratio averaged 2 : 1, typical of this model . Spleens at 45 wpt contained an average of 6 × 106 CD4+ T cells, providing ample cell numbers to support HIV-1 infection and to detect ca-vRNA or HIV-1 viral DNA (vDNA) with reasonable sensitivity in individual mice. In addition, lineage negative, HLA-DRhi CD11c+ myeloid and CD123+ plasmacytoid dendritic cells remained detectable at 45 wpt. Overall, TKO-BLT mice maintained high-levels of human engraftment over 45 weeks with no clinical signs of GVHD.
TKO-BLT mice were next assessed for their ability to support HIV-1 infection over an extended period. Groups of TKO-BLT mice were infected with HIV-1JR-CSF at 12 wpt and all sustained p24 antigen in plasma over 22 weeks of infection (Fig. 1c). Starting at 7 weeks post infection (wpi), antigenemia remained relatively stable with no significant decline over 15 weeks, suggesting that peak viremia had resolved and the proviral reservoir was established by 7 wpi. In summary, TKO-BLT mice remained healthy, maintained high-levels of human engraftment, and supported HIV-1 infection over the extended periods required to test cure strategies.
Daily-injected antiretroviral therapy regimens suppress viremia and cell-associated viral RNA
Cure studies require viremia suppression below a stringent limit of detection (LOD) for a period of time sufficient to establish latency. Previously, small plasma volumes from mice restricted the LOD to 800 copies of vRNA/ml. Now more sensitive kits permit a LOD of 140 copies/ml. Reduction of vRNA to levels below this LOD indicate that latent HIV-1 provirus remains as the primary source of virus upon therapy removal. As depicted in Fig. 2a, mice were infected with HIV-1JR-CSF for 6 weeks. At 6 wpi mice were distributed into treatment groups with relatively equivalent levels of plasma vRNA. Half of the mice received a daily subcutaneous regimen of ART and half received control injections. Viremia was monitored for up to 6 weeks of therapy and in some groups therapy was removed to assess for recrudescence as diagrammed (Fig. 2a). Four ART regimens were selected based on preliminary pharmacokinetic data from C57BL/6 and TKO-BLT mice to determine dose ranges that would provide efficacious plasma drug levels (data not shown). The lowest dose regimen, Regimen 1, achieved significant suppression of viremia compared with MOCK controls after 14 days of administration but did not suppress vRNA below the LOD that would be required for cure studies. Continued treatment for an additional 2 weeks did not further reduce viremia so the group was terminated at day 28 (Fig. 2b). Mice receiving Regimen 2 achieved plasma vRNA levels below the level of 800 copies/ml reported in other humanized mouse studies [1,10,14–18] by day 14 and became undetectable by the more sensitive assay employed in our study by day 28 (Fig. 2c). Suppression was maintained until day 42 when the mice were removed from therapy to test for recrudescence. Viremia recrudesced to untreated levels 2 weeks later (day 56, Fig. 2c). We expected higher dose regimens to drop viremia below the 140 copies/ml LOD even more quickly than Regimen 2 but this was not the case. The original Regimen 3, (Regimen 3a), produced rapid but no better suppression of viremia than regimen 2 by day 14 (Fig. 2d) and mice receiving this dosage showed minor signs of toxicity. Thus, the dosages of FTC and dolutegravir were cut in half on day 21, which alleviated the toxicity and reduced viremia below 140 copies/ml after an additional 2 weeks of administration (Fig. 2d). Therapy interruption at day 36 in all the treated animals resulted in recrudescence to levels similar to untreated by day 56 (Fig. 2d). Surprisingly, the highest dose Regimen, Regimen 4, did not prove to be more effective so therapy was discontinued at day 21 and the mice were euthanized at day 36, after virus recrudescence (Fig. 2e). Thus, the daily subcutaneous ART regimens varied in their ability to suppress viremia below stringent LODs in TKO-BLT mice, with Regimen 2 appearing optimal among the doses tested. Removal of ART-therapy resulted in rapid viral recrudescence in TKO-BLT mice even when plasma vRNA levels were below the LOD.
