JOURNAL OF VIROLOGY,
Copyright © 2000, American Society for Microbiology. All Rights Reserved.
July 2000, p. 6087–6095 Vol. 74, No. 13
Simian Immunodeficiency Virus Rapidly Penetrates the Cervicovaginal
Mucosa after Intravaginal Inoculation and Infects
Intraepithelial Dendritic Cells
JINJIE HU,1† MURRAY B. GARDNER,2,3AND CHRISTOPHER J. MILLER1,2,4*
California Regional Primate Research Center,1Center for Comparative Medicine,2Department of Medical Pathology,
School of Medicine,3and Department of Pathology, Microbiology and Immunology, School of
Veterinary Medicine,4University of California, Davis, California 95616
Received 1 February 2000/Accepted 31 March 2000
Despite recent insights into mucosal human immunodeficiency virus (HIV) transmission, the route used by
primate lentiviruses to traverse the stratified squamous epithelium of mucosal surfaces remains undefined. To
determine if dendritic cells (DC) are used by primate lentiviruses to traverse the epithelial barrier of the
genital tract, rhesus macaques were intravaginally exposed to cell-free simian immunodeficiency virus
SIVmac251. We examined formalin-fixed tissues and HLA-DR?-enriched cell suspensions to identify the cells
containing SIV RNA in the genital tract and draining lymph nodes within the first 24 h of infection. Using
SIV-specific fluorescent in situ hybridization combined with immunofluorescent antibody labeling of lineage-
specific cell markers, numerous SIV RNA?DC were documented in cell suspensions from the vaginal
epithelium 18 h after vaginal inoculation. In addition, we determined the minimum time that the SIV inoculum
must remain in contact with the genital mucosa for the virus to move from the vaginal lumen into the mucosa.
We now show that SIV enters the vaginal mucosa within 60 min of intravaginal exposure, infecting primarily
intraepithelial DC and that SIV-infected cells are located in draining lymph nodes within 18 h of intravaginal
SIV exposure. The speed with which primate lentiviruses penetrate mucosal surfaces, infect DC, and dissem-
inate to draining lymph nodes poses a serious challenge to HIV vaccine development.
Development of a vaccine to prevent transmission of human
immunodeficiency virus (HIV) in heterosexuals remains one of
the most pressing challenges facing modern medicine. Vaccine
development efforts are likely to advance only when the biol-
ogy of heterosexual HIV transmission is better understood. In
order for HIV to be transmitted to women through vaginal
intercourse, the virus must cross the epithelial barrier of the
genital tract. Studies in the simian immunodeficiency virus
(SIV)-rhesus macaque model have demonstrated that removal
of the cervix and upper genital tract does not alter susceptibil-
ity to atraumatic vaginal SIV inoculation (18), so target cells in
the vaginal mucosa are the only known requirement for genital
SIV transmission. It has been shown that unidentified SIV-
infected cells are present in the lamina propria of the cervico-
vaginal mucosa 48 h after vaginal inoculation (32) and because
putative dendritic cells (DC) were similarly located in adjacent
tissue sections, the researchers concluded that DC were target
cells in vaginal SIV transmission. It has recently been shown
that SIV-infected T cells and macrophages are in the organized
lymphoid tissue of the tonsils of rhesus macaques 96 h after
tonsillar SIV inoculation (33) and that SIV infects activated
and quiescent T cells in the cervix at 72 h after vaginal inocu-
lation (PI) (35). Despite these insights into mucosal HIV trans-
mission, the route used by primate lentiviruses to traverse the
stratified squamous epithelium of mucosal surfaces remains
The gross and histologic anatomy of the genital tracts of
women and female rhesus macaques is very similar. In both
species, the mucosa of the vagina is composed of a stratified
squamous epithelium and an underlying highly vascular lamina
propria. The architecture of the ectocervix is similar to that of
the vagina, while the endocervix (which is not normally ex-
posed to material in the vaginal lumen) is composed of a
simple columnar epithelium covering a highly vascular lamina
propria. M cells have not been demonstrated in the vagina or
cervix; the intraepithelial antigen-presenting cells in the lower
female genital tract are the CD1a?intraepithelial DC or Lang-
erhans cells (LC) (4, 21).
