Matthew D. Marsden,aMichael Kovochich,aNuttee Suree,bSaki Shimizu,bRoshni Mehta,aRuth Cortado,bGregory Bristol,a Dong Sung An,band Jerome A. Zackc
Department of Medicine, Division of Hematology and Oncology, David Geffen School of Medicine at UCLA, Los Angeles, California, USAa; UCLA School of Nursing, Los Angeles, California, USAb; and Department of Medicine, Division of Hematology and Oncology, Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at UCLA, Los Angeles, California, USAc
Even after extended treatment with powerful antiretroviral drugs, HIV is not completely eliminated from infected individuals. Latently infected CD4ⴙT cells constitute one reservoir of replication-competent HIV that needs to be eliminated to completely
purge virus from antiretroviral drug-treated patients. However, a major limitation in the development of therapies to eliminate this latent reservoir is the lack of relevantin vivomodels that can be used to test purging strategies. Here, we show that the hu-manized BLT (bone marrow-liver-thymus) mouse can be used as both an abundant source of primary latently infected cells for ex vivolatency analysis and also as anin vivosystem for the study of latency. We demonstrate that over 2% of human cells recov-ered from the spleens of HIV-infected BLT mice can be latently infected and that this virus is integrated, activation inducible, and replication competent. The non-tumor-inducing phorbol esters prostratin and 12-deoxyphorbol-13-phenylacetate can each induce HIVex vivofrom these latently infected cells, indicating that this model can be used as a source of primary cells for test-ing latency activators. Finally, we show activation-inducible virus is still present followtest-ing suppression of plasma viral loads to undetectable levels by using the antiretroviral drugs zidovudine, indinavir sulfate, and didanosine, demonstrating that this model can also be used to assess thein vivoefficacy of latency-purging strategies. Therefore, the HIV-infected BLT mouse should provide a useful model for assessment of HIV latency activators and approaches to eliminate persistentin vivoHIV reservoirs.
H
ighly active antiretroviral therapy (HAART) is capable ofin-hibiting HIV replication and reducing plasma viral loads to undetectable levels for years, thereby preventing disease progres-sion (20, 23, 24). However, replication-competent HIV persists in HAART-treated patients, and if therapy is stopped for any reason
then viral loads rapidly increase (13). Latently infected CD4⫹T
cells represent one of the primary cellular reservoirs capable of maintaining infection during HAART (17, 22, 43). This latent
reservoir consists of resting CD4⫹T cells harboring an integrated
provirus expressing little or no viral gene products. If these cells become stimulated, then productive infection ensues and infec-tious virus is released from the cell. The latent reservoir is formed soon after primary infection (15) and consists of a rare population
of approximately 106 cells in total within each treated patient,
translating to around 1 per million resting CD4⫹T cells (14).
Latently infected cells have a long half-life, 42 months, which is probably sufficient to maintain lifelong infection unless methods to eliminate them more rapidly are developed (21). Most sug-gested methods for depleting these cells involve activating the vi-rus in some way to induce HIV replication, leading to cell death due to immune responses or viral cytopathic effects (16, 30, 36). If this were performed in the continued presence of HAART, then spread of the recently activated virus to new cells would be pre-vented.
Historically, attempts to develop effective latency-purging strategies have been hampered by a limited number of relevant primary cell models for HIV latency and a complete lack of small
animal models suitable forin vivoassessment of these strategies
(reviewed in references 31 and 32). More recently, a number of primary cell models have been developed to investigate the mo-lecular mechanisms regulating HIV latency and aid in
optimiza-tion of eliminaoptimiza-tion strategies (6, 12, 44, 45). However, tractablein
vivo systems are still lacking. We have previously used the
SCID-hu (Thy/Liv) (severe combined immunodeficient mouse/
human thymus/liver) mouse model as a source of primary in-fected cells for studying latency (3, 8–11, 27, 37). In this model, human fetal liver and thymus tissue (Thy/Liv) are implanted un-der the kidney capsule of an immunodeficient mouse (33, 35). The Thy/Liv implant forms a conjoined organ similar in structure and function to a human thymus, allowing normal T cell thymopoiesis to take place. The implant can support productive infection with HIV in a process that also generates a high frequency of latently infected cells (up to around 10% of CD4 single-positive cells) (10).
