JOURNAL OF VIROLOGY, Mar. 2007, p. 2700–2712
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 81, No. 6
Human Immunodeficiency Virus Type 1 Pathobiology Studied in
Santhi Gorantla,1Hannah Sneller,1Lisa Walters,1John G. Sharp,3Samuel J. Pirruccello,4John T. West,5
Charles Wood,5Stephen Dewhurst,6Howard E. Gendelman,1,2and Larisa Poluektova1*
Center for Neurovirology and Neurodegenerative Disorders and Department of Pharmacology and Experimental Neuroscience1and
Departments of Internal Medicine,2Genetics, Cell Biology and Anatomy,3and Pathology and Microbiology,4University of
Nebraska Medical Center, Omaha, Nebraska; Nebraska Center for Virology and School of Biological Sciences,
University of Nebraska-Lincoln, Lincoln, Nebraska5; and Department of Microbiology and Immunology and
James P. Wilmot Cancer Center, University of Rochester Medical Center, Rochester, New York6
Received 14 September 2006/Accepted 13 December 2006
The specificity of human immunodeficiency virus type 1 (HIV-1) for human cells precludes virus infection in
most mammalian species and limits the utility of small animal models for studies of disease pathogenesis,
therapy, and vaccine development. One way to overcome this limitation is by human cell xenotransplantation
in immune-deficient mice. However, this has proved inadequate, as engraftment of human immune cells is
limited (both functionally and quantitatively) following transplantation of mature human lymphocytes or fetal
thymus/liver. To this end, a human immune system was generated from umbilical cord blood-derived CD34?
hematopoietic stem cells in BALB/c-Rag2?/??c
irradiation of newborn pups resulted in uniform engraftment characterized by human T-cell development in
thymus, B-cell maturation in bone marrow, lymph node development, immunoglobulin M (IgM)/IgG produc-
tion, and humoral immune responses following ActHIB vaccination. Infection of reconstituted mice by CCR5-
coreceptor utilizing HIV-1ADAand subtype C 1157 viral strains elicited productive viral replication and
lymphadenopathy in a dose-dependent fashion. We conclude that humanized BALB/c-Rag2?/??c
represent a unique and valuable resource for HIV-1 pathobiology studies.
?/?mice. Intrapartum busulfan administration followed by
A genetically modified immunodeficient mouse with trunca-
tion or knockout of the interleukin-2 (IL-2) receptor (common
cytokine receptor) gamma chain (?c) provides a unique plat-
form for permanent engraftment of human hematopoietic
stem cells (HSCs). The attenuation of cell signaling pathways
via ?cfor IL-2, -4, -7, -9, -15, and -21 cytokines, which are
involved in survival, differentiation, and function of lympho-
cytes, impairs the development of mouse lymphatic compart-
ments. This provides a niche for human lymphoid and myeloid
cell reconstitution and results in the development of a func-
tional human immune system (HIS).
Development of a functional HIS in mice has been reported
to occur in a variety of genetic backgrounds, including NOD/
(these are referred to hereafter as HIS mice). Previous work
has described the generation of all major human lymphoid and
myeloid cell types in this model, following HSC engraftment,
but the utility of the model has been limited due to variability
of HIS reconstitution (18, 27). Attempts to reduce the vari-
ability of reconstitution in BALB/c-Rag2?/??c
volved high-dose total body irradiation of newborn pups and
administration of granulocyte-macrophage colony-stimulating
factor (GM-CSF) (18) and myeloablative regimens consisting
null(11, 14, 31), BALB/c-Rag2?/??c
?/?(7, 15, 24), and NOD/LtSz-scid IL-
null(13, 26). For our studies we used newborn BALB/c-
?/?mice, which we engrafted with CD34?HSCs
of busulfan administration to pregnant females followed by
irradiation of newborn pups, or busulfan alone (32).
Current humanized mouse models include transplantation
of mature peripheral blood lymphocytes (PBL) (hu-PBL mice
) or fetal thymus/liver (SCID-hu mice ). Both models
do not permit the analysis of human immunodeficiency virus
type 1 (HIV-1) replication within the context of an intact
human immune system (29). We therefore conducted experi-
ments to examine HIV-1 replication in mice that were en-
grafted with a functionally active HIS.
The CCR5-utilizing subtype B viral strain ADA (6) and the
subtype C isolate 1157 (33) were used for infection of HIS
mice. Animals were observed for 8 to 10 weeks after infection.
Over this time period, HIS mice infected with HIV-1ADAat
high dose developed an inversion of the CD4/CD8 T-cell ratio
in peripheral blood as well as lymph node infiltration with
CD8?and immunoglobulin G [IgG]-producing cells similar to
those observed in HIV-1-infected humans. Only 50% of ani-
mals infected with subtype C HIV-1 had detectable viremia
and HIV-1 p24-positive cells in lymphoid tissues. There was no
detectable change in the CD4/CD8 T-cell ratio in the periph-
eral blood of these mice. We conclude that HIS mice retain
human cell engraftment for more than 7 months. They can be
experimentally infected with HIV-1 and, following HIV-1 in-
fection, animals develop changes in engrafted human lymphoid
tissues, which resemble those found in HIV-1-positive human
subjects. Collectively, these data establish the HIS mouse as a
more authentic model system for studies of HIV-1 pathogen-
esis than previously available mouse models (21, 22). This
system affords significant advantages in investigating HIV-1
* Corresponding author. Mailing address: 985880 Nebraska Medical
Center, Omaha, NE 68198-5880. Phone: (402) 559-8926. Fax: (402)
559-3744. E-mail: firstname.lastname@example.org.
