Disseminated and sustained HIV infection in CD34+ cord blood cell-transplanted Rag2-/-gamma c-/- mice.
ABSTRACT Because of species selectivity, HIV research is largely restricted to in vitro or clinical studies, both limited in their ability to rapidly assess new strategies to fight the virus. To prospectively study some aspects of HIV in vivo, immunodeficient mice, transplanted with either human peripheral blood leukocytes or human fetal tissues, have been developed. Although these are susceptible to HIV infection, xenoreactivity, and short infection spans, resource and ethical constraints, as well as biased HIV coreceptor tropic strain infection, pose substantial problems in their use. Rag2(-/-)gamma(c)(-/-) mice, transplanted as newborns with human CD34(+) cells, were recently shown to develop human B, T, and dendritic cells, constituting lymphoid organs in situ. Here we tested these mice as a model system for HIV-1 infection. HIV RNA levels peaked to up to 2 x 10(6) copies per milliliter of plasma early after infection, and viremia was observed for up to 190 days, the longest time followed. A marked relative CD4(+) T cell depletion in peripheral blood occurred in CXCR4-tropic strain-infected mice, whereas this was less pronounced in CCR5-tropic strain-infected animals. Thymus infection was almost exclusively observed in CXCR4-tropic strain-infected mice, whereas spleen and lymph node HIV infection occurred irrespective of coreceptor selectivity, consistent with respective coreceptor expression on human CD4(+) T cells. Thus, this straightforward to generate and cost-effective in vivo model closely resembles HIV infection in man and therefore should be valuable to study virus-induced pathology and to rapidly evaluate new approaches aiming to prevent or treat HIV infection.
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ABSTRACT: Continued efforts to define the immunogenic properties of the HIV-1 envelope glycoproteins (Env) are needed to elicit effective antibody (Ab) responses by vaccination. HIV-1 is a highly neutralization-resistant virus due to conformational and glycan shielding of conserved Ab determinants on the virus spike. Elicitation of broadly neutralizing Abs that bind poorly accessible epitope regions on Env is therefore extremely challenging and will likely require selective targeting of specific sub-determinants. To evaluate such approaches there is a pressing need for in vivo studies in both large and small animals, including mice. Currently, most mouse immunization studies are performed in the BALB/c strain; however, the C57BL/6 strain offers improved possibilities for mechanistic studies due to the availability of numerous knock-out strains on this genetic background. Here, we compared Env immunogenicity in BALB/c and C57BL/6 mice and found that the magnitude of the antigen-specific response was somewhat lower in C57BL/6 than in BALB/c mice by ELISA but not significantly different by B cell ELISpot measurements. We then established protocols for the isolation of single Env-specific memory B cells and germinal center (GC) B cells from immunized C57BL/6 mice to facilitate future studies of the elicited response at the monoclonal Ab level. We propose that these protocols can be used to gain an improved understanding of the early recruitment of Env-specific B cells to the GC as well as the archiving of such responses in the memory B cell pool following immunization.Viruses 09/2014; 6(9):3400-3414. · 3.28 Impact Factor
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ABSTRACT: Epstein-Barr virus (EBV) is a ubiquitous herpesvirus infecting more than 90% of the adult population of the world. EBV is associated with a variety of diseases including infectious mononucleosis, lymphoproliferative diseases, malignancies such as Burkitt lymphoma and nasopharyngeal carcinoma, and autoimmune diseases including rheumatoid arthritis (RA). EBV in nature infects only humans, but in an experimental setting, a limited species of new-world monkeys can be infected with the virus. Small animal models, suitable for evaluation of novel therapeutics and vaccines, have not been available. Humanized mice, defined here as mice harboring functioning human immune system components, are easily infected with EBV that targets cells of the hematoimmune system. Furthermore, humanized mice can mount both cellular and humoral immune responses to EBV. Thus, many aspects of human EBV infection, including associated diseases (e.g., lymphoproliferative disease, hemophagocytic lymphohistiocytosis and erosive arthritis resembling RA), latent infection, and T-cell-mediated and humoral immune responses have been successfully reproduced in humanized mice. Here we summarize recent achievements in the field of humanized mouse models of EBV infection and show how they have been utilized to analyze EBV pathogenesis and normal and aberrant human immune responses to the virus.Pathogens (Basel, Switzerland). 01/2013; 2(1):153-176.