Latency generation in the implant occurs when CD4⫹ CD8⫹
(double-positive) thymocytes become infected with HIV and then differentiate into single-positive cells with greatly reduced tran-scriptional activity, resulting in a concomitant reduction in HIV transcription (10). One limitation of the SCID-hu (Thy/Liv) model is that peripheral reconstitution with human cells is not robust, with low levels of human cells present in peripheral blood and spleen and latently infected splenocytes detected in fairly small numbers and in only some infected animals. This restricts
the use of the SCID-hu (Thy/Liv) model as anin vivosystem for
testing latency-purging strategies and as a source of latently in-fected cells that are nonthymic in origin.
Several alternative murine models for HIV infection, including
humanized Rag2⫺/⫺␥c⫺/⫺mice (4, 40) and the recently
devel-oped BLT (bone marrow-liver-thymus) mouse, now exist. The BLT mouse represents a significant improvement over the SCID-hu (Thy/Liv) mouse by providing robust peripheral
recon-Received20 September 2011Accepted20 October 2011
Published ahead of print9 November 2011
Address correspondence to Jerome A. Zack, jzack@ucla.edu.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JVI.06366-11
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stitution with multiple human hematopoietic lineages (34). These BLT mice are produced by transplanting human fetal liver and thymus tissue under the kidney capsule of a nod-SCID-common gamma chain-knockout (NSG) mouse. Endogenous hematopoi-etic progenitors are then depleted by irradiation or chemical
treat-ment, and new human CD34⫹hematopoietic stem cells are
intro-duced by intravenous injection. Human immune cells in BLT mice are present in multiple organs, including the Thy/Liv im-plant, peripheral blood, lymph nodes, gut, lungs, bone marrow,
and liver (34, 38). Moreover, the human CD4⫹cells in these mice
can also be infected with HIV (18, 19, 38, 39).
We demonstrate here that latent HIV infection can form in peripheral human cells in BLT mice. This virus is integrated, acti-vation inducible, and replication competent, and can serve as an
abundant source of latent virus forex vivoinvestigation.
Further-more, when plasma viral loads are suppressed to undetectable levels with antiretroviral drugs, activation-inducible virus can still
be detected, suggesting that in vivo testing of latency-purging
strategies is feasible in this model.
MATERIALS AND METHODS
Construction of BLT mice.Fetal tissue for these studies was obtained from either the UCLA CFAR Gene and Cellular Therapy Core or Ad-vanced Biosciences Resources Inc. SCID-hu (Thy/Liv) mice were gener-ated as previously described (7, 33) using NOD.Cg-PrkdcscidIl2rgtm1Wjl (NSG) mice. Myeloablation was achieved either by irradiation of mice with 270 rads using a cobalt-60 source or by two injections of 25 mg/kg (of body weight) busulfan (Sigma) spaced 2 days apart (total, 50 mg/kg). This was performed at the time of implantation or at some point over the following 4 months depending on the series. Immediately following abla-tion, mice were infused via retro-orbital injection with between 105and 106human fetal liver-derived CD34⫹cells, which were isolated as
previ-ously described (38).
Infection procedures and antiretroviral therapy.BLT mice were in-fected with HIV strain NL4-3 (1) or NLHSAs (25) 2 to 4 months following CD34⫹cell infusion depending on the series. Infections were performed by retro-orbital injection with 50 ng or 200 ng of HIV p24 in a 150-l volume. Some mice were treated with zidovudine (AZT)-indinavir sulfate or AZT-indinavir sulfate-didanosine (ddI) as previously described (2, 42). Briefly, indinavir and AZT were added to the drinking water (pH 3) at concentrations of 1.5 and 0.4 mg/ml, respectively, with mice receiving an estimated daily dose of 4.5 mg of indinavir and 1.2 mg of AZT. ddI was administered by daily intraperitoneal injection of 1 mg/day.
Cell/tissue harvesting.During eye bleeds, a micropipette (VWR) that had been precoated internally with 1.5l of a solution composed of 10 parts 500 mM EDTA, 2 parts 25% human serum albumin (Baxter Health-care), and 3 parts sterile water was inserted retro-orbitally to remove 50l of blood. This blood was immediately placed in a tube containing 1l of 200 mM EDTA and then centrifuged at 845⫻gfor 5 min. The plasma was removed and stored at⫺80°C before viral load quantification by reverse transcription (RT)-PCR. Red blood cells in the cell pellet were lysed using ammonium chloride solution (StemCell Technologies) according to manufacturer’s guidelines. Peripheral blood mononuclear cells (PBMC) were then used for flow cytometric analysis.