DC are potent antigen-presenting cells found in all tissues,
but they are especially common in lymphoid organs. Many DC
can be identified by expression of a 55-kDa, intracytoplasmic
actin-bundling protein, designated fascin (P55). The DC des-
ignation includes both mature and immature DC. LC are a
type of immature, major histocompatibility complex (MHC)
class II?, and CD4?DC that reside in stratified squamous
epithelia and characteristically express CD1a and frequently
coexpress P55 (7). LC are located within the ectocervical and
vaginal squamous epithelium of humans (4). These cells are
also abundant in the squamous epithelia of the rhesus ma-
caque lower genital tract, and they extend dendritic processes
to the lumen of the vagina (21, 25). LC are common in the skin,
where upon stimulation, they migrate to the draining lymph
node in as little as 30 min, with maximal migration generally
occurring within 24 h of stimulation (1, 8, 9, 11, 12, 30, 34). In
mice, antigen absorption in the vagina occurs via CD4?LC
(28), and these cells then migrate and are detectable in the
draining lymph node as early as 4 h after application of antigen
to the vaginal mucosa (27).
To determine if DC are used by primate lentiviruses to
traverse the epithelial barrier of the genital tract, rhesus ma-
caques were intravaginally exposed to cell-free SIV. Detailed
* Corresponding author. Mailing address: Virology and Immunol-
ogy Unit, California Regional Primate Research Center, University of
California, Davis, CA 95616. Phone: (530) 752-8584. Fax: (530) 752-
2880. E-mail: firstname.lastname@example.org.
† Present address: Laboratory of Molecular Microbiology, National
Institute of Allergy and Infectious Diseases, Rockville, MD 20852.
studies were conducted to identify the infected cells in the
genital tract and draining lymph nodes within the first 24 h of
infection. In addition, we determined the minimum time that
the SIV must remain in contact with the genital mucosa for the
virus to move from the vaginal lumen into the mucosa. We now
show that SIV enters the vaginal mucosa within 60 min of
intravaginal exposure, infecting primarily intraepithelial DC
and that SIV-infected cells are located in draining lymph nodes
within 18 h of intravaginal SIV exposure. The speed with which
primate lentiviruses penetrate mucosal surfaces, infect DC,
and disseminate to draining lymph nodes poses a serious chal-
lenge to HIV vaccine development.
MATERIALS AND METHODS
Animals and virus stocks. All animals used in this study were colony-bred,
multiparous female rhesus macaques (Macaca mulatta) from the California Re-
gional Primate Research Center. The animals were housed in accordance with
American Association for Accreditation of Laboratory Animal Care standards.
The investigators adhered to the “Guide for the Care and Use of Laboratory
Animals” prepared by the Committee on Care and Use of Laboratory Animals
of the Institute of Laboratory Resources, National Resource Council. When
necessary, the animals were immobilized with ketamine. Prior to use, the animals
were negative for serum antibodies to HIV type 2, SIV, type D retrovirus, and
simian T-lymphotropic virus type 1. The uncloned and pathogenic SIVmac251
stock used in these study was produced by short-term culture in rhesus macaque
peripheral blood mononuclear cells (PBMC) and had a titer of 10550% tissue
culture infective doses (TCID50) per ml (19). SIVmac251 is dualtropic, replicat-
ing in both T-cell lines and primary rhesus macaque macrophages in vitro (19).
The virus was carefully instilled into the vaginal canal with a 1-ml tuberculin
syringe (with no needle) to ensure no damage to the vaginal epithelium, as was
confirmed by histology (Fig. 1).
Sample collection. An overview of the steps in processing tissues from the
genital tract is shown in Fig. 1. At necropsy, the vagina was dissected free and
opened by cutting along the longitudinal axis; 5-mm-wide pieces of tissue were
taken from the vaginal fornix, midcanal, and introitus by making two parallel
transverse cuts through the entire vaginal wall. Of these tissue samples, com-
prising approximately 10% of the total surface area of the vaginal mucosa, half
were fixed in 10% buffered formalin and the other half were quick-frozen for
PCR analysis of SIV proviral DNA. The formalin-fixed tissue was embedded in
paraffin, sectioned every 6 ?m, and examined by in situ hybridization (ISH) for
cells containing SIV RNA (SIV RNA?cells). The remaining fresh tissue (ap-
proximately 90% of the total surface area of the vaginal mucosa) was placed
aseptically into RPMI 1640 medium, supplemented with antibiotics, and kept on
ice until processing.