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ABSTRACT: Broadly neutralizing antibodies (bNAbs) against HIV-1 provide both effective pre-exposure prophylaxis and treatment of HIV-1 infection in murine and nonhuman primate models, suggesting their potential use in humans. Although much is known about the role of variable domains in the neutralization breadth and potency of these bNAbs, the contribution of Fc domains to their activities is, by contrast, poorly characterized. Assessment of the in vivo activity of several bNAbs revealed that FcγR-mediated effector function contributes substantially to their capacity to block viral entry, suppress viremia, and confer therapeutic activity. Enhanced in vivo potency of anti-HIV-1 bNAbs was associated with preferential engagement of activating, but not inhibitory FcγRs, and Fc domain-engineered bNAb variants with selective binding capacity for activating FcγRs displayed augmented protective activity. These findings reveal key roles for Fc effector function in the in vivo activity of anti-HIV-1 bNAbs and provide strategies for generating bNAbs with improved efficacy.Cell 09/2014; 158(6):1243-53. · 33.12 Impact Factor
Disseminated and sustained HIV infection in CD34?
cord blood cell-transplanted Rag2?/??c?/?mice
Stefan Baenziger*, Roxane Tussiwand†, Erika Schlaepfer*, Luca Mazzucchelli‡, Mathias Heikenwalder§,
Michael O. Kurrer¶, Silvia Behnke¶, Joachim Frey?, Annette Oxenius**, Helen Joller††, Adriano Aguzzi§,
Markus G. Manz†‡‡, and Roberto F. Speck*‡‡
*Division of Infectious Diseases and Hospital Epidemiology,††Division of Clinical Immunology,§Institute of Neuropathology, and¶Department of Pathology,
University Hospital Zurich, Raemistrasse 100, 8091 Zurich, Switzerland; **Institute of Microbiology, Swiss Federal Institute of Technology, 8093 Zurich,
Switzerland;†Institute for Research in Biomedicine, Via Vincenzo Vela 6, 6500 Bellinzona, Switzerland;‡Institute for Pathology, 6600 Locarno, Switzerland;
and?Institute of Veterinary Bacteriology, University of Berne, 3001 Berne, Switzerland
Edited by Richard A. Flavell, Yale University School of Medicine, New Haven, CT, and approved August 28, 2006 (received for review May 31, 2006)
Because of species selectivity, HIV research is largely restricted to
in vitro or clinical studies, both limited in their ability to rapidly
assess new strategies to fight the virus. To prospectively study
some aspects of HIV in vivo, immunodeficient mice, transplanted
with either human peripheral blood leukocytes or human fetal
tissues, have been developed. Although these are susceptible to
HIV infection, xenoreactivity, and short infection spans, resource
and ethical constraints, as well as biased HIV coreceptor
tropic strain infection, pose substantial problems in their use.
Rag2?/??c?/?mice, transplanted as newborns with human CD34?
cells, were recently shown to develop human B, T, and dendritic
cells, constituting lymphoid organs in situ. Here we tested these
mice as a model system for HIV-1 infection. HIV RNA levels peaked
to up to 2 ? 106copies per milliliter of plasma early after infection,
and viremia was observed for up to 190 days, the longest time
followed. A marked relative CD4?T cell depletion in peripheral
blood occurred in CXCR4-tropic strain-infected mice, whereas this
was less pronounced in CCR5-tropic strain-infected animals. Thy-
mus infection was almost exclusively observed in CXCR4-tropic
strain-infected mice, whereas spleen and lymph node HIV infection
occurred irrespective of coreceptor selectivity, consistent with
respective coreceptor expression on human CD4?T cells. Thus, this
straightforward to generate and cost-effective in vivo model
closely resembles HIV infection in man and therefore should be
valuable to study virus-induced pathology and to rapidly evaluate
new approaches aiming to prevent or treat HIV infection.