Humanized mouse models have the advantage of access to organs to assess the effect of treatments on tissue vRNA levels. To evaluate ca-vRNA in the tissues, TKO-BLT were infected for 8 weeks prior to receiving daily subcutaneous doses of the most efficacious regimen, Regimen 2, for 4 weeks. After treatment tissues were assessed for ca-RNA levels. HIV-1 ca-RNA was detectable but significantly decreased in treated mice compared with untreated mice (Fig. 2f). Thus, daily subcutaneous ART decreased but did not eliminate tissue vRNA despite suppression of viremia below detectable levels. This result is similar to what is seen in humans  and other humanized mouse models .
HIV-1 suppression in triple knockout-BLT mice using antiretroviral therapy-supplemented mouse chow
As daily subcutaneous injections are labor intensive over the extended periods required to perform cure studies and sharp use is contraindicated when working with HIV-1, we next sought to induce HIV-1 latency using a previously reported free-fed, ART-supplemented mouse chow formulation . As illustrated in Fig. 3a, mice were infected and infection was allowed to progress for 6 weeks. At 6 wpi, mice were assigned to either ART-treated (ART FOOD) or untreated (MOCK) groups based on equivalent levels of plasma p24 (Fig. 3b). After 4 weeks of treatment (10 wpi) the mice were tested for viremia and half the mice in each group were euthanized to assess splenic vDNA (Fig. 3a). Viremia was significantly suppressed in the treated animals compared with controls with seven of ten mice below the LOD of 800 copies/ml and five below 300 copies/ml (Fig. 3c left). The mice receiving ART also had significantly less vDNA in CD4+ T cells harvested from spleen compared with untreated animals after 4 weeks of therapy (Fig. 3c right). To assess recrudescence following treatment interruption, the remaining mice were changed back to regular mouse chow for 2 weeks (Fig. 3a). Mice that had received ART all recrudesced and had vRNA and vDNA levels similar to the untreated group (Fig. 3d). Therefore, oral ART suppressed viremia in TKO-BLT mice while maintaining a detectable vDNA reservoir capable of reestablishing viremia upon treatment withdrawal.
Oral antiretroviral therapy in HIV-1-infected triple knockout-BLT mice prevents T-cell depletion and inversion of the CD4+ : CD8+ ratio
Ideally, a model for HIV-1 cure studies would replicate the immunological benefits of ART, primarily the preservation of CD4+ T-cell counts and a high CD4+ : CD8+ ratio. To assess these parameters in TKO-BLT mice receiving oral ART, we examined the blood cell frequencies (Fig. 4a) and counts (Fig. 4b) of ART-treated compared with untreated animals. Blood was tested prior to ART initiation at 6 wpi, at the end of ART at 10 wpi, and 2 weeks after treatment interruption (12 wpi). Cell frequencies and counts of human leukocytes (hCD45+), CD3+ T cells, CD4+ and CD8+ T cells, and CD19+ B cells were all similar between groups at the initiation of ART (Fig. 4a and b). Overall human cell frequencies were significantly higher in ART-treated animals at the end of 4 weeks of therapy (10 wpi), as were CD4+ T-cell frequencies (Fig. 4a). Notably, within the CD3+ T-cell compartment the CD4+ frequency remained high compared with the CD8+ T-cell frequency, that is, there was a higher CD4+ : CD8+ ratio of 2.52 ± 0.28 in the ART group compared with the untreated group (ratio of 1.52 ± 0.62) (Fig. 4a). Similarly, at the completion of ART at 10 wpi, cell counts had significantly increased for all cell types in treated mice compared with untreated mice, including B cells, which was surprising as this cell type is not a target for HIV-1 and typically declines over time in TKO-BLT mice even in the absence of infection (Fig. 1a). These increases appeared related to viral suppression as within 2 weeks of ART withdrawal cell numbers had declined and were no longer significantly different from untreated HIV-1-infected animals (Fig. 4b).