ISH. ISH for SIV RNA was performed with digoxigenin-labeled riboprobes as
previously described (7) with some modifications. The riboprobe cocktail in-
cluded seven in vitro transcription products that span most of the SIVmac239
genome. To detect the bound riboprobe, the slides were incubated with perox-
idase-conjugated anti-digoxigenin sheep antisera. The peroxidase signal was en-
hanced using the indirect Tyramide Signal Amplification kit (NEN Life Science
Products, Boston, Mass.) and Nitro Blue Tetrazolium (NBT)-5-bromo-4-chloro-
3-indolylphosphate (BCIP) as the substrate. In addition, ISH was performed on
selected tissue sections using35S-labeled SIV riboprobes as previously described
(2) with some modifications. Radioactive probes had a specific activity of 3 ? 108
cpm/?g by in vitro transcription labeling of the SIV gag and env genes. The
hybridization solution (24) contained radiolabeled SIV probes at a total concen-
tration of 8 ? 106cpm/50 ?l. Fifty microliters of riboprobe cocktail in hybrid-
ization buffer was layered over each tissue section. The slides were coated with
LM-1 autoradiographic emulsion (Amersham) and allowed to develop at 4°C for
4 to 10 days. Controls for ISH included (i) SIV-infected and uninfected trans-
formed human T-cell lines, (ii) matched tissues from SIV-uninfected rhesus
monkeys, (iii) matched tissues from SIV-infected rhesus monkeys with high virus
loads (positive control), (iv) tissue sections (or cytospin slides) hybridized with
SIV sense riboprobes, and (v) omission of probe. Using this ISH procedure, we
consistently detected SIV RNA expression in T-cell lines or primary rhesus
macaque PBMC beginning at 12 h after initiation of in vitro infection; however,
SIV RNA?cells were not detected earlier in the inoculated cultures (data not
shown). Thus, the ISH technique used in these studies detected productively
Combined ISH and immunohistochemistry. In paraffin-embedded tissues,
SIV-infected cell types were identified with a combination of radioisotope ISH
and immunohistochemistry. Following ISH with35S-labeled riboprobes, the sec-
tions were washed and immunostained with the appropriate monoclonal anti-
bodies (MAbs). Anti-CD3 (Dako Corporation, Carpenteria, Calif.) was used to
detect T cells; Ham-56 (Dako) was used to detect macrophages. The antibodies
were detected using the ABC protocol with AEC as the chromogen (Vector
Labs, Burlingame, Calif.). The slides were coated with autoradiographic emul-
sion and developed as described above.
Processing of the fresh tissues. To separate vaginal epithelium from the
lamina propria, fresh tissue samples were cut into 1-cm2pieces, placed into
RPMI 1640 medium containing 1.2 U of dispase II (Sigma Chemical Co., St.
Louis, Mo.) per ml for 90 min at 37°C, and agitated in a shaking water bath. The
epithelium was removed manually with fine forceps and placed in digestion
medium containing 100 U of DNase per ml and 0.01% trypsin for 1 h at 37°C
using a magnetic stirring bar to agitate the suspension. Large pieces of tissue
were removed, and cells were collected from the supernatant by centrifugation
with Lymphocyte Separation Medium (LSM; Organon-Teknika, Durham, N.C.).
The lamina propria was sliced into very thin pieces and incubated overnight in
FIG. 1. Procedure for processing fresh genital tract tissues from animals at necropsy. Note that approximately 90% of the vaginal mucosa was used to produce cell
suspensions, while 10% of the tissue was processed for histology or quick-frozen for PCR analysis. Abbreviations: IHC, immunohistochemistry; ISH/IHC, combination
of ISH and IHC; IFA, immunofluorescent antibody labeling.