Spleens and Thy/Liv implants were forced through a steel mesh to disaggregate tissue and then passed through a 40-m filter to produce a single cell suspension. For depletion of heat-stable antigen (HSA), the cells were stained with a biotinylated anti-mouse CD24 (HSA) antibody (eBioscience), and then HSA-positive (HSA⫹) cells were depleted using antibiotin microbeads and an autoMACS separator (Miltenyi Biotec) ac-cording to the manufacturer’s instructions.
Viral load measurement.Viral RNA was extracted from plasma using the High Pure viral RNA kit (Roche) and stored at⫺80°C before use. To generate RNA standards, a section ofgagwithin pNL101 was amplified
with the primers NG1CF (position 366 to 398) (5=-GGAGAATTAGATA AATGGGAAAAAATTCGGTTA-3=) and NG1CR (position 679 to 648) (5=-GCCTTTTTCTTACTTTTGTTTTGCTCTTCCTC-3=) and cloned into pCR4TOPO (Invitrogen). The insert was excised using SpeI and BsrGI, and the resultant 600-bp fragment was gel purified. Using this fragment as the template, RNA was produced with T7 RNA polymerase (Promega Riboprobe transcription kit) and quantitated spectrophoto-metrically atA260. The transcribed RNA served as an RT control. This RNA was serially diluted in THE RNA Storage buffer (Ambion) with 0.4 U/l RNasin and 5 ng/l of Hind III-cleaved lambda DNA (as carrier), to make a stock of 500,000 copies/l. Before each RT run, a vial of control RNA was diluted in the above buffer to make five standards of 100,000 to 10 copies per 2l.
The oligonucleotides used for RT-PCR were NG1F (position 453 to 480) (5=-GAGCTAGAACGATTCGCAGTTAATCCTG-3=), NG1R (posi-tion 570 to 534) (5=-ATAATGATCTAAGTTCTTCTGATCCTGTC TGAAGGGA-3=), and NG1Z probe (position 482 to 520) (6-carboxyfluorescein [FAM]-5=-CCTTTTAGAGACATCAGAAGGCTGT AGACAAATACTGGG-3=-Black Hole Quencher [BHQ]). The Super-Script III kit (Invitrogen) was used for the reverse transcription step. Reactions were run in one low-profile, no-skirt, 96-well plate. The anneal-ing step consisted of 5l of template RNA plus 3l of a mixture consisting of 1.5 parts 20M NG1R, 0.5 parts RNasin Plus (Promega), 16 parts 5⫻ RT buffer, and 12 parts water. The resulting 8l was heated to 70°C for 2 min and then 60°C for 5 min and was then cooled to room temperature. The RT step was run by adding 2l of a mixture of 8 parts water, 4 parts 5⫻buffer, 5 parts dithiothreitol, 2 parts 25 mM deoxynucleoside triphos-phates (dNTPs) (Invitrogen), and 1 part SuperScript III. This was heated to 55°C for 30 min and then at 85°C for 5 min and was then cooled to room temperature.
An additional set of DNA standards from a cassette of pNL101 was also run during the PCR step to control for the efficiency of the RT step. The DNA standards included 20, 200, 2,000, 20,000, and 200,000 copies of template DNA. An oligonucleotide mix designated NG-FRZ consisting of 2.5M NG1F, 7.5M NG1R, and 2.5M NG1Z was made up in water at 5 times the final reaction concentration. Fifteen microliters of the PCR mix consisting of 38.5 parts water, 44 parts 25 mM MgCl2, 50 parts NG-FRZ oligonucleotides, 5 parts 500 mM Tris buffer, pH 8.3, 8.5 parts 1 M KCl, 2.5 parts 25 mM dNTPs, and 1.25 parts PlatinumTaqwas added to all wells and mixed. The real-time PCR was 5 min activation at 95°C and 45 cycles of 95°C for 15 s and 60°C for 1 min on a Bio-Rad CFX96 ther-mocycler.