Next, we examined the effect of oral ART on the frequency and number of CD4+ T cells in spleen. Mice after 4 weeks of ART (at 10 wpi) contained significantly higher frequencies and numbers of CD4+ T cells in spleen compared with untreated mice (Fig. 4c), indicating that ART had penetrated the tissues and suppressed HIV-1. Evaluation of spleens 2 weeks after ART interruption (12 wpi) showed that while frequencies of CD4+ T cells remained higher than untreated mice, the cell counts had declined and were no longer different from controls (Fig. 4d). Overall, the results demonstrated that oral ART in the TKO-BLT model afforded immunological benefits in both blood and tissues analogous to those seen in humans.
The human hematopoietic graft and latent HIV-1 reservoir is maintained over extended periods of antiretroviral therapy-mediated viral load suppression in triple knockout-BLT mice
Previous humanized mouse studies reported administration of ART for periods of 14 weeks with no assessment of virus recrudescence  or for 9 weeks followed by virus recrudescence for 3 weeks . To determine if we could significantly extend these periods in our model we administered ART to mice at 4 wpi, and continued therapy for 18 weeks, followed by treatment interruption for 4 weeks. Viremia was monitored as shown in Fig. 5a. HIV-1 viremia declined to below 200 copies/ml in 29 of 47 mice and below 800 copies/ml in 43 of the 47 mice after 4 weeks of treatment (Fig. 5a, 8 wpi). After 18 weeks (22 wpi) only two of 22 mice had viremia above 800 copies/ml and 16 of 22 had levels below the 200 copies/ml LOD. The two mice above 800 copies/ml at 22 wpi had both been undetectable at previous time points and may have been undergoing viral load ‘blips’ similar to those seen in ART-treated human patients . Further optimization of the ART dose in chow will be required to determine if the percentage of mice below the more stringent LOD can be increased or if viral suppression can be achieved more quickly. The reduced number of mice at 22 wpi was not a result of mortality but assignment of half of the mice to a separate study. The reassigned mice had equivalent virus suppression levels (data not shown).
After 18 weeks on ART (22 wpi), half of the mice were euthanized for analysis and half underwent treatment interruption for 4 weeks to assess viral recrudescence. Viremia increased to pretherapy levels after 2 weeks (24 wpi) and remained high after 4 weeks (26 wpi) without ART. At that point, the remaining mice were euthanized for tissue analysis (Fig. 5a, 26 wpi). HIV-1 DNA in the CD4+ splenocytes of mice was significantly reduced immediately after 18 weeks of ART (22 wpi) compared with mice that underwent ART interruption for 4 weeks (Fig. 5b, 26 wpi). Two mice at 22 wpi, immediate posttherapy, had vDNA levels below the assay's LOD but all mice undergoing ART interruption had detectable cellular vDNA and plasma vRNA. These results indicated that, as in humans, ART did not purge the latent HIV-1 reservoir from the mice (Fig. 5a, b). Assessment of the number of human leukocytes in the spleens at 22 and 26 wpi (Fig. 5c) and the frequency of total CD3+, CD4+, and CD8+ T-cell splenocytes (Fig. 5d) revealed no significant differences compared with HIV-1 negative controls. At the time of euthanasia, all mice appeared healthy.
These data demonstrated that TKO-BLT mice administered oral ART over an extended period of 18 weeks followed by treatment interruption and viral recrudescence to a total of 26 weeks of infection (39–43 weeks posthumanization) not only remain healthy and well engrafted with a high CD4+ : CD8+ ratio but are able to achieve long-term viral suppression and significant contraction, but not loss, of the HIV-1 proviral reservoir that rapidly reactivates upon therapy removal.