6088 HU ET AL.J. VIROL.
complete RPMI 1640 medium with 50 ?M ?-mercaptoethanol, 0.5 mg of colla-
genase (type II; Sigma) per ml, 0.1 mg of DNase per ml, and 20 ?g of cipro-
floxacin HCl per ml in a shaking water bath at 37°C. After vigorous pipetting of
the tissue pieces, the supernatant was strained through a 100-mesh stainless steel
sieve, and the resulting cell suspension was washed and layered over a discon-
tinuous Percoll (Sigma) density gradient (75 and 40% [vol/vol]) and centrifuged
at 2,000 rpm for 30 min. Cells at the interface between the 40 and 75% Percoll
layers were collected, and separate aliquots of the cell suspensions from the
vaginal lamina propria and stratified squamous epithelium were frozen for PCR
or used to prepare cytospin slides. The bulk of the cell suspensions was stained
with anti-HLA-DR MAb and further purified by cell sorting (see below). Be-
cause of the relatively small size of the cervix, no attempt was made to separate
the cervical epithelium from the underlying lamina propria. A 5-mm-wide piece
of tissue was taken from the cervix so that both endocervix and ectocervix were
included in the fixed sample. Cell suspensions were produced from the remaining
90% of the cervix as described above for the vaginal lamina propria. For each of
the cell suspensions produced (vaginal epithelium, vaginal lamina propria, and
cervix), approximately 10% of the cells were frozen for PCR analysis, 10% were
used to make cytospin slides of the unsorted cells, and the remainder was sorted
to produce cell suspensions enriched for cells expressing high levels of MHC
class II molecules, as described below.
Flow cytometric sorting of HLA-DR (hi) cells. To enrich for DC, the bulk of
the cell suspensions from vaginal epithelium, vaginal lamina propria, and cervix
were stained with phycoerythyrin-conjugated anti-HLA-DR MAb (Becton Dick-
inson Corporation, San Jose, Calif.). The cells that stained very brightly for the
MHC class II molecule HLA-DR [HLA-DR (hi) cells] were concentrated using
the enrich mode of a FACS Vantage cell sorter (Becton Dickinson). The result-
ing HLA-DR (hi)-enriched cell suspensions (HLA-ECS) were divided; the bulk
of each suspension was used to produce cytospin slides, but approximately 10%
of each suspension was frozen and analyzed by PCR. DC, particularly CD1a?
LC, were the most common cell type (approximately 80%) in the HLA-ECS
cytospin slides of vaginal epithelium; however, CD3?T cells were also found in
this enriched cell population. In the HLA-ECS cytospin slides of the vaginal
lamina propria, P55?DC and CD1a?LC (see below) were the most common
cell types (approximately 60%). In addition, however, CD3?T cells and macro-
phages were present at much higher frequencies on HLA-ECS cytospin slides of
the vaginal lamina propria than on the HLA-ECS cytospin slides of the epithe-
lium. In the HLA-ECS cytospin slides of cervix, more than 50% of the cells were
LC and DC. Many CD3?T cells were also present in the cervical HLA-ECS
cytospins. In the cytospin slides of slides of the vaginal epithelium, a few epithe-
lial cells were also present, while in the slides from the cervix and vaginal lamina
propria, both epithelial and stromal cells were present in low numbers.
PCR analysis. Nested PCR was carried out on genomic DNA from PBMC,
frozen tissues, and cell suspensions using SIV gag-specific primer pairs as previ-
ously described (7). The frozen tissues were cut into 2- to 5-mm3blocks and
digested with 200 ?g of proteinase K per ml in PCR lysis buffer. The genomic
DNA, isolated using the QIAmp DNA isolation kit (Qiagen, Chatsworth, Calif.),
was quantitated by spectrophotometry, and 0.6 ?g of DNA (equivalent to 105
cells) was in each aliquot used for PCR, and 20 to 40 aliquots of DNA from each
tissue sample were analyzed. In addition, we tested at least 106cells from both
sorted and unsorted cell suspensions for the presence of proviral DNA.
Combined ISH and immunofluorescent antibody labeling. The immunophe-
notype of SIV RNA?cells in cytospin slides was determined by ISH combined
with immunofluorescent antibody staining. The slides were incubated overnight
with the riboprobe cocktail at 52°C and then incubated with peroxidase-conju-
gated anti-digoxigenin sheep antisera. MAbs for cell markers (Table 1) were
applied to the cytospin slides, as described previously (7). An anti-P55 (fascin)
MAb (Dako Inc.) or affinity-purified anti-CD3 rabbit sera (Dako Inc.) identified
DC or T cells, respectively. Bound primary reagents were detected by an anti-
mouse immunoglobulin G (IgG) subclass-specific Texas red conjugate or an
anti-rabbit IgG-biotin conjugate. LC were identified with a biotinylated anti-
CD1a MAb (Becton-Dickinson), and bound MAb was detected with streptavi-
din-Texas red (Vector Labs). Bound riboprobe was detected with a Direct Tyra-
mide Signal Amplification FITC kit (NEN Life Science Products). Care was
taken to ensure that reagents used to detect the SIV riboprobe or the cell
markers did not react with the anti-HLA-DR MAb that was used to sort the cells
Identification of SIV RNA?cells in the lower genital tract
and draining lymph nodes 18 and 24 h after intravaginal SIV
exposure. Four adult female rhesus macaques were inoculated
intravaginally with cell-free SIVmac251. Two of these animals
were euthanized 18 h PI, and the other two animals were
euthanized 24 h PI. Proviral SIV gag sequences were detected
by nested PCR in samples of vagina and cervix from all animals
(Table 2). In addition, the mesenteric and iliac lymph nodes of
animal 23319, culled at 18 h PI, contained detectable SIV
provirus; while animal 24294, culled at 24 h PI, also had de-
tectable, but low-level, provirus in inguinal, cervical, and mes-
enteric lymph nodes, palatine tonsil, and ileum.