?Published ahead of print on 20 December 2006.
pathobiology as well as exploring new therapeutic and vaccine
MATERIALS AND METHODS
Mice. Rag2?/??c?/?mice were obtained from the Central Institute of Exper-
imental Animals (Kawasaki, Japan) and were bred and maintained under spe-
cific-pathogen-free conditions in accordance with ethical guidelines for care of
laboratory animals at the University of Nebraska Medical Center as set forth by
the National Institutes of Health.
CD34?cell isolation. Human cord blood was obtained, with parental written
informed consent, from healthy full-term newborns (Department of Gynecology
and Obstetrics, University of Nebraska Medical Center). After density gradient
centrifugation, CD34?cells were enriched using immunomagnetic beads accord-
ing to the manufacturer’s instructions (CD34?selection kit; Miltenyi Biotec Inc.,
Auburn, CA). Numbers and purity of CD34?cells were evaluated by fluores-
FIG. 1. Human cell reconstitution in mice myeloablated by different protocols and treated with GM-CSF. (A) Determination of optimal doses of
irradiation. A significant level of engraftment was achieved at an irradiation dose of between 500 and 650 cGy. A dose of 400 cGy was not effective (not
shown). (B) Effect of GM-CSF administration. Newborn pups were irradiated at 650 cGy and injected subcutaneously with GM-CSF every day for 3
weeks (0.1 or 1 ?g/dose/mouse) starting at 1 or 2 weeks of age. The number of human cells in the circulation was analyzed 4 weeks after last injection.
(C) Effects of intrapartum busulfan administration. Pregnant dams were injected subcutaneously with 15 mg/kg of busulfan solution once, at day 18
postcoitus, and pups were irradiated after birth with 500 cGy or 400 cGy. In the last group busulfan was injected at days 15 and 18 and pups were not
irradiated (B ? 2). The animals were segregated by litter; each litter received the same CD34?cell sample. Individual mouse profiles are presented in
panels A, B, and C. Numbers above bars correspond to the mouse number (A) and doses of GM-CSF (B). (D) Statistical analysis of the efficacy of
different myeloablative procedures presented in panels B and C. The groups were compared to irradiated mice alone. Results are expressed as means ?
SEMs. (E) Statistical analysis of the effects of GM-CSF in irradiated animals.*, P ? 0.05 by ANOVA.
VOL. 81, 2007 HIV-1 IN HIS MICE2701
cence-activated cell sorting (FACS). CD34?cell purity was ?90%. Cells were
either frozen or immediately transplanted into newborn mice at 104/mouse
intrahepatically. From two to seven littermates were reconstituted with one cord
blood sample derived from one donor. The numbers of animals reconstituted
were dependent on the number of CD34?cells isolated from cord blood.
Irradiation and busulfan administration. On the day of birth, newborn mice
were irradiated twice using a C9 cobalt 60 source (Picker Corporation, Cleveland,
OH) at a 3- to 4-h interval. Titrated sublethal doses of 650 cGy, then reduced to 500
and 400 cGy, were used after administration of busulfan to pregnant dams. Busulfan
was dissolved in dimethyl sulfoxide (both from Sigma Chemical Co., St. Louis, MO)
and administered at a concentration of 15 mg/kg as a 20% dimethyl sulfoxide
solution subcutaneously to pregnant dams at day 18 postcoitus. At 4 to 12 h postir-
radiation, mice were transplanted with CD34?cells in 20 ?l phosphate-buffered
saline intrahepatically using a 30-gauge needle. Newborns always received cells from
single donors. Mice were weaned at 3 weeks of age.
Virus stocks. The CCR5 coreceptor-utilizing HIV-1ADAstrain was propagated
in monocyte-derived macrophages cultured for 7 days in the presence of mac-
rophage colony-stimulating factor (a generous gift from Wyeth, Inc., Cambridge,
MA) (6). Subtype C HIV-1C1157(CCR5) (33), NL4-3 (CXCR4), and UG 029A
(CCR5) viral strains were propagated in phytohemagglutinin (PHA)-stimulated
peripheral blood mononuclear cells in the presence of interleukin-2 (BD Bio-
science, San Jose, CA) (PHA/IL-2 lymphoblasts). Viral preparations were
screened and found to be negative for endotoxin (?10 pg/ml) (Associates of
Cape Cod, Woods Hole, MA) and mycoplasma (Gen-Probe II; Gen-Probe, San
Diego, CA). The viral titers were assayed on PHA/IL-2 lymphoblasts and mono-
cyte-derived macrophages. Titers were 106and 10550% tissue culture infectious
doses (TCID50)/ml for HIV-1C1157and HIV-1ADA, respectively.
HIV-1 infection in hu-PBL-BALB/c-Rag2?/??c?/?and HIS (BALB/c-
Rag2?/??c?/?) mice. BALB/c-Rag2?/??c?/?mice were injected with 30 ? 106
human PBL obtained from HIV-1, HIV-2-, and hepatitis B virus-seronegative
donors as described previously (6, 23). One week after reconstitution, HIV-1
strains ADA, NL4-3, and UG 029A (subtype B) and C1157 (subtype C) were
injected intraperitoneally (i.p.) at 102and 104TCID50. Two weeks after infection,
the levels of HIV-1 p24 protein in serum and the numbers of CD4?and CD8?
cells in spleens were analyzed.
HIS (BALB/c-Rag2?/??c?/?) mice at 16 to 20 weeks were infected i.p. with
HIV-11157(n ? 14) and HIV-1ADA(n ? 3) at 102and 5 ? 102TCID50,
respectively. Since only a single mouse showed detectable levels of HIV-1 p24 in
plasma at 2 weeks following this initial exposure to virus, the animals were
reinfected with 103and 3.5 ? 104TCID50, respectively.