Cell culture.Cells were cultured in a humidified incubator at 37°C in 5% CO2. Base culture media (RF10) consisted of RPMI medium 1640 (Invitrogen) containing 10% fetal bovine serum (Omega Scientific) and 100 units/ml of penicillin plus 100g/ml of streptomycin (Pen/Strep; Invitrogen). The majority of stimulations were performed by incubating 500,000 cells/well in a 48-well plate. For costimulated conditions, wells were precoated with 300l of goat anti-mouse antibody (50g/ml) in phosphate-buffered saline (PBS). Plates were incubated overnight at 4°C, washed 3 times with PBS, and then 1g/ml of OKT3 antibody was added to each well. The plates were incubated for a further 45 min at 37°C and washed 3 times with PBS, and then 2 ml of cells suspended in RF10 was added to the coated wells. Final concentrations of 1g/ml of anti-CD28 antibody and 20 units/ml of interleukin 2 (IL-2) were then added to the cultures. Corresponding unstimulated wells were incubated in RF10 only. Raltegravir (1M), saquinavir (1M), prostratin, or 12-deoxyphorbol-13-phenylacetate (DPP) were included in certain cultures. For some stim-ulations described in Table 1, a smaller format assay was used. These cells were incubated at 200,000 cells/well in a 200-l volume in wells of a 96-well plate. Costimulation was achieved using a 2:1 cell/bead ratio of Dynabeads Human T-Activator CD3/CD28 reagent (Invitrogen) and 20 units/ml of IL-2. During harvesting, supernatants were diluted in 2% Triton X-100 in PBS and the p24 concentration determined using the HIV
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p24 antigen enzyme-linked immunosorbent assay (ELISA) kit (Beckman Coulter) according to the manufacturer’s instructions.
Flow cytometry.Cell surface staining was performed by suspending cell pellets in 50l of a 1:1 mix of PBS and human AB serum (Sigma). The following human-specific fluorescent conjugated antibodies were then added: CD4-fluorescein isothiocyanate (Beckman Coulter), CD3-phycoerythrin (Beckman Coulter), CD8-allophycocyanin (Beckman Coulter), and CD45-phycoerythrin cyanine 7 (PC7; BD Biosciences). Cells were incubated at 4°C for 15 min. After incubation, the cells were washed with PBS and suspended in 2% paraformaldehyde and then stored at 4°C before analysis.
For intracellular flow cytometry, cells were pelleted, then resuspended in 2% paraformaldehyde, and incubated for 30 min at 4°C. Cells were then washed with PBS and resuspended in 0.02% Tween 20 in PBS before incubation for 20 min at room temperature. After incubation, cells were washed again with PBS and then resuspended in 1:1 PBS-human AB serum containing KC57-RD1 (Beckman Coulter) and CD45-PC7. Cells were then incubated for 20 min at 4°C, washed with PBS, and resuspended in 2% paraformaldehyde. Data were acquired using a FACSCalibur (Becton Dickinson) flow cytometer and analyzed using FlowJo (v7) software.
RESULTS
Detection of latent virus in HIV-infected BLT mice.The BLT mice used in this study were generated by creating Thy/Liv im-plants in one or both kidneys, followed by ablation of murine hematopoietic progenitors by using either irradiation or busulfan treatment. Differences in the specific procedure for generating the mice did not lead to variation in human cell reconstitution, with the majority of mice from each series producing multiple human hematopoietic lineages that were readily detectable in peripheral blood (Fig. 1).
Initial experiments were conducted by infecting BLT mice in-travenously with the NLHSAs reporter virus (25). This virus is a modified version of HIV NL4-3 that encodes the murine heat-stable antigen (HSA; also known as CD24) selectable marker in
place ofvpr, which allows specific depletion of cells actively
ex-pressing HIV (9). At week 5 postinfection, the mice were sacrificed and splenocytes obtained then immunomagnetically depleted of
HSA⫹(productively infected) cells. Cells were either left
unstimu-lated or costimuunstimu-lated using antibodies specific for CD3 and CD28
in the presence of interleukin 2 (IL-2). The HIV integrase inhibi-tor raltegravir was added to all cultures to inhibit virus spread and also to ensure that recently infected cells containing nonintegrated virus would not confound the analysis of genuine integrated latent proviruses. At day 2 poststimulation, some cells were harvested and analyzed for intracellular Gag expression by flow cytometry
(Fig. 2A). Only the CD45⫹cells in costimulated, infected cultures
were positive for intracellular Gag protein. Cultures from the same animals were incubated for a further 3 days in the presence of raltegravir, and then HIV p24 capsid protein accumulation in the supernatant was quantified by p24 ELISA (Fig. 2B). A substantial increase in p24 was evident in the infected, costimulated cultures compared to mock infected or unstimulated cultures. The low level of p24 detected in the unstimulated, infected cultures may be due to contaminating activated cells, some nonspecific stimula-tion of latently infected cells during the sorting and subsequent culture procedures, or the presence of rare productively infected cells that escaped HSA depletion because the infecting virus was in the short phase between integration and viral reporter gene ex-pression when the HSA sort was performed. Therefore, latent vi-rus is present in the spleens of BLT mice at levels that can be readily quantified by intracellular Gag staining.