By ISH on formalin-fixed tissues, we detected SIV RNA?
cells in the vaginal epithelium and lamina propria of all four
animals at both 18 and 24 h PI (Fig. 2). In general, the SIV
RNA?cells were located in the lower levels of the vaginal
epithelium. In serial sections from the same blocks, HLA-DR?
cells were located in the same position within the epithelium
(not shown). SIV RNA?cells were present in the iliac lymph
nodes of all three animals examined. In the iliac and obturator
lymph nodes of animal 24659, culled at 18 h PI, SIV RNA?
cells were found in the subcapsular sinus (Fig. 2). This finding
indicates rapid dissemination of SIV RNA?cells from the
genital tract through lymphatic vessels to the draining lymph
nodes. However, in all tissues examined, the frequency of SIV
RNA?cells detected by ISH was low (five or less SIV RNA?
cells per tissue section). Combined ISH-immunohistochemis-
try on the formalin-fixed tissues demonstrated that the major-
ity of the SIV RNA?cells in the cervicovaginal epithelium and
lamina propria were not T cells or macrophages (Fig. 3). Rare
SIV-infected macrophages were found in the paracortex of the
iliac lymph node from one animal at 18 h PI (Fig. 3E).
Because there are only 5 to 20 LC in most histologic sections
of ectocervix or vagina (21), we reasoned that the probability of
successfully double labeling an infected LC by standard ISH on
TABLE 1. MAbs used to determine the immunophenotype of cells in cytospins from rhesus macaque tissues
SpecificityDetection systemSource of primary antibody
Anti-CD3 (rabbit antisera)
Anti-CD68 (mouse IgG1 MAb)
Anti-CD1a (biotinylated mouse MAb)
Anti-p55 (mouse IgG1 MAb)
Horse anti-rabbit IgG-Texas red
Horse anti-mouse IgG1-Texas red
Biotinylated horse anti-mouse IgG1-streptavidin-Texas red
aNote that the anti-HLA-DR antibody used to sort the cells is a mouse IgG2a molecule.
TABLE 2. SIV infection in genital tract as determined by PCR
DNA isolated from cell
aResult of nested PCR for SIV gag on DNA extracted from frozen tissue.
bResult of nested PCR for SIV gag on DNA extracted from the cells isolated
cLow, ?10% of the DNA aliquots tested were positive by PCR; Neg., the
DNA aliquots tested were negative by PCR; Mod., ?10 and ?40% of the DNA
aliquots tested were positive by PCR; ND, not done.
VOL. 74, 2000 DENDRITIC CELLS ARE TARGETS IN VAGINAL SIV TRANSMISSION6089
paraffin-embedded sections was low. In addition, we have
never achieved satisfactory staining in formalin-fixed tissues
with available DC-specific antibodies. Thus, we elected to
make cell suspensions from the bulk of the fresh genital tract
and enrich those suspensions by cell sorting for HLA-DR (hi)
cells, including DC and LC. Mononuclear cell suspensions
were prepared from fresh samples of vagina and cervix of three
animals culled at 18 or 24 h PI (Table 3 and Fig. 1). The
majority of the cell suspension from each tissue (vaginal epi-
thelium, vaginal lamina propria, and cervix) was then enriched
for cells expressing high levels of MHC class II HLA-DR
molecules by fluorescence-activated cell sorting. These HLA-
DR (hi)-enriched cell suspensions (HLA-ECS) were centri-
fuged onto microscope slides using a cytocentrifuge. Cytospin
slides were also prepared from the unsorted cell suspensions of
each tissue. The cytospin slides of the HLA-ECS had dramat-
ically higher numbers of SIV RNA?cells than the unsorted
samples (Table 3). Because SIV RNA?cells were rare in the
cytospin slides of unsorted cell suspensions, we undertook
combined ISH-immunofluorescent antibody double-label stud-
ies only on the cytospin slides produced from the HLA-ECS
(Fig. 4). In the HLA-ECS of the vaginal epithelium, 50 to 65%
of SIV RNA?cells were p55?DC and 65 to 90% of SIV
RNA?cells were CD1a?LC. No SIV-infected CD3?T cells
or macrophages were detected in any cytospin slides from the
vaginal epithelium. In the HLA-ECS cytospin slides of the
vaginal lamina propria, 30 to 50% of SIV RNA?cells were
P55?DC and 60 to 80% of SIV RNA?cells were CD1A?LC.