mirror infection in humans. HIV is a human-specific virus, and
consequently laboratory rodents as mice or rats are not susceptible
to infection (1). Although non-human primates such as chimpan-
zees can be infected, they do not develop HIV-associated immu-
nodeficiency (2, 3), whereas sooty mangabeys, rhesus macaques,
and baboons are susceptible to only HIV-related simian immuno-
deficiency virus (4). Therefore, HIV research in non-human pri-
mates, although of importance, remains restricted by biological as
engineer rodents to become HIV targets (e.g., artificial expression
of human CD4, CCR5, or CXCR4) have largely failed, because,
even if infection in vitro was achieved, HIV replication in vivo was
limited or absent (1, 6, 7). Thus, substitute xenochimeric models
have been developed by transplanting immunodeficient mice with
hu) (10, 11). Both hu-PBL-SCID and SCID-hu mice sustain HIV
infection and replication in vivo. However, in hu-PBL-SCID mice
xenoreactivity and successive loss of human leukocytes limit infec-
tion to a relatively short time frame, and, possibly driven by
activation-induced CCR5 expression on human cells, infection is
skewed toward HIV strains with the respective coreceptor tropism
(12, 13). In SCID-hu mice, because of the transfer of human
ince the beginning of the HIV pandemic, research has been
hampered because of the lack of assessable animal models that
hematopoietic stem cell-containing tissue, HIV infection can be
human fetal organs is restricted for practical and ethical reasons,
and HIV pathology in these mice is mainly limited to the tissue
implants, with naı ¨ve T cells being preferentially susceptible to
CXCR4-tropic strain infection (1, 14, 15). Given these limitations
and the fact that no primary immune responses were generated,
hu-PBL-SCID and SCID-hu mice did not fully match the demand
for a small animal model that closely mirrors infection in humans.
Recently we found that injection of human cord blood
CD34?cells into newborn Rag2?/??c?/?mice leads to devel-
opment of human T, B, and dendritic cells, successive forma-
tion of primary and secondary lymphoid organs, and some in
vivo immune responses (16, 17). We here evaluated these mice
as a model system for both CXCR4- and CCR5-tropic HIV
and CD4?T Cells in Rag2?/??c?/?Mice. Depending on the use of
chemokine receptors in combination with CD4 for cellular entry,
HIV has been classified into CXCR4- or CCR5-tropic strains
(18–20). In humans, CD4?thymocytes and CD4?T cells broadly
express CXCR4, whereas CCR5 expression is restricted to a
fraction of mainly CD4?T memory cells (21). Similarly, most of
thymic and peripheral CD4?T cells in CD34?cord blood cell-
transplanted Rag2?/??c?/?mice expressed CXCR4, whereas
that almost exclusively displayed a memory phenotype as deter-
mined by CD45RO expression (Fig. 1 and data not shown). Thus,
de novo generated human CD4?thymocytes and T cells in CD34?
cord blood cell-transplanted Rag2?/??c?/?mice closely resemble
chemokine receptor expression patterns observed in humans and
therefore should be valid targets for HIV strains with respective
Long-Term and High-Titer CCR5- and CXCR4-Tropic HIV Infection in
Human CD34?Cord Blood Cell-Transplanted Rag2?/??c?/?Mice.
CD34?cord blood cell-transplanted Rag2?/??c?/?mice with a
mean human peripheral blood CD45?and CD4?cell chimerism of
Author contributions: M.G.M. and R.F.S. contributed equally to this work; S. Baenziger,
analyzed data; and S. Baenziger, M.G.M., and R.F.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
Abbreviations: PBL, peripheral blood leukocyte; hu-PBL-SCID mice, human PBL SCID mice;
SCID-hu mice, SCID human thymus?liver mice.
‡‡To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or roberto.
© 2006 by The National Academy of Sciences of the USA
www.pnas.org?cgi?doi?10.1073?pnas.0604493103 PNAS ?
October 24, 2006 ?
vol. 103 ?
no. 43 ?