Flow cytometry. Peripheral blood samples were collected from the subman-
dibular vein by using lancets (MEDIpoint, Inc., Mineola, NY) in EDTA-coated
tubes. Blood leukocytes and suspensions of spleen cells were tested for mouse
CD45 and human pan-CD45, CD45RO, CD45RA, CD3, CD4, CD8, CD11c,
CD14, CD19, CD25, CD56, CD123, and HLA-DR markers as four-color com-
binations (fluorescein isothiocyanate conjugated, phycoerythrin [PE]-cyanin 5.1
conjugated, PE conjugated, and allophycocyanin conjugated). Antibodies as well
as isotype controls were obtained from BD PharMingen, San Diego, CA, and
staining was analyzed with a FACSCalibur using CellQuest software (BD Im-
munocytometry Systems, Mountain View, CA). Results were expressed as per-
centages of total numbers of gated lymphocytes.
Isolated bone marrow cells and thymocytes were stained with fluorescein isothio-
cyanate-conjugated antibodies (CD8, CD10, and CD33), PE-conjugated antibodies
(CD4, CD11c, CD19, and CD235a), PE-Texas red (ECD)-conjugated antibodies
(CD3, CD4, CD24, and CD34), PC5-conjugated antibodies (CD2, CD8, CD34, and
CD117), and PE-cyanin 7-conjugated antibodies (CD3 and CD45). T-cell receptor
(TCR) beta chain expression profiles were analyzed with the IOTest Beta Mark
TCR V beta repertoire kit from Beckman Coulter according to the manufacturer’s
specifications. Data were collected on a Beckman Coulter FC500 flow cytometer
(Beckman Coulter, Miami, FL) using Beckman Coulter Cytomics CXP software
(Applied Cytometry Systems, Dinnington, United Kingdom). Results were ex-
pressed as percentages of total human CD45?cells.
Immunohistochemistry. Tissue samples (spleen, lymph node, thymus, liver,
lung, gut, kidney, and brain) were fixed with 4% paraformaldehyde overnight and
embedded in paraffin. Five-micrometer-thick sections were stained with mouse
monoclonal antibodies for vimentin (clone 3B4, 1:50), CD68 (clone KP-1, 1:50),
HLA-DR (clone CR3/43, 1:100), CD8 (clone 144, 1:50), CD20 (clone L26, 1:50),
HIV-1 p24 (clone Kal-1, 1:10), and CD3 (1:100, rabbit polyclonal), all from Dako
(Carpinteria, CA). Biotinylated anti-IgG, -IgM, and -IgA antibodies were used at
a 1:200 dilution (Vector Laboratories, Burlingame, CA).
Immunoglobulin and HIV-1 p24 measurements. The plasma levels of IgG,
IgM, and IgA were determined by enzyme-linked immunosorbent assay (ELISA)
(Bethyl, Montgomery, TX). The levels of HIV-1 p24 in the plasma were deter-
mined by ELISA (Beckman Coulter, Inc., Brea, CA) according to the manufac-
ELISA and Western blot tests. Plasma samples collected from infected ani-
mals were analyzed for HIV-1-specific human antibodies by Genetic Systems
HIV-1/HIV-2 enzyme immunoassay (Bio-Rad Laboratories, Hercules, CA) and
by Western blotting with HIV-1 nitrocellulose strips (Calypte, Alameda, CA).
The strips were incubated overnight in 1:5 diluted serum samples with shaking at
4°C, and horseradish peroxidase-conjugated anti-human IgM and IgG was used
as a secondary antibody. The strips were developed using a chemiluminescent
substrate (Pierce Biotechnology, Inc., Rockford, IL) and were exposed to X-ray
film. HIV-1-positive and -negative human sera were used as positive and negative
controls, respectively, at a dilution of 1:1,000.
ActHIB vaccination and humoral immune responses. One-fifth (2 ?g) of a
single human dose of Haemophilus influenzae type b (HIB) conjugate vaccine
ActHIB (Aventis Pasteur Inc., Swiftwater, PA) was injected into mice i.p. at 24
to 27 weeks of age. A second injection was delivered 2 weeks later. Mice were
killed 3 weeks after the last injection, and the levels of HIB-specific IgG were
determined by ELISA according to the manufacturer’s instructions (The Binding
site, Inc., San Diego, CA).
Statistical analysis. Data were analyzed using Excel software; statistical tests
employed were the Student’s t test (for pairwise comparisons) and one-way
analysis of variance (ANOVA) for comparisons of multiple groups. A P value of
FIG. 2. Thymus and spleen morphology of reconstituted mice. Light microscopic images of paraffin-embedded thymus (23-week-old mouse)
and spleen (21-week-old mouse) sections are shown. (A) The thymus tissue contains well-defined cortical and medullary areas. (B) Splenic sections
were stained for human CD3, CD20, CD8, and CD68 and mouse CD45 cell markers (diaminobenzidine [DAB]). Sections were counterstained with
hematoxylin. Magnification, ?20. Mouse CD45?cells surround follicles occupied by human CD3?T cells and a smaller number of CD8?cells
(inset; magnification, ?200), CD20?B cells and small numbers of IgM-producing plasma cells (inset; magnification, ?200) were distributed in the
marginal zone and red pulp.
2702 GORANTLA ET AL.J. VIROL.
FIG. 3. Thymocyte development, human TCR V? repertoire in spleens, and B cells in bone marrow of 18-week-old HIS mice. (A) Dot
plots show CD45?human thymocytes stained for CD4?, CD8?, CD2?, and CD3?(two left panels). The results demonstrate that the
majority of human cells isolated represent CD4 and CD8 dual-positive common thymocytes and that there is also generation of both CD4
and CD8 single-positive, mature T cells. The two plots on the right demonstrate the characteristic increase in thymocyte CD3 density that
occurs during thymic maturation/selection. (B) The TCR repertoire of human T cells in the spleen of the same mouse (no. 152) is shown.