HIV-1 cure strategies require a small animal model that recapitulates latent HIV-1 infections in humans being treated with ART. Humanized mice have become well established for the study of HIV-1 infection and testing of therapeutics . They are particularly valuable for studies that rely on both an authentic HIV-1 infection and/or a genuine human immune system . The BLT method of humanization produces robust multilineage reconstitution in multiple lymphoid tissues complete with innate and adaptive immune responses. HIV-1 infection in the humanized mouse simulates human infection, including high virus titers, tissue dissemination, proviral reservoir seeding, immune dysfunction, and CD4+ T-cell loss .
The utility of BLT-humanized mice has been shown for in-vivo studies evaluating the cell types harboring latent virus [7,8,23], the role of interferon in reservoir size and persistence , the ability of a histone deacetylase inhibitor to reactivate latent HIV-1  as well as short-term evaluations of reservoir purging strategies [10,16]. However, the failure of these mice to thrive in long-term experiments has restricted their utility for cure studies. Here, we demonstrate that using TKO-BLT mice allows the extension of studies for more than 4 months longer than previously reported in other BLT models. This longevity makes it feasible to test cure therapies in immunologically competent and healthy mice that survive and remain highly reconstituted with human cells for at least 45 weeks posttransplantation. As the mice remained healthy at study's end suggests that experiments may be extended even further.
In summary, we demonstrate that upon HIV-1 infection of the TKO-BLT mouse, subcutaneous or oral ART can be administered to suppress viremia below stringent LODs and that viral suppression results in CD4+ T-cell count recovery similar to that seen in humans. Additionally, the mice contain abundant cells for ex-vivo analysis such as assessing latency reversal agent-induced ca-RNA increases or therapeutic reductions in provirus. Importantly, HIV-1 recrudesces rapidly after ART interruption facilitating observations of delays in recrudescence because of interventions that reduce viable provirus and serves as the most stringent test for functional cure. Finally, the immune responses in this model  make it suitable to test strategies that utilize endogenous immune-modalities to clear the HIV-1 reservoir, particularly when such experiments require extended timelines.
We thank Drs Stefano Boi, Alyssa B. Evans, and Ron Swanstrom for critical reading of the manuscript. K.J.L. designed and performed experiments and analyzed and interpreted data; C.P. designed experiments and analyzed data; K.S., R.J.M., D.L.P., N.W.C., S.N., J.Z., J.G., M.W., and E.S.V.D. performed experiments; K.J.L., R.J.M., K.P. and B.R. humanized the mice; U.D., G.K., and K.J.H. designed experiments and interpreted data. K.J.H. and K.J.L. wrote the paper.
Support for this work was provided by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institute of Health, USA, by the Mayo Clinic, by Gilead Sciences. and the Deutsche Forschungsgemeinschaft, project TRR60.
Conflicts of interest
There are no conflicts of interest.