29.4 ? 18.2% and 2.7 ? 3.0%, respectively (detailed analysis of
human cell engraftment in representative animals is given in Table
1, which is published as supporting information on the PNAS web
site), were infected i.p. with either CCR5-tropic YU-2 (n ? 15) or
CXCR4-tropic NL4-3 (n ? 19) HIV strains at 10–28 (mean 16.4 ?
6.7) weeks of age. Plasma levels of viral RNA were measured at
per milliliter of plasma, whereas thereafter viremia mostly stabi-
lized at lower levels and was maintained for up to 190 (YU-2) and
120 (NL4-3) days, the longest time followed (Fig. 2A). No HIV
of the latter ones, all four became HIV RNA-positive.
In a subgroup of both CCR5- and CXCR4-tropic strain-infected
and CD8?cell counts in peripheral blood over time: partial CD4?
T cell depletion occurred in three of four mice infected with
CCR5-tropic strains beyond 125 days of infection, whereas all
CXCR4-tropic strain-infected animals showed a more pronounced
relative peripheral blood CD4?T cell depletion already early in
infection, i.e., beyond day 25, after the initial rise of plasma HIV
RNA (Fig. 2B).
HIV strains recovered from either YU-2- or NL4-3-infected
chimeric Rag2?/??c?/?mice were fully functional because cocul-
tured mouse spleen cells propagated infection in primary human
peripheral blood leukocytes (PBL) in vitro (Fig. 6, which is pub-
lished as supporting information on the PNAS web site). Together
these findings indicate that CD34?cord blood cell-transplanted
Rag2?/??c?/?mice have an overall high susceptibility to both
CXCR4- and CCR5-tropic HIV and develop high and sustained
viral titers, comparable to titers found in HIV-infected individuals.
HIV Spreading in Lymphoid Organs of Human CD34?Cell-Transplanted
Rag2?/??c?/?Mice Resembles HIV Infection in Humans. To visualize
the major cell types productively infected with HIV in lymphoid
organs, serial sections were taken and stained with anti-HIV p24
capsid antigen and anti-human CD3, revealing that, as expected,
most infected cells were human T cells (Fig. 3A). Although CD68?
macrophages, CD11c?dendritic cells, and CD14?monocytic cells
were generated from human CD34?cells in mice (Fig. 3 B–D and
Table 1) (17), p24-expressing, and thus productively HIV-infected
non-T cells such as CD68?macrophages, were only occasionally
detected (Fig. 3 C and D), suggesting that non-T cells are a minor
source of HIV in this model.
From 18 days after infection, spleens and lymph nodes of both
NL4-3- and YU-2-infected animals contained p24?cells. In thymi,
however, p24 expression was detected in NL4-3-infected animals,
but only infrequently or not at all in YU-2-infected animals,
consistent with the expression of CXCR4 but not CCR5 on human
CD4?thymocytes (Figs. 1 and 4).
In both CXCR4- and CCR5-tropic HIV-infected mice, p24?
multinucleated giant cells were formed in lymph node and spleen
sections (Fig. 4B), a phenomenon previously reported in brain and
lymphoid tissues of HIV-infected individuals.
organs of human CD34?cell-transplanted Rag2?/??c?/?mice
closely resembles HIV infection in humans.
Human CD34?Cell-Transplanted Rag2?/??c?/?Mice Mount No or Very
Limited B Cell Responses to HIV Infection. To determine human B
cell responses in Rag2?/??c?/?mice beyond 3 weeks of infection,
plasma samples of n ? 23 were tested for total human IgG levels,
and n ? 25 were analyzed for HIV-specific IgM and IgG by
Western blot. As expected from our previous observations (17),
total IgG levels accounted for a mean of 0.136 g?liter (range
0.0295–0.5 g?liter), an amount ?80 times less than that found in
healthy human adults. Only one animal infected with YU-2 at 23
weeks of age (11% human CD45?cells in peripheral blood) and
analyzed at 42 days after infection produced a detectable IgG
response, but no measurable IgM response, against p34, gp41
(weak), p52, p58, and gp160 (Fig. 5).