TCR repertoire profiles for two additional mice from different litters (mice no. 144 and 147) are also shown. (C) B cells in bone marrow of
the same mouse no. 152. Plots show CD45-positive human cells (black) and CD45-negative mouse cells (light gray) on a side light scatter
(left). The middle plot shows background staining of the human cell population with IgG1 control antibodies for human CD45-positive
events. The right plot demonstrates phenotypically normal human B-cell precursors (arrowhead) for human CD45-positive events. These
precursors are CD10 and CD24 positive and show differentiation (arrows) to mature B cells as evidenced by a loss of CD10 expression and
a decrease in CD24 antigen density.
VOL. 81, 2007 HIV-1 IN HIS MICE2703
?0.05 was considered statistically significant. All results are presented as means ?
standard errors of the means (SEMs).
Improvement of reconstitution by myeloablation with busul-
fan and administration of GM-CSF. Different myeloablation
procedures were evaluated in order to optimize human im-
mune cell reconstitution in mice. Irradiation doses ranging
from 500 to 750 cGy were sufficient for engraftment of human
CD34?cells in newborn pups (Fig. 1A and B). At 10 to 14
weeks of age, 18 of 75 engrafted mice contained ?10% of
human cells in their peripheral circulation. The highest doses
of irradiation led to significant retardation of animal growth,
induction of seizures, and abnormalities of gait secondary to
Injection of busulfan to pregnant dams (day 18 postcoitus) at
15 mg/kg followed by 500- or 400-cGy irradiation of newborn
pups resulted in high levels of human immune cell reconstitu-
tion. Here, after 8 to 10 weeks, 36 out of 42 engrafted mice
contained ?10% of human cells in their peripheral circulation.
One-third of these animals had ?40% of human cells in their
blood (Fig. 1C and D).
In order to increase the numbers of engrafted human im-
mune cells in irradiated mice, GM-CSF was administered for 3
weeks at a dose of either 0.1 or 1 ?g/mouse/day, starting at 1 or
2 weeks after reconstitution with CD34?HSCs (Fig. 1B and
E). The number of circulating human cells was analyzed at 4 to
5 weeks after the last GM-CSF administration. The GM-CSF
dose of 1 ?g/mouse/day resulted in a statistically significant
increase in the number of CD3?T cells in circulation, as well
as in the number of myeloid CD11c?cells; the latter effect did
not, however, reach statistical significance.
Representative immunohistology data from the thymuses
and spleens of mice reconstituted with human CD34?HSCs
are shown in Fig. 2. There was no discernible difference in
tissue morphology between the various groups of animals/my-
eloablative regimens. The thymus and spleen contained large
numbers of human lymphocytes but few human CD68?cells.
Spleens contained follicular structures composed of human T
and B cells. CD8?T cells were limited in numbers, while
IgM-producing plasma cells were present only in the “marginal
zone” and red pulp (Fig. 2). IgG-producing cells were rare
(data not shown).
FACS analysis of representative thymuses and TCR V?
chain repertoire analysis of human T cells in the spleens of
18-week-old mice (Fig. 3A and B) confirmed the development
of polyclonal human T cells de novo. Human B-cell maturation
and differentiation in mouse bone marrow were also confirmed
by FACS (Fig. 3C; Table 1).
A human immune system was sustained in 7.5-month-old
animals. The proportions of human cells phenotyped as
CD45?, CD3?, CD19?, CD11c?, and CD123?in the spleens
of nine selected animals (three from each myeloablation pro-
tocols) were 18.8% ? 5.3% (range, 7.5% to 31%), 6% ? 1.9%
FIG. 4. Profiles of peripheral blood from reconstituted mice. (A) Ki-
netics of mouse CD45 and human T, B, and CD11c-positive cells in
peripheral blood. Error bars indicate SEMs. (B) Human IgG and IgM
levels in peripheral blood at 5 and 6 months postreconstitution. The three
groups of mice represent animals that were myeloablated using high-dose
irradiation alone (650 cGy; n ? 8), a combination of busulfan plus lower-
dose irradiation (B ? 500 cGy; n ? 11), or busulfan alone (B ? 2; n ? 5).
In panel A, “a” denotes statistically significant differences between mice
irradiated and pretreated with busulfan (B ? 500 cGy) and the other two
groups, and “b” denotes statistically significant differences between mice
myeloablated by the combination of irradiation and busulfan (B ? 500
cGy) and animals treated with busulfan alone (B ? 2) (P ? 0.05 by
ANOVA). In panel B,*denotes statistically significant differences be-
tween IgG levels in 5- and 6-month-old mice.
TABLE 1. Human bone marrow cell subset distributions in engrafted mouse bone marrowa
% of human CD45?cells positive for:
(precursor B cell)
aPercentage of human CD45?cells positive for the indicated antigen(s) in bone marrow isolated from two representative mice engrafted with CD34?human
hematopoietic stem cells.
2704 GORANTLA ET AL.J. VIROL.
(0.9% to 14.9%), 4.3% ? 2.3% (0.12% to 17.4%), 0.6% ?