SIV-infected macrophages were not detected, but SIV-in-
fected CD3?T cells were 10% of the SIV RNA?cells in the
vaginal lamina propria of animal 24659. Because of the small
size of the cervix, no attempt was made to separate the epi-
thelium and lamina propria (Fig. 1). Thus, the cytospin slides
prepared from the cervix consisted of mononuclear cells from
both sites and these slides contained numerous SIV-infected
cells. In the HLA-ECS cytospin slides of the cervix, 50 to 65%
of the SIV RNA?cells were DC and 60% of the SIV RNA?
cells were LC. SIV-infected T cells and macrophages were not
detected in the HLA-ECS cytospin slides of cervix obtained at
18 and 24 h PI.
Minimum length of vaginal SIV exposure required for mu-
cosal penetration and establishment of systemic infection. To
confirm the rapid penetration of SIV into the genital tract,
female rhesus macaques were inoculated intravaginally with 1
ml of SIVmac251 (105TCID50). The inoculum was allowed to
remain undisturbed for a defined period of time (15, 30, or 60
min), and then the vaginal cavity was gently lavaged with 250
ml of dilute acetic acid solution (pH 2.8) (white vinegar; Heinz
Inc.). This pH has been shown to inactivate HIV (13) and SIV
(C. J. Miller, unpublished data) in vitro. The volume and pH of
the lavage were sufficient to inactivate and flush the inoculum
from the vagina. The experimental design involved five ex-
periments as shown in Fig. 5. Animals in experiment A were
exposed once to SIV and then the inoculum was lavaged from
the vagina. Animals in experiment B were exposed once to SIV
and then the inoculum was lavaged from the vagina. After 4 h,
the animals were again inoculated with SIV and then the sec-
ond inoculum was flushed from the vagina. In order to control
for the possibility that the prior vinegar lavage increased sus-
ceptibility to a subsequent SIV exposure, experiment C was
performed. In experiment C, the vaginal cavity of two mature
female rhesus macaques was gently lavaged with 250 ml of
dilute acetic acid. Four hours later, the animals were inocu-
lated intravaginally with 1 ml of SIVmac251. The inoculum was
allowed to remain undisturbed for 15 min, and then the vaginal
cavity was gently lavaged with 250 ml of dilute acetic acid. As
positive controls for vaginal transmission, two groups of ani-
mals were intravaginally inoculated with the SIVmac251 stock
and the inoculum was allowed to remain undisturbed in the
vagina. Six animals were intravaginally exposed once to SIV
(experiment D), while eight animals were exposed once to SIV
and then after 4 h the animals were again inoculated with SIV
(experiment E). Blood samples were collected at 7, 14, 28, 56,
86, and 116 days PI, and the infection status of the inoculated
animals was assessed by virus isolation (19) nested PCR to
detect provirus in PBMC (described above), and a SIV-specific
antibody enzyme-linked immunosorbent assay to detect anti-
SIV serum antibodies (22). Based on the results of these ex-
periments (Table 4), we conclude that SIV can penetrate from
the vaginal lumen into the vaginal mucosa within 30 to 60 min
FIG. 2. SIV-infected cells detected by ISH in tissues of rhesus macaques 18 h
after vaginal SIV inoculation. (A) SIV RNA?cells (arrows) in the vaginal
mucosa (animal 24659, 18 h PI). Note that one cell is in the middle layer of the
epithelium and the other is in the lamina propria. (B) SIV RNA?cell in the
subcapsular sinus of the iliac lymph node, which drains the vagina (animal 24659,
18 h PI). Two double-headed arrows denote the subcapsular sinus of the lymph
node. ISH was done with NBT-BCIP as the chromogen and nuclear fast red
6090 HU ET AL.J. VIROL.
By using cytospin slides of HLA-ECS, our analysis focused
on the antigen-presenting cells of the genital tract, including
immature CD1a?LC and mature P55?DC. Thus, we were
able to definitively demonstrate that DC in general, and LC in
particular, are target cells for primary SIV infection in the
vaginal epithelium and lamina propria of rhesus macaques.