We also performed a limited analysis of T cell responses: spleen
cells of each two NL4-3- and YU-2-infected animals killed at
52–115 days after infection were loaded in vitro with HIV-specific
peptides covering a broad range of HLA types, followed by flow
cytometric measurement of intracellular IFN-? production. How-
ever, no relevant IFN-? production could be detected, suggesting a
lack of T cell response, or a T cell response below the detection
cell-transplanted Rag2?/??c?/?mice mount no substantial or only
very occasional B cell responses and, likewise, insufficient T cell
To investigate infectious agents prospectively, suitable in vivo
laboratory models are needed. This poses a problem in research
depict representative receptor expressions (shaded histograms) and respective isotype controls (open histograms) on CD4?gated control human PBL and cells
isolated from tissue of a mouse at 16 weeks after birth and transplantation of CD34?cells.
CCR5 and CXCR4 expression on human CD4?cells in CD34?cell-transplanted Rag2?/??c?/?mice resembles expression patterns in humans. Histograms
www.pnas.org?cgi?doi?10.1073?pnas.0604493103Baenziger et al.
involving such human-specific pathogens as HIV. To this end,
xenochimeric models have been developed by transplanting immu-
nodeficient mice with cellular targets of HIV, i.e., either human
PBL (8, 9) or pieces of human fetal tissues such as liver and thymus
containing hematopoietic cells (SCID-hu) (10, 11); although suit-
able to study some aspects of HIV in vivo, both models are limited
by systematic issues (1). Human PBL transplanted into SCID mice
(hu-PBL-SCID) are activated within the xenogeneic environment,
T cells become successively anergic, and, because of lack of both
continuing hematopoiesis and appropriate hu-PBL maintenance,
the xenograft is nonfunctional within several weeks (1, 9). In
contrast, mice transplanted with human fetal liver and thymus
(SCID-hu) de novo generate and maintain human cells, especially
T cells, within the human thymus graft (10). However, SCID-hu
mice are laborious and costly to generate, and availability of
transplantable human fetal organs is restricted for practical and
ethical reasons, limiting broader use of these mice in laboratories.
Recently, major advances in generating xenogeneic mouse models
that continuously produce T cells and all other major cell types of
the human adaptive immune system in respective mouse organs
from human hematopoietic stem and progenitor cells were
achieved (reviewed in refs. 22–24). We demonstrated that newborn
human cord blood CD34?cell-transplanted Rag2?/??c?/?mice
develop de novo human T, B, and dendritic cells, form primary and
secondary lymphoid organs in situ, and mount some immune
responses upon tetanus toxoid vaccination or infection with EBV
(17). Similar results were obtained by transplanting NOD?
SCID?c?/?mice with either human cord blood or mobilized
peripheral blood CD34?cells (25–28).
We here establish cord blood CD34?cell-transplanted
Rag2?/??c?/?mice as a tool to study HIV infection and
pathogenesis in vivo. In this model system, CXCR4 and CCR5
expression on in vivo generated human CD4?T cells in
respective lymphoid organs closely resembles HIV coreceptor
RNA plasma levels in animals successfully infected with CCR5-tropic (YU-2; n ? 13) and CXCR4-tropic (NL4-3; n ? 18) HIV strains. Black triangles and connector
levels (copies per milliliter of plasma; right y axis) and relative CD4?and CD8?T cell levels (percentage of human CD45?blood cells; left y axis) in individual mice
over time (uninfected, n ? 2; YU-2- and NL4-3-infected, n ? 4 each), showing more pronounced CD4?T cell depletion in CXCR4-tropic infected animals.
Long-term and high-titer CCR5- and CXCR4-tropic HIV infection in human CD34?cord blood cell-transplanted Rag2?/??c?/?mice. (A) Quantitative HIV
Baenziger et al. PNAS ?
October 24, 2006 ?
vol. 103 ?
no. 43 ?
expression in humans (Fig. 1) (21). Accordingly, efficient in
vivo infection of human cells with both CXCR4-tropic (NL4-3)
and CCR5-tropic (YU-2) HIV strains was reliably achieved
(Figs. 2–4), leading to replication of fully functional HIV (Fig.