Induction of latent virus with PKC agonists.We previously demonstrated in SCID-hu (Thy/Liv) mice that the latently in-fected cells can be activated by stimulation with protein kinase C (PKC) agonists such as prostratin (9, 27). To determine whether latently infected cells from the BLT mouse can be similarly
acti-vated in this manner to produce virusex vivo, mice were infected
with wild-type HIV strain NL4-3 and cells from Thy/Liv implants were isolated at week 7 postinfection. Cells from 3 infected mice were pooled and then exposed to different concentrations of pros-tratin in the presence of raltegravir. At day 2 postinfection, the cells were harvested and stained for intracellular Gag expression (Fig. 3A). Latent virus was induced by prostratin in a
dose-dependent manner beginning at approximately 0.1 to 1M
[image:3.585.41.288.78.195.2]con-centrations, which is consistent with that required to activate HIV from latency in patient samples (29) and SCID-hu (Thy/Liv) model thymocytes (27). We then assessed whether prostratin or 12-deoxyphorbol-13-phenylacetate (DPP), a second PKC activa-tor (5), was capable of inducing latent virus from BLT splenocytes. For this assay, splenocytes isolated from BLT mice infected with NLHSAs were isolated at week 7 postinfection and left un-stimulated, coun-stimulated, treated with prostratin, or treated with DPP (Fig. 3B). Latent virus was induced to express in
TABLE 1Summary of latency detection in BLT micea
Virus ART regimen
Viral load suppressionb
No. of mice with detectable latency in spleen/total no. of mice
NLHSAs None NA 4/4
NL4-3 None NA 11/11
NL4-3 AZT⫹indinavir ⬍90% suppression 7/8 NL4-3 AZT⫹indinavir⫹ddI ⬎90% suppression or
undetectable
2/4
aSplenocytes from infected mice were costimulated in the presence of raltegravir, and
upregulation of virus expression was identified by either Gag detection by intracellular flow cytometry or p24 detection in the culture supernatants at days 2 to 5
poststimulation. These experiments included mice from 7 different series, with each series generated using tissue from different human donors. Those mice with undetectable latency also had low to undetectable levels of virus in unstimulated cells, indicating a low overall level of infection.
bNA, not applicable.
FIG 1Reconstitution of human immune cells in BLT mice. Representative flow cytometry analysis of peripheral blood mononuclear cells obtained from BLT mice following staining for human CD45, CD3, CD4, and CD8.
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[image:3.585.301.543.577.693.2]splenocytes by both prostratin and DPP treatments in each of the infected mice. Therefore, latently infected cells from BLT mice can be induced to express virus by PKC activators in a manner similar to the induction of latently infected cells from HAART-treated patients.
Characterization of latent virus byex vivotreatment with antiretroviral drugs.To demonstrate that the virus induced to
expressex vivofrom BLT mice is predominantly integrated and
also replication-competent, mice were infected with NLHSAs and the spleen and Thy/Liv implants obtained at week 7 postinfection. Cultures from these tissue samples were costimulated via CD3 and CD28 ligation plus IL-2 without drug or in the presence of either
raltegravir or the HIV protease inhibitor saquinavir. At day 4
postinfection, cells were stained for the presence of Gag⫹cells.
Stimulation in raltegravir- or saquinavir-treated cultures
pro-duced similar percentages of Gag⫹splenocytes (Fig. 4A),
demon-strating that most of the virus induced to express in this system is already integrated. Hence, functional preintegrated HIV from re-cent infection either is not present or is present at very low levels in the cells. Thy/Liv implants from the same two mice each had a
slightly higher frequency of Gag⫹cells in saquinavir-treated
cul-tures than in raltegravir-treated culcul-tures (Fig. 4B), indicating a modestly elevated level of preintegrated virus in this tissue. In both
tissues, an increase in Gag⫹cells was observed in untreated
stim-FIG 2Generation ofin vivolatency using the BLT humanized mouse model. BLT mice were infected with a modified version of HIV NL4-3 that encodes the murine heat-stable antigen (HSA) selectable marker in place ofvpr(NLHSAs). At week 5 postinfection, splenocytes were obtained and immunomagnetically depleted of HSA⫹(productively infected) cells. Cells were either left unstimulated or costimulated via CD3 and CD28 ligation in the presence of IL-2. Raltegravir
was added to all cultures to prevent new integration events and inhibit virus spread during the assay. (A) At day 2 poststimulation, some cells were harvested and analyzed for intracellular Gag expression by flow cytometry. Boxed numbers represent the percentages of human cells that are HIV Gag⫹. (B) At day 5
poststimulation, supernatants were harvested and HIV p24 levels were analyzed by ELISA. CS, costimulated; US, unstimulated. Mean values from duplicate wells for each condition are presented.