0.3% (0.03% to 2.45%), and 0.9% ? 0.8% (0.1% to 3.6%),
respectively. The proportion of human CD45?cells in bone
marrow was 6.6% ? 4.5% (1.79% to 31%), and both myeloid
(CD117?CD33?) and erythroid (CD117?CD33?) progeni-
tors were detected (Table 1). Furthermore, all tested animals
also retained lineage-negative CD34?HSCs in the bone
In order to characterize the kinetics of repopulation and
maturation of the HIS, the mouse peripheral blood was ana-
lyzed. Three groups of animals were evaluated: (i) eight new-
born pups that received irradiation alone, (ii) 11 pups whose
mothers were treated with busulfan following 500 cGy of irra-
diation on the day of delivery, and (iii) 5 pups whose mothers
received busulfan during pregnancy at days 15 and 18 postcoi-
tus. These animals were observed for survival and for the
presence of human cells in blood (Fig. 4A; Table 2). High-
dose-irradiated mice showed poor survival compared to mice
treated with a combination of busulfan and irradiation. There
was no mortality in the group of animals treated only with
busulfan (Table 2). These animals remained fertile, delivered
pups, and also retained engrafted human cells (data not
The first wave of human cells in mouse blood represented, in
significant measure, expansion of CD19?B cells. Maximal
numbers of human T cells in circulation were not observed
until 18 to 20 weeks postengraftment (Fig. 4). After 22 weeks,
the number of T cells in the peripheral blood stabilized and in
some animals even increased. The proportion of CD19?B
cells diminished at 18 weeks of age. Recipients that were only
irradiated had a slower expansion of human cells than the
animals that received the combination of busulfan pretreat-
ment and reduced-dose irradiation.
Significant IgG production was first observed in 5-month-old
mice (Fig. 4B). In two animals high IgM levels (1,231 and 1,580
?g/ml) were detected at 5 months of age, which subsequently
declined (to 669 and 1,244 ?g/ml), coinciding with the increased
IgG production (from 44 to 150 and from 9.5 to 33 ?g/ml, re-
spectively) at 6 months. IgA levels were low in all mice tested.
Taken together, these findings show that myeloablation proto-
col, which included busulfan administration, significantly im-
proved mouse survival, as well as human immune cell reconstitu-
tion in BALB/c-Rag2?/??c
minimal effects. Full maturation of the HIS in the engrafted mice,
as reflected by the ability to produce IgG, required 5 to 6 months
?/?mice. GM-CSF administration had
FIG. 5. Viral replication and cytopathic effects of different HIV-1
strains in hu-PBL-Rag2?/??c
mice. Adult Rag2?/??c
human PBL (30 ? 106cells/mouse). One week later mice were infected
i.p. by the indicated strains of HIV-1 at a dose of 102TCID50. Plasma and
splenocytes were collected 2 weeks after infection and analyzed for the
levels of HIV-1 p24 antigen in circulation (A) and for the presence of
human CD4 and CD8 cells (B). All viruses were tested in three different
experiments with mice that were reconstituted with PBL derived from
three different donors, and similar results were obtained. Error bars in-
dicate SEMs. (C to E) HIS BALB/c-Rag2?/??c
of age were infected with HIV-1 via the i.p. route, using either the C1157
and then exposed to a higher-dose inoculum two weeks later. Blood samples
for the levels of HIV-1 p24 antigen (C and D), as well as for number of
human CD4 and CD8 cells (D and E). (D) Only HIV-1ADA-infected mice
showed inversion of the CD4/CD8 ratio. Open symbols represent CD4/CD8
ratios in peripheral blood; closed symbols represent HIV-1 p24 concentra-
evidence of productive infection (n ? 7), and mice exposed to C1157 with
HIV-1ADA-infected mice showed statistically significant changes in the num-
ber of circulating CD8 cells.*, P ? 0.05.
?/?mice and HIS BALB/c-Rag2?/??c
?/?mice (n ? 4 per group) were injected with
?/?mice at 18 to 24 weeks
TABLE 2. Survival rates and efficacy of engraftment
total (% surviving)
No. of graft failuresb
Irradiation (650 cGy)
Busulfan (15 mg/kg) ?
irradiation (500 cGy)
Busulfan (2 doses of
5/5 (100) 1 (18)
aNumber surviving ?30 weeks.
bLoss of the graft is defined as the presence of ?1% circulating human cells
cDifference between irradiation alone (650 cGy) and combination of busulfan
with a lower dose of irradiation, P ? 0.04 by chi test.
VOL. 81, 2007 HIV-1 IN HIS MICE2705
and might be reminiscent of some aspects of pre- and postnatal
development of human immunity.
HIV-1 infection of hu-PBL-Rag2?/??c
fection was first tested in Rag2?/??c
with human peripheral blood lymphocytes (hu-PBL-Rag2?/?
?/?mice. HIV-1 in-
30 ? 106PBL/mouse. One week after reconstitution, the
HIV-1 strains ADA, NL4-3, UG 029A (subtype B), and C1157
(subtype C) were independently injected i.p. at a dose of 102or
104TCID50. Two weeks after infection, levels of HIV-1 p24
?/?mice). Animals at 5 weeks of age were injected i.p. with
FIG. 6. Pathomorphology of cervical lymph nodes from HIV-1-infected mice. (A) Low- and high-magnification images of two cervical lymph
nodes of control mouse 127 are shown. Paraffin-embedded 5-?m sections were stained for human CD3, CD20, CD8, HLA-DR, IgG, and vimentin
and mouse CD45 markers. The lymph node contained medullae (characterized by the presence of dendritic cells), a paracortical T-cell region with
very few CD8-positive cells, marginal zone B cells, and few IgG-secreting cells. (B) Cervical lymph nodes from HIV-1C1157-exposed mouse no. 73
are shown. No p24 antigen-positive cells were detected in this lymph node, although HIV-1 p24-positive cells were found in spleen from this animal
(inset; magnification, ?200). (C) Enlargement of the cervical lymph node from mouse no. 20 infected with HIV-1ADA. This panel also shows
infiltration of this lymph node with CD8?and IgG-producing cells. The right column in each of the panels provides the phenotypic profile of
peripheral blood cells from each of the animals, as well as their serum IgG and IgM profiles. Brown, DAB; purple, Permanent Red.