6). Although CXCR4-tropic strains infected all lymphoid
organs, CCR5-tropic strain infection was largely restricted to
extrathymic tissues (Fig. 4). Both viral strains led to long-term,
high-titer infection with an initial viremic peak of up to 2 ? 106
HIV RNA copies per milliliter of plasma, followed by a
chronic phase with somewhat lower RNA levels for up to 190
days, the longest time followed (Fig. 2).
In both CXCR4- and CCR5-tropic strain-infected animals,
productively HIV-infected cells, i.e., p24?cells, were mostly
CD3?and only occasionally non-T cells such as CD68?macro-
phages (Fig. 3 and data not shown). These findings are remi-
niscent of data acquired from ex vivo isolated lymphoid tissue of
HIV-infected individuals, where productive macrophage infec-
tion by both CXCR4- and CCR5-tropic HIV strains is very
infrequently observed (29–31) and likely increases only in end-
stage disease with occurrence of opportunistic infections (32).
Irrespective of coreceptor selectivity, HIV-infected, multinu-
cleated giant cells were formed (Fig. 4B), a phenomenon pre-
viously observed in brain and lymphoid tissues of HIV-infected
individuals, likely associated with high viral replication, spread-
ing infection, and CD4?T cell loss (33, 34).
Together, these findings are clearly distinct from previous
observations on HIV infection in hu-PBL SCID or SCID-hu
mice: although both CXCR4- and CCR5-tropic viruses infect
hu-PBL-SCID mice, CCR5-tropic viruses are more aggressive,
likely because of xenogeneic activation and CCR5 up-regulation
of transferred mature T cells, and, with consecutive graft failure,
infection is limited to few weeks; in contrast, SCID-hu mice
develop few human CCR5-carrying cells, and CXCR4-tropic
HIV replication is limited rather exclusively to the fetal tissue
grafts; furthermore, in both SCID models, by virtue of their
nature, no HIV dissemination to respective lymphoid organs
In humans, CCR5-tropic HIV strains are primarily transmit-
ted and dominate infection over extended times. In approxi-
mately half of late-stage HIV patients, CXCR4-tropic strains
emerge, either as cause or consequence of accelerated immu-
CD34?cell-transplanted Rag2?/??c?/?mice resembles
HIV infection in humans. (A) Representative p24-
stained thymus and lymph node sections of YU-2- and
NL4-3-infected mice analyzed at 37 and 23 days after
infection, respectively. No or very rare p24 staining is
observed in thymi of CCR5-tropic YU-2-infected ani-
mals. (B) Representative tissue section of spleen (Left)
and lymph node (Right) of a YU-2-infected animal at
52 days after infection showing multinucleated p24?
giant cells (enlarged Insets Right).
HIV spreading in lymphoid organs of human
cells such as CD68?macrophages. (A) Histologies show consecutive spleen sec-
days after infection. (B) Anti-human CD68 staining on paraffin-embedded ma-
terial. (C) Merged anti-CD68 (green), HIV-p24 (red), and DAPI (blue) staining of
cryoembedded spleen (6-?m sections) showing that p24?cells mainly localize in
white pulp areas (darker area; see also Fig. 4B), whereas CD68?cells mostly
localize at adjacent margins and red pulp areas. (D) Consecutive spleen cryosec-
tion staining and respective merged presentation showing a rare CD68 and p24
area with the double-positive cell. (B–D) Representative spleen sections from a
YU-2-infected animal 23 days after infection.
www.pnas.org?cgi?doi?10.1073?pnas.0604493103Baenziger et al.