FIG 3Stimulation of latent virus with protein kinase C activators. (A) BLT mice were infected with wild-type HIV, and then the Thy/Liv implants were removed at week 7 postinfection. Thymocytes from three BLT mice were pooled and then stimulated in triplicate with the indicated concentration of prostratin for 2 days before quantitation of Gag⫹cells by intracellular flow cytometry. Raltegravir (1M) was present in all cultures throughout the assay period. Prostratin activated
HIV from latency in a dose-dependent manner in this assay.ⴱ,P⬍0.05 (pairedttest) (B) BLT mice infected with strain NLHSAs were sacrificed at week 7 postinfection, and then splenocytes were either cultured unstimulated or stimulated with costimulation (CS), 1M prostratin (Pro), or 1M 12-deoxyphorbol-13-phenylacetate (DPP). Raltegravir (1M) was added to all cultures. At day 4 poststimulation, cells were assayed for intracellular Gag expression. Prostratin and DPP each activated HIV from latency in splenocytes under these conditions.
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[image:4.585.45.540.68.274.2] [image:4.585.135.459.515.656.2]ulated cultures, demonstrating that the latent virus is replication competent. Control unstimulated cultures for each
animal/con-dition uniformly contained⬍0.1% Gag⫹cells (not shown).
Latency in HAART-treated BLT mice.One significant advan-tage of a humanized mouse model that produces peripheral HIV latency is the possibility that it may be used for preclinical assess-ment of strategies for purging persistent HIV reservoirs from HAART-treated patients. An important step in this direction is the treatment of infected mice with antiretroviral drugs to pro-duce an environment that more closely emulates the situation in these patients. Therefore, we infected BLT mice with HIV strain NL4-3 and at 3 weeks postinfection began administration of the antiretroviral drugs AZT, indinavir, and ddI to the mice. This particular regimen was used because we have found it to be effec-tive at suppressing HIV replication in the SCID-hu (Thy/Liv) mouse (2, 42). Mice were bled at weekly time points postinfection in order to perform cell subset analysis and plasma viral load quantitation. Maximum viral loads varied significantly from ani-mal to aniani-mal, ranging from approximately 5,000 to over 200,000
HIV RNA copies per 100l of blood (Fig. 5A). Antiretroviral
treatment reduced viral loads to below the limit of detection (10
RNA copies/100l plasma) within 1 week of treatment initiation
in 3 of 4 treated infected animals (Fig. 5A). Protection of CD4⫹T
cells from depletion by the antiretroviral therapy was evaluated by
comparing the CD4/CD8 ratios in the CD45⫹CD3⫹population
of the peripheral blood mononuclear cells. A significant reduction in CD4/CD8 ratio in the infected untreated mice compared with the ART-treated mice was evident at both 1 and 2 weeks posttreat-ment initiation (Fig. 5B).
At week 5 postinfection, the mice were sacrificed and the spleens removed. Splenocytes were either left unstimulated or were costimulated in the presence of raltegravir for 4 days and then p24 protein in the culture supernatants was quantified by ELISA. Differing levels of latent virus production occurred in cul-tures from untreated infected mice in contrast to low background
expression in unstimulated cultures (Fig. 6A). None of the un-stimulated cultures from the infected ART-treated mice had de-tectable p24 levels in the supernatant (Fig. 6B). However, 2 of 4 costimulated samples from infected ART-treated animals did have detectable p24 protein in the supernatant (Fig. 6B), although at lower levels than those of corresponding costimulated samples from the untreated infected animals. Importantly, these stimula-tions were performed in the presence of a drug that inhibits viral spread (raltegravir), which provides a more stringent but less sen-sitive procedure for detecting latent virus than other available methods such as limiting dilution coculture (14).
To date, we have tested for the presence of activation-inducible virus in the spleens of 24 mice that were constructed using human donor tissue from 7 individuals (Table 1). These included mice with untreated, partially suppressed (by treatment with only AZT and indinavir in the animals’ drinking water), or completely sup-pressed viral loads. The majority of these infected mice showed
evidence of latency whenⱕ500,000 splenocytes were assessed for
upregulation of HIV during costimulation by either intracellular Gag staining or p24 accumulation in the supernatant.