2706 GORANTLA ET AL.J. VIROL.
were measured in plasma, and CD4?and CD8?cells were
enumerated in spleens. As shown in Fig. 5A and B, inoculation
of animals with a dose of 102TCID50of HIV-1C1157resulted in
a significant viremia with depletion of CD4?T lymphocytes in
spleen. HIV-1ADAalso replicated and was detected in the
mice, albeit to a lower level than HIV-1C1157. Viral strains
NL4-3 and UG 029A elicited a depletion of human CD4?T
lymphocytes. The higher-dose inoculum (104TCID50) yielded
similar results for all of the strains (data not shown). The
subtype C isolate C1157 and HIV-1ADAwere selected for
infection of HIS mice (Fig. 5C and D).
HIV-1 replication and tissue histopathology in HIS mice.
Low doses of viruses (102TCID50for HIV-1C1157[n ? 14] and
5 ? 102TCID50for HIV-1ADA[n ? 3]) were injected i.p. in 16-
to 20-week-old mice. At 2 weeks after infection, only one
mouse inoculated with HIV-1C1157had a detectable level of
HIV-1 p24 in the circulation (200 pg/ml). In light of this ap-
parent failure to infect the majority of mice with a low-dose
inoculum, the animals were then reinfected at higher doses of
viruses (103and 3.5 ? 104TCID50for HIV-1C1157and HIV-
1ADA, respectively). Blood samples were collected every 2 to 4
weeks thereafter to quantify HIV-1 p24 and the number of
human cells in circulation (CD3, CD19, CD4, and CD8). Eight
weeks after exposure to HIV-1C1157, only 7 of 14 infected
animals (50%) had detectable levels of HIV-1 p24 in blood,
and 6 of them had HIV-1 p24?cells in their lymphoid tissues
(detected at 8 to 10 weeks following virus infection and local-
ized predominantly to the lymph nodes and spleen rather than
the thymus). As shown in Fig. 5C, by 8 weeks after viral infec-
tion, the level of HIV-1 p24 in the circulation decreased to
undetectable levels in some mice. All animals that underwent
productive infection by HIV-1C1157(as evidenced by detection
of HIV-1 p24 antigen in blood and/or p24 antigen-positive cells
in lymphoid tissue) retained CD4?T lymphocytes in circula-
tion without a significant elevation of CD8?T lymphocytes.
Only animals infected with high viral inoculum of HIV-1ADA
showed an increased proportion of CD8?T lymphocytes in
circulation, reduced CD4?T cells, and an inversion of the
CD4/CD8 ratio (Fig. 5D and E). All HIV-1ADA-infected mice
had HIV-1 p24?cells in spleen and lymph nodes.
Cervical lymph node (CLN) histology for control (unin-
fected) and HIV-1-infected mice, as well as human immune
cell profiles in blood, is shown in Fig. 6. All animals developed
“miniature” human-like nodes with a central medullar area
occupied by dendritic cells, a cortical area with T and B cells,
and limited numbers of immunoglobulin-secreting plasma
cells. A few CD8?T lymphocytes were also present in CLN
tissues from uninfected mice. In mice infected with HIV-
1C1157, an enlargement of CLN tissue was detectable, accom-
panied by infiltration with CD3?cells of the medullar region
and the presence of HIV-1 p24-positive cells in spleen (but not
in lymph nodes) (Fig. 6B). Significant hyperplasia, infiltration
with CD8 cells, IgG-producing cells, and abnormal structures
were also observed in CLNs from two HIV-1ADA-infected
mice (mice 11 and 20) (Fig. 6C and 7). A younger mouse (F1)
infected at 16 week of age with single high dose of HIV-1ADA
and killed 5 weeks later exhibited significant infiltration of
lymph nodes by CD8?cells and the presence of human CD68?
cells in the mesenteric lymph node capsule (Fig. 8). None of
the infected mice were found to produce detectable levels of
HIV-1-specific antibodies, as assessed by the Genetic Systems
HIV-1/HIV-2 enzyme immunoassay or by Western blot assays
(data not shown).
Humoral immune response to ActHIB in HIS mice. To con-
firm the competence of the humoral immune system in the HIS
mice, three mice (24 to 27 weeks of age) were vaccinated with
two doses of ActHIB (2 ?g/mouse) delivered i.p. with a 2-week
interval in between. Plasma samples were collected 3 weeks
after the second dose. The humoral immune responses were
analyzed by ELISA. All immunized animals developed HIB-
FIG. 7. Representative serial sections of an enlarged mediastenal lymph node and spleen lymphoid follicle from mouse no. 11 infected with
HIV-1ADA. (A) Distribution of CD20?and HIV-1 p24?cells and infiltration with IgG-producing cells and absence of IgM-producing cells in lymph
node. Magnification, ?40. (B) Distribution of CD8?/HIV-1 p24?cells in a spleen lymphoid follicle; IgM- and IgG-producing cells are also present.
CD8?cells are brown (DAB), and HIV-1 p24?cells are pink (Permanent Red). Magnification, ?40. The levels of HIV-1 p24 protein in peripheral
blood 2 weeks after infection in this animal were undetectable, but after reinfection, HIV-1 p24 antigen was detected in blood at 16 pg/ml (25
weeks) and at 45 pg/ml (29 weeks).