1. Denton PW, Olesen R, Choudhary SK, Archin NM, Wahl A, Swanson MD, et al. Generation of HIV latency in humanized BLT mice
. J Virol
2. Marsden MD, Kovochich M, Suree N, Shimizu S, Mehta R, Cortado R, et al. HIV latency in the humanized BLT mouse
. J Virol
3. Brainard DM, Seung E, Frahm N, Cariappa A, Bailey CC, Hart WK, et al. Induction of robust cellular and humoral virus-specific adaptive immune responses in human immunodeficiency virus-infected humanized BLT mice
. J Virol
4. Lavender KJ, Pang WW, Messer RJ, Duley AK, Race B, Phillips K, et al. BLT-humanized C57BL/6 Rag2-/-γc-/-CD47-/- mice are resistant to GVHD and develop B and T-cell immunity to HIV infection
5. Greenblatt MB, Vbranac V, Tivey T, Tsang K, Tager AM, Aliprantis AO. Graft versus host disease in the bone marrow, liver and thymus humanized mouse model
. PloS One
6. Cummins NW, Sainski AM, Dai H, Natesampillai S, Pang YP, Bren GD, et al. Prime, shock, and kill: priming CD4 T cells from HIV patients with a BCL-2 antagonist before HIV reactivation reduces HIV reservoir size
. J Virol
7. Honeycutt JB, Wahl A, Archin N, Choudhary S, Margolis D, Garcia JV. HIV-1 infection, response to treatment and establishment of viral latency in a novel humanized T cell-only mouse (TOM) model
8. Arainga M, Edagwa B, Mosley RL, Poluektova LY, Gorantla S, Gendelman HE. A mature macrophage is a principal HIV-1 cellular reservoir in humanized mice after treatment with long acting antiretroviral therapy
9. Lavender KJ, Messer RJ, Race B, Hasenkrug KJ. Production of bone marrow, liver, thymus (BLT) humanized mice on the C57BL/6 Rag2-/-γc-/-CD47-/- background
. J Immunol Methods
10. Halper-Stromberg A, Lu CL, Klein F, Horwitz JA, Bournazos S, Nogueira L, et al. Broadly neutralizing antibodies and viral inducers decrease rebound from HIV-1 latent reservoirs in humanized mice
11. Malnati MS, Scarlatti G, Gatto F, Salvatori F, Cassina G, Rutigliano T, et al. A universal real-time PCR assay for the quantification of group-M HIV-1 proviral load
. Nat Protoc
12. Seung E, Tager AM. Humoral immunity in humanized mice: a work in progress
. J Infect Dis
2013; 208 (suppl 2):S155–S159.
13. Jangalwe S, Shultz LD, Mathew A, Brehm MA. Improved B cell development in humanized NOD-scid IL2Rγnull mice transgenically expressing human stem cell factor, granulocyte-macrophage colony-stimulating factor and interleukin-3
. Immun Inflamm Dis
14. Choudhary SK, Archin NM, Cheema M, Dahl NP, Garcia JV, Margolis DM. Latent HIV-1 infection of resting CD4(+) T cells in the humanized Rag2-/- γ-/-(c) mouse
. J Virol
15. Nischang M, Sutmuller R, Gers-Huber G, Audige A, Li D, Rochat MA, et al. Humanized mice recapitulate key features of HIV-1 infection: a novel concept using long-acting antiretroviral drugs for treating HIV-1
. PloS One
16. Denton PW, Long JM, Wietgrefe SW, Sykes C, Spagnuolo RA, Snyder OD, et al. Targeted cytotoxic therapy kills persisting HIV infected cells during ART
. PLoS Pathog
17. Tsai P, Wu G, Baker CE, Thayer WO, Spagnuolo RA, Sanchez R, et al. In vivo analysis of the effect of panobinostat on cell-associated HIV RNA and DNA levels and latent HIV infection
18. Cheng L, Ma J, Li J, Li D, Li G, Li F, et al. Blocking type I interferon signaling enhances T cell recovery and reduces HIV-1 reservoirs
. J Clin Invest
19. Yukl SA, Shergill AK, Ho T, Killian M, Girling V, Epling L, et al. The distribution of HIV DNA and RNA in cell subsets differs in gut and blood of HIV-positive patients on ART: implications for viral persistence
. J Infect Dis
20. Havlir DV, Bassett R, Levitan D, Gilbert P, Tebas P, Collier AC, et al. Prevalence and predictive value of intermittent viremia with combination HIV therapy
21. Garcia VJ. Humanized mice for HIV and AIDS research
. Curr Opin Virol
22. Karpel ME, Boutwell CL, Allen TM. BLT humanized mice as a small animal model of HIV infection
. Curr Opin Virol
23. Honeycutt JB, Thayer WO, Baker CE, Ribeiro RM, Lada SM, Cao Y, et al. HIV persistence in tissue macrophages of humanized myeloid-only mice during antiretroviral therapy
. Nat Med