DISCUSSION
The presence of latently infected cells is one of the factors that allow persistence of HIV during HAART (21). Efforts to flush this latent virus from infected individuals have been hampered by a
lack of availablein vivosmall animal models to test and optimize
purging methodologies (32). Our group has previously used the
SCID-hu (Thy/Liv) mouse as anex vivosource of latently infected
cells that can be used to study the molecular mechanisms regulat-ing latency activation (8–10). However, the poor peripheral re-constitution that occurs in this model limited its utility for directly
testingin vivolatency-purging strategies. The more recently
de-veloped BLT mouse model (34) has the potential to address this key limitation by providing robust multilineage reconstitution in different tissues. While the specific definition of HIV latency can
FIG 4Latency in BLT splenocytes is predominantly in the postintegration form. (A) Splenocytes were isolated from infected BLT mice and then costimulated for 4 days in the presence of the indicated drug. Levels of viral spread were measured under culture conditions without any antiretroviral drugs (No drug), and an increase in Gag⫹cells was observed, demonstrating the presence of replication-competent HIV. Addition of the integrase inhibitor raltegravir (Ral) or the
protease inhibitor saquinavir (Sqv) prevented this spread but still allowed expression of virus from latently infected cells. The frequency of Gag⫹cells in
raltegravir-treated cultures was similar to that in saquinavir-treated cultures, demonstrating that preintegration latency is not present at high levels in these cells. (B) Cells isolated from the Thy/Liv implant were treated in the same way as described for panel A. Control unstimulated cell cultures were included for all spleen and Thy/Liv implant conditions, and these were uniformly negative for Gag expression (not shown).
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[image:5.585.98.489.64.244.2]vary depending on context, virological latency in model systems generally requires the presence of cells harboring a nonexpressing, integrated HIV provirus can be induced to produce replication-competent virus upon stimulation. We describe here that using this virological definition, HIV-infected BLT mice can indeed serve as a model for HIV latency.
Following intravenous infection with HIV, up to several
per-cent of splenocytes harbored activation-inducible virus that could be readily detected either by intracellular staining for Gag protein or by quantification of HIV p24 protein in the culture superna-tants by ELISA (Fig. 2). The majority of T cells in this mouse model have a naïve phenotype (34, 38); therefore, it is likely that most of the latently infected cells are naïve T cells, as is the case with SCID-hu (Thy/Liv) mice. Importantly, these experiments
FIG 5Suppression of viremia and protection of CD4⫹T cells with combination antiretroviral therapy. BLT mice were infected with wild-type NL4-3 for 3 weeks
and then treated for 2 weeks with antiretroviral therapy (ART) consisting of AZT, indinavir sulfate, and ddI. (A) HIV plasma viral loads were quantified for treated and control mice. HIV RNA copies per 100l of plasma are presented. Shaded areas indicate treatment with ART. (B) Mice were bled at the indicated postinfection time points, and PBMC were isolated for cell subset quantitation by flow cytometry. The CD4/CD8 ratios within the human T cell population (CD45⫹CD3⫹) are presented. A significant decline in CD4/CD8 ratio was observed in the infected untreated animals, indicating that the ART was protective
against CD4⫹T cell depletion.
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[image:6.585.38.536.71.561.2]were conducted in the presence of the integrase inhibitor raltegra-vir, which prevented confounding of the analysis by viral spread during the assay or integration of any preintegrated viral genomes that may have been present in recently infected cells. In order for
these primary cells to be useful forex vivotesting of new latency
activators, they would ideally respond not only to strong stimula-tory signals provided by costimulation via ligation of CD3 and CD28 but also to signals produced by other potential latency ac-tivators. Therefore, we tested the ability of the non-tumor-inducing phorbol ester prostratin to activate latent virus from BLT thymocytes and splenocytes. Prostratin was capable of inducing virus expression in this model to a level similar to or greater than that induced by costimulation (Fig. 3). Moreover, a second PKC activator (DPP) was also able to stimulate HIV from latency in BLT splenocytes. These data are consistent with the ability of pros-tratin to activate latent virus from HAART-treated patients (29). Hence, the BLT mouse can potentially be used as an abundant source of primary latently infected cells for use in screening novel
latency activatorsex vivo.
Latently infected cells are rare in patients (14), making the development and optimization of latency-purging strategies heavily dependent on HIV latency models. Primary cell models are generally preferable in this context because they are considered to be more likely to accurately reflect patient-derived latently in-fected cells than continuously proliferating cell lines do. Depend-ing on the precise frequency of latently infected human cells present, which varies from mouse to mouse, we have found that as many as several million latently infected cells can be obtained from a single HIV-infected BLT mouse spleen.