VOL. 81, 2007 HIV-1 IN HIS MICE2707
specific IgG (25), and only one of four nonvaccinated mice had
detectable Hib-specific IgG levels (Table 3). FACS analyses of
blood, spleen, and bone marrow and morphological analysis of
the thymus and spleen from one of these vaccinated mice
(mouse 120) are presented in Fig. 9. The thymus of this animal
was populated with human T cells, macrophages, and a small
number of CD20?cells (not shown). The spleen was populated
with human T, B, myeloid, and plasmacytoid antigen-present-
FIG. 8. Immunopathology of lymphoid tissues in HIV-1ADA-infected HIS mouse no. F1. Representative serial sections of cervical (A) and mesenteric
(B) lymph nodes and spleen (C) of a mouse infected at 16 weeks of age with HIV-1ADAare shown. The distributions of CD3?, CD20?, CD68?, and
HIV-1 p24?cells are shown, together with infiltration by CD8?cells. IgM-producing cells were present; however, IgG-producing cells were rare. In
double-stained panels CD8?cells are brown (DAB) and HIV-1 p24?cells are pink (Permanent Red). Magnification, ?100. The level of HIV-1 p24
protein in peripheral blood 2 weeks after infection was 225 pg/ml, and it dropped to 29 pg/ml by the fifth week after infection.
2708 GORANTLA ET AL.J. VIROL.
ing cells. A higher expression of HLA-DR was also detected,
and the engrafted human T and B cells were found to be
clustered in areas that were reminiscent of germinal-like cen-
ters. These findings confirm that HIS mice retain a functional
immune system and are able mount humoral immune re-
sponses at ages of over 24 weeks.
Mice engrafted with human immune cells (mature periph-
eral blood lymphocytes or fetal thymus/liver transplant under
kidney capsule) have emerged as a powerful model for study-
ing the pathogeneses of species-specific virus pathogens, in-
cluding HIV-1, human T-lymphotropic virus type 1, and hu-
man herpesviruses such as varicella-zoster virus and human
herpesvirus 6 (8, 12, 20). The ability to engraft human HSCs in
small animals is extremely valuable, since it offers a new model
system for studies on the ontogeny of the human immune
system and for the analysis of the interplay of species-specific
pathogens. Recent progress in the generation of humanized
mice with long-term reconstitution of human functional lym-
phoid tissues by transplantation of HSCs was reviewed by Mac-
chiarini et al. (17) and Legrand et al. (16). The best results so
far, in terms of the longevity of engraftment and the functional
properties of a HIS in a murine environment, have been
achieved in mice with a deletion or truncation of IL-2R?c:
derived from a range of different sources, including umbilical
cord blood, mobilized HSCs from peripheral blood, and fetal
liver. However, to date, none of the published protocols has
resulted in reproducible, efficient, and uniform engraftment of
human immune cells in these mice. The first part of the present
work was therefore dedicated to the development of an im-
proved method for the reconstitution of mice with HSCs.
We used BALB/c-Rag2?/??c
constitution experiments (27) but were unsuccessful in creating
stable chimerism using the published irradiation dose of 375
cGy. The irradiation needed to create a niche for the engraft-
ment of human cells was between 550 and 700 cGy. Even a
high dose of 650 cGy did not guarantee higher levels of en-
graftment. Moreover, such doses induced significant health
nullmice. Such animals can be engrafted with HSCs
null, and NOD/LtSz-
?/?newborn pups in our re-
problems and reduced the survival of animals. We therefore
combined lower-dose irradiation with busulfan-mediated my-
eloablation. We based our busulfan regimen on a protocol that
was previously used for successful allotransplantation of bone
marrow in C57BL/6 mice (7).
Busulfan, a common component of pretransplant condi-
tioning regimens in human bone marrow transplantation (9),
selectively destroys quiescent stem cells (2, 4, 10). When com-
bined with a lower (400-cGy) dose of irradiation, the intrapar-
tum busulfan injection yielded efficient, stable, and reproduc-
ible engraftment of HSCs. The reduced irradiation dose in this
combination regimen resulted in improved outcomes in terms
of survival and neurologic damage (seizures or balance abnor-
In order to assess the kinetics of maturation of HIS in
immunoglobulin levels in peripheral blood. During the early
postengraftment period, a significant expansion of CD19?cells
was observed, as previously noted in NOD/SCID/?c
(11, 13). This may be related, in part, to the presence of
immature B-cell precursors in cord blood and to ongoing stim-
ulation of these cells by the murine environment. However, we
observed that the expansion of these cells gradually tapered
off, and the number of human B cells in circulation began to
decline by 22 to 28 weeks after engraftment. At a slightly
earlier time point (16 to 20 weeks), the frequency of human T
cells in peripheral blood reached a stable peak, and lymph
nodes and spleen were populated. Thus, all components of an
HIS were present by 16 weeks of age. However, full functional
maturation of the HIS was not complete until 5 to 6 months of
age, as reflected by the ability to stably produce IgG and to
mount a humoral immune response to ActHIB vaccination.
The second aspect of this study focused on an evaluation of
HIV-1 infection in HIS mice. Researchers interested in the
development of a mouse model for HIV-1 infection previously
have used either (i) immune-deficient mice reconstituted with
human peripheral blood lymphocytes (hu-PBL mice) (21) or
(ii) immune-deficient mice engrafted with fetal human thymus/
liver under the kidney capsule (Thy/Liv SCID-hu mice) (1, 19,
22). There was considerable initial enthusiasm for the use of
these models to study HIV-1 pathobiology. However, impor-
tant technical limitations have precluded their widespread use
and broad applicability. These limitations include reduced sur-
vival of the engrafted human immune cells, graft-versus-host
reactions, an incomplete human T-cell receptor repertoire,
deficiency of human antigen-presenting cells, and incomplete
peripheral lymphocyte reconstitution. Perhaps most impor-
tantly, the functional properties of a human immune system
were compromised, and HIV-1 infection led to very rapid
depletion of human CD4?T lymphocytes without most of the
other hallmarks of HIV-1 infection in human subjects.