nodeficiency, a matter still under debate (35). Previously, it was
demonstrated that CXCR4-tropic HIV strains led to pro-
nounced cell depletion in human fetal thymus grafts in SCID-hu
mice (14) and replicated more efficiently in primary human
thymocytes in vitro (36). Interestingly, CXCR4-tropic HIV in-
fection in CD34?cell-transplanted Rag2?/??c?/?mice, led to
more rapid blood CD4?cell loss than CCR5-tropic infection
(Fig. 2B). Thus, these findings suggest a causative rather than
secondary role of CXCR4 virus in accelerated immunodefi-
ciency, possibly in part by destruction of emerging thymocytes
An ultimate goal in the use of substitute xenogeneic small
animal models is the generation of robust human primary
adaptive immune responses to rapidly test potential new vaccine
candidates, a matter unfortunately not met so far in human
hematopoietic stem and progenitor cell-transplanted animals (1,
23, 24). However, some limited evidence for primary immune
responses in the xenogeneic setting were reported: (i) SCID-hu
mice were resistant to opportunistic infections, suggesting some
direct or indirect immune function of the human grafts (10); (ii)
we have demonstrated that human CD34?cell-transplanted
Rag2?/??c?/?mice generate some low-level specific IgG re-
sponses to tetanus toxoid upon repeated vaccination beyond 12
weeks of age, and, after EBV infection, some mice showed
inverted CD4:CD8 ratios, and CD8 T cells proliferated in vitro
when stimulated with autologous EBV-transformed target cells
(17); (iii) ovalbumin-specific human IgM and IgG responses
were observed in NOD?SCID?c?/?mice (27). Although in the
data presented here all HIV-infected Rag2?/??c?/?mice tested
produced human IgG at levels on average 80-fold lower than
healthy human adults, only 1 of 25 mounted a detectable
HIV-specific IgG response (Fig. 5). Similarly, although we had
limited data, we did not detect HIV-specific T cell responses, as
determined by IFN-? detection upon in vitro restimulation. Low
or absent immune responses to HIV might in part be due to
preferential destruction of virus reactive cells by HIV (38);
however, in this setting the more likely explanation might be
inefficient MHC selection of human T cells in the mouse thymus
and possible lack of some cross-reactive cytokines and chemo-
kines in the xenogeneic environment (16, 17, 23, 24). Thus,
generating primary HIV-specific immune responses remains a
challenge that might be solved by adding human MHC, cyto-
kines, chemokines, or stromal cell compounds to the recipient
In summary, the data presented here establish newborn
human CD34?cell-transplanted Rag2?/??c?/?mice (17) as a
tool to study HIV infection and pathogenesis in vivo. Upon
CCR5-tropic or CXCR4-tropic HIV challenge these mice de-
velop long-term, high-titer, and lymphoid organ disseminated
infection closely resembling HIV infection in humans. This
straightforward to generate, cost-effective, ethically unproblem-
atic, and easy to monitor in vivo model should thus be valuable
to study virus-induced pathology, as well as pharmacologic or
genetic approaches aiming to prevent or treat HIV infection.
Materials and Methods
Cord Blood Samples.Human cord blood was obtained with written
approval of the local ethical board. CD34?cells were enriched
by using immunomagnetic beads (Miltenyi Biotec, Bergisch
Gladbach, Germany) as described (17). Cells were either frozen
or transplanted immediately. Animals used in this study received
50,000–600,000 (mean 227,500 ? 140,000) CD34?selected cells.
Mice. Human CD34?cell-reconstituted mice were generated as
described in accordance with the guidelines of the Institute for
Research in Biomedicine (Bellinzona, Switzerland) animal fa-
cility (17). Rag2?/??c?/?mice were originally kindly provided by
M. Ito (Central Institute for Experimental Animals, Kawasaki,
HIV-1 Infection. Viral stocks were obtained by calcium phosphate
transfection (Promega, Madison, WI) of 293T cells with pNL4-3
or pYU-2. pYU-2 and pNL4-3 were obtained from B. H. Hahn
(University of Alabama at Birmingham, Birmingham, AL) and
M. A. Martin (Laboratory of Molecular Microbiology, National
Institute of Allergy and Infectious Diseases, Bethesda, MD),
respectively, through the National Institutes of Health AIDS
Research and Reference Reagent Program. Forty-eight hours
after transfection, virus was harvested, filtered (0.22 ?m), and
frozen at ?80°C until use. Mice were injected i.p. with 0.2 ml of
PBS containing either YU-2 or NL4-3 at a tissue culture-
infecting dose 50 of 2 ? 106. Infection was performed in a
Biosafety Level 3 laboratory in accordance with the Institute for
Research in Biomedicine and the Institute of Veterinary Bac-
teriology (University of Berne) animal facility guidelines.