Ap-proaches that could potentially benefit fromex vivouse of
la-tently infected cells generated in the BLT mouse include screening of molecule libraries for new latency activating agents (44), optimizing nanoparticle-based methods for deliv-ery of latency activators (28), and testing modified versions of existing latency activators (41). Other methodologies for en-hancing reservoir depletion, such as the specific killing of
re-cently activated latently infected cells, could also be assessedex
vivousing these cells. These may include stimulation in the
pres-ence of anti-HIV envelope immunotoxins (9) or genetically mod-ified HIV-specific cytotoxic T lymphocytes (26), both of which
have the potential to accelerate the decay of recently activated latently infected cells once viral proteins have been induced to express.
The use of different antiretroviral drugs duringex vivo
stimu-lation also allowed us to verify that the induced virus is indeed replication competent and that only a minor portion of preinte-grated virus in splenocytes is capable of expressing upon stimula-tion (Fig. 4). This is consistent with the previously described labile nature of preintegrated viral intermediates in resting cells (46).
HIV latency in patients is of clinical importance primarily because it allows the virus to be maintained during HAART. The turnover and natural life span of immune cells in general in animal models could be different from the situation in hu-mans, and this may be particularly true in humanized mice, in which long-term homeostatic signals are unlikely to be opti-mally regulated. However, if viral loads in infected BLT mice can be suppressed to undetectable levels with antiretroviral drugs while maintaining a population of latently infected cells, thenin vivotesting of purging strategies intended to deplete the latent reservoir should be possible. Therefore, we infected BLT mice with wild-type NL4-3 and allowed the infection to pro-ceed for 3 weeks. The mice were then treated with an antiret-roviral therapy regimen consisting of AZT, indinavir, and ddI, which we have previously shown to be effective in the SCID-hu (Thy/Liv) mouse (2, 42). In 3 of 4 treated mice, the viral loads
were suppressed to undetectable levels (⬍10 copies per 100l
of plasma) within 1 week of treatment (Fig. 5A). Antiretroviral
therapy also protected CD4⫹T cells from depletion in these
mice (Fig. 5B). Activation-inducible virus was detectable in the spleens of two mice with completely suppressed viral loads (Fig. 6). Importantly, the assay used for detection of activation-inducible virus utilized only 500,000 cells and did not allow viral spread. This provides a stringent but relatively insensitive detection method for latent virus. For example, the latently infected cells in HAART-treated mice described in Fig. 6B were too infrequent to enumerate using intracellular flow cytometry specific for Gag protein. Therefore, in future work, we intend to use limiting dilution coculture approaches similar to those utilized for latency quantification in HAART-treated patients (14) to assess the effect of reservoir purging strategies on
la-FIG 6Presence of activation-inducible virus in the spleens of ART-treated mice with undetectable viral loads. Splenocytes were obtained from the mice described in the legend to Fig. 5 and either left unstimulated (US) or costimulated (CS) for 4 days in the presence of raltegravir. The level of p24 protein present in the supernatants was quantified to detect release of viral particles. (A) HIV p24 levels in cultures of splenocytes from mice that were not treated with antiretrovirals. (B) Levels of p24 protein in splenocyte cultures from ART-treated animals. Statistical comparisons were performed using the Wilcoxon rank-sum test.
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[image:7.585.138.455.64.216.2]tency in the BLT mice. The decline of latently infected cells following a relatively short course of HAART described in Fig. 6 most likely suggests that the turnover rate of T cells in the BLT mice may be higher than that in adult humans, which may be due in part to inadequate interaction with human major histo-compatibility proteins in the periphery, which is needed to maintain cell survival.
Overall, we detected latent virus in the majority of HIV-infected BLT mice tested, including those HIV-infected with wild-type HIV or the NLHSAs reporter virus. Infected mice treated with only AZT-indinavir, or a combination of AZT-indinavir-ddI also contained readily detectable activation-inducible virus (Table 1). Therefore, we conclude that the BLT mouse can be used as an
abundant source of latently infected cells forex vivoassessment of
latency activators and also has potential as anin vivomodel for
preclinical testing of latency-purging strategies.
ACKNOWLEDGMENTS
We thank Patricia Avancena and Alvin Welch for technical assistance. Saquinavir was obtained through the AIDS Research and Reference Re-agent Program, Division of AIDS, NIAID, NIH. Raltegravir was obtained from Merck & Co.
This work was supported by NIH grant numbers AI70010 and U19AI096113, project 3.4, to J.Z. and 1R01HL086409 to D.S.A. and the UCLA CFAR (grant P30 AI28697).
Our laboratory has received funding at various times from Amgen, Johnson and Johnson Research, and Merck.
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