We show here that engrafted human immune cells in BALB/
that the dynamics of virus infection are substantially differ-
ent in this model than in previously described models such
as hu-PBL mice. The HIS mice are less susceptible to low-
dose infection with HIV-1 and undergo a slower, more pro-
tracted course of CD4?T-cell depletion compared to hu-
PBL mice. This may be because HIS mice contain a larger
fraction of naive human T cells than hu-PBL mice. Impor-
?/?mice, we monitored human cells and
?/?mice are susceptible to HIV-1 infection but
TABLE 3. Anti-ActHIB responses
Age (wk) at
% of human cells
aAll vaccinated animals mounted an antigen-specific IgG response consistent
with protective humoral immunity.
bVaccinated after HIV-1C1157infection (HIV-1 p24, 81.3 pg/ml at end point).
cExposed to C1157 but did not develop detectable evidence of productive
virus infection (no HIV-1 p24 could be detected in blood and tissues).
dThree more nonvaccinated mice (no. 127, 131, and 132) were used as con-
trols, and they did not show detectable HIB-specific antibodies.
VOL. 81, 2007HIV-1 IN HIS MICE2709
tantly, the large number of naive T cells in HIS mice is
consistent with what is known about the T-cell population in
normal human subjects, and this highlights the biological
relevance of this new model.
In HIS mice the cytotoxicity of tested viral isolates did not
correspond to the virus behavior in hu-PBL mice, where a
rapid depletion of CD4?cells occurs after administration of
HIV-1C1157, as well as NL4-3 and UG 029A. In contrast, the
same low doses of HIV-1 did not induce rapid viremia and
depletion of CD4?cells in HIS mice. Indeed, we were able to
demonstrate the stable infection of HIS mice with HIV-1C1157,
for at least 8 to 10 weeks following virus inoculation. This may
FIG. 9. FACS profiles and/or morphology of peripheral blood, spleen, bone marrow, and thymus from 30-week-old mouse no. 120, which was
HIV-1C1157infected and then vaccinated with ActHIB. (A to C) FACS profiles for peripheral blood (A), spleen (B), and bone marrow (C);
antibodies were specific for the indicated human (h) or mouse (m) cell surface proteins. (D) Paraffin-embedded sections of two thymus lobes
stained for T cells and CD68-positive cells. Magnification, ?20. (E) Spleen sections stained for CD3, CD20, CD68, and HLA-DR markers.
Magnification, ?20. (F) Magnified views of selected regions of the same areas in panel A. Magnification, ?200.
2710 GORANTLA ET AL.J. VIROL.
provide a model for analysis of the early stages of natural
HIV-1 infection in humans.
Infection of HIS mice with a high dose of HIV-1ADAin-
duced significant activation of the engrafted human immune
system, with expansion of CD8?cells, activation of B cells, and
pathomorphologic changes in lymph nodes. These changes
were similar to those observed in advanced HIV-1 infection in
human subjects and again serve to underscore the relevance of
the HIS mouse model for HIV-1 infection. At the same time,
it is important to note that we were not able to detect humoral
responses against HIV-1 in the HIS mice. This was unex-
pected, since the animals were capable of mounting an effec-
tive humoral immune response against a different antigen. It is
possible that reconstitution of mice with HSCs may fail to
provide key cells required for the efficient generation of HIV-
1-specific humoral immune responses (such as human follicu-
lar dendritic cells) (27). It is also possible that induction of
humoral immune responses to HIV-1 in HIS mice may require
a higher level of virus replication (30) or that virus replication
in HIS mice may interfere with B-cell maturation and preclude
the generation of a HIV-1-specific antibody response, or it may
simply be the case that seroconversion and development of
HIV-1-specific humoral immune responses may require more
than 10 weeks (3, 5, 28). Further studies will be needed to
distinguish these possibilities.
While our studies were in progress, Watanabe and col-
leagues reported on the outcome of HIV-1 infection in human
HSC-transplanted NOD/SCID-IL-2R?nullmice, following ex-
posure to both low and high doses of HIV-1JRCSF, HIV-1MNp,
or SHIV-C/1 (30). These studies showed that virus replication
persisted for up to 40 days after inoculation and that it resulted
in inversion of CD4/CD8 T-cell ratios in the spleen, at least in
some cases. The present work corroborates these findings but
also extends them considerably by (i) developing an improved
and more reproducible engraftment procedure, (ii) demon-
strating stable HIV-1 infection of HIS mice for up to 10 weeks
following virus inoculation, and (iii) showing changes in lym-
phoid architecture that resemble those found in HIV-1-infected
humans. These findings establish unequivocally the utility of the
HIS model. Overall, we conclude that HIS mice (BALB/c-Rag2?/
cells) are a unique and valuable resource to study early stages of
HIV-1 infection in its human host.
?/?mice engrafted with CD34?human hematopoietic stem
This work was supported by grants from the National Institutes of
Health (P20RR15635 to L.P., C.W., and H.E.G. and 2R37 NS36126,
P01 NS31492, P01 NS43985, and NS034239 to H.E.G.).
We thank Mamoru Ito (Laboratory of Immunology, Central Insti-
tute for Experimental Animals, Kawasaki, Japan) for providing the
ing the animals’ genetic backgrounds. We thank Linda Wilkie and
Victoria Smith for support with FACS and Dawn Eggert for assistance
in the breeding of the animals.
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