Analysis. Plasma HIV RNA concentrations were determined by
Cobas Amplicor RT-PCR assay (Roche Diagnostics, Basel,
Switzerland). p24 antigen levels in cell culture supernatants were
quantified by ELISA as described (39). Human cell engraftment
in mice was measured by flow cytometry as described (17).
Anti-CXCR4 (12G5) and anti-CCR5 (2D7) antibodies were
from BD Pharmingen (San Diego, CA). Immunohistochemical
IgM responses is shown. (Left) Three-step dilution of plasma from an HIV-
infected patient (no antiviral therapy) showing full IgG seroconversion.
(Right) Undiluted plasma of an animal showing a measurable IgG response
against p34, gp41, p52, p58, and gp160 (lane A) and undiluted plasma of an
animal showing no response (lane B).
Humoral immune response in HIV-infected human CD34?cell-
Baenziger et al. PNAS ?
October 24, 2006 ?
vol. 103 ?
no. 43 ?
stainings were performed in an automated Ventana Discovery
Module (Ventana, Strasbourg, France). Paraffin sections were
incubated in a 1:5 dilution of mouse monoclonal antibody to
HIV-1 p24 (clone Kal-1; DAKO Diagnostics, Zug, Switzerland).
Staining for CD3 (SP7; Lab Vision, Fremont, CA) was per-
formed according to Ventana protocols. Immunofluorescent
stainings and microscopy were done as described (40). Briefly,
diluted 1:10 (DAKO Diagnostics); (ii) 5 ?g?ml Cy3-conjugated
goat anti-mouse Ig (Amersham, Little Chalfont, U.K.); (iii) 5
?g?ml mouse IgG (Sigma, Buchs, Switzerland); (iv) FITC-
conjugated mouse anti-human CD68 (1:20; DAKO Diagnos-
5 ?g?ml Alexa Fluor 488-conjugated goat anti-rabbit Ig (Mo-
lecular Probes, Leiden, The Netherlands) and 0.5 ?mol?liter
DAPI (Sigma). To assess nonspecific binding, tissue from un-
transplanted and?or uninfected transplanted mice was stained as
LC-Partigen plates (Behring, Marburg, Germany). HIV-specific
Western blot analysis was done by using a commercially available
kit (New Lav BlotI; Bio-Rad, Hercules, CA). HIV-specific T cell
responses were evaluated as previously described (41): briefly,
106splenocytes from HIV-infected and uninfected mice were
pulsed with pools of overlapping peptides covering the HIV gag
[National Institutes of Health HIV-1 Consensus B Gag (15-mer)
Peptides Set: pool I, catalog nos. 7872–7933; pool II, catalog nos.
7934–7994] or HIV nef [National Institutes of Health HIV-1
Consensus B Nef (15-mer) Peptides Complete Set, catalog no.
5189] or with a 17-peptide pool [National Institutes of Health
catalog nos. 7891, 7920, 7926, 7945, 7976, 7980, 7983 (gag), 5507,
5527, 5544, 5576 (pol), 6287, 6412, 6420 (env), 5172, 5183 (nef),
and 6078 (vpr)] at a concentration of 2 ? 10?6M per peptide.
One hour later, brefeldin was added for 5 h. Splenocytes were
stained with mAb against CD4, CD8, and intracellularly against
IFN-? by using the Cytofix?Cytoperm permeabilization?fixation
kit (BD Pharmingen).
We thank the staff of Ospedale San Giovanni (Bellinzona, Switzerland),
for cord blood collection. We thank Friederike Burgener and Helen
Steinmann for technical assistance in processing histopathology tissues.
HIV peptides were generously provided by the National Institutes of
Health AIDS Research and Reference Reagent Program. This work was
supported in part by the EMDO Foundation (R.F.S.), the Baugarten-
stiftung (R.F.S.), Swiss National Science Foundation Grants 3100A0-
108352?1 (to R.F.S. and M.G.M.) and 3100A0-102221 (to M.G.M.), and
the Bill and Melinda Gates Foundation (M.G.M.).
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