These infected DC were detected in the first 18 to 24 h after
vaginal SIV exposure. However, even in the HLA-ECS cyto-
spin slides, the two types of DCs we identified accounted for
only 50 to 90% of the SIV RNA?cells in the samples. Thus, a
considerable number of cells other than DCs are infected in
FIG. 3. SIV-infected cells in tissues of rhesus macaques as detected by combined ISH and immunohistochemistry. SIV RNA?cells in formalin-fixed sections of
vagina at 24 h PI are shown. (A) SIV RNA?cells (arrows) in the vaginal epithelium and uninfected CD3?T cells (red, some are denoted by arrowheads) in the vaginal
epithelium and lamina propria (animal 24294, 24 h PI). (B) Higher-magnification view of panel A. Note that the SIV RNA?cells are not CD3?T cells. The solid black
line demarcates the basal lamina. (C) SIV RNA?cell (arrow) in the basal layer of the vaginal epithelium and uninfected HAM 56?macrophages (red, some are denoted
by arrowheads) in the vaginal epithelium and lamina propria (animal 24294, 24 h PI). (D) Higher-magnification view of panel C. Note that the SIV RNA?cell is not
a macrophage and the macrophages are not SIV RNA?cells. The solid black line demarcates the basal lamina. (E) SIV RNA?macrophage (arrow) in the paracortex
of an iliac lymph node (animal 24294, 24 h PI). Note in panels A and C that the vaginal epithelium is intact at 24 h PI, consistent with the atraumatic virus inoculation
procedure. ISH using35S-labeled SIV riboprobes combined with immunohistochemistry using AEC as the chromogen and Meyer’s hematoxylin counterstain were used.
VOL. 74, 2000DENDRITIC CELLS ARE TARGETS IN VAGINAL SIV TRANSMISSION 6091
the first 24 h of SIV exposure. Aside from a single animal in
which SIV-infected CD3?T cells were identified in the vaginal
lamina propria HLA-ECS, we were unable to immunopheno-
type the SIV-infected cells that were not LC or DC in the
limited sample material available. The unidentified SIV RNA?
cells consisted of two morphologic types, small, round cells
with scant cytoplasm and round nuclei or medium-sized, irreg-
ularly round to oval cells with abundant cytoplasm and kidney-
shaped nuclei. Based on morphologic criteria, these other cell
types are lymphocytes and macrophages, respectively. The
finding of SIV RNA?macrophages in the iliac lymph node at
18 h PI (Fig. 3E) also supports this interpretation. Thus, we
conclude that the initial target cells for SIV during vaginal
transmission include large numbers of LC, DC, T lymphocytes,
and macrophages. It has been shown that vaginal LC take up
antigen from the vaginal lumen and then migrate to the T-cell-
rich paracortex of the draining lymph nodes (27). The data
presented support the conclusion that intraepithelial DC are
critical initial target cells after intravaginal SIVmac251 inocu-
lation. DC may play a similar role in heterosexual transmission
of HIV to women.
Our ability to document DC infection immediately after
mucosal SIV exposure contrasts with the results of several
other groups (32, 33, 35). The different results can be explained
largely on the basis of methodological differences in the stud-
ies. The kinetics of DC antigen uptake from the vagina and
subsequent migration to draining lymph node (27, 28) led us to
focus our analysis on events occurring in the genital mucosa
within 24 h of SIV exposure. The ISH assay used in this study
was not able to detect SIV in T-cell lines until 12 h after in vitro
infection. Thus, the time points after inoculation that we chose
to examine were an attempt to balance the need to allow viral
RNA expression to reach detectable levels and the need to
obtain the tissue samples before substantial DC migration oc-
curred. The cell sorting strategy, designed to enrich our sam-
FIG. 4. Immunophenotypic characterization of SIV RNA?cells in HLA-ECS cytospin slides of vaginal epithelium at 18 h PI. Panels A to C show A single,
high-magnification field in a cytospin slide from animal 23319 (18 h PI). (A) Viewed through an appropriate band-pass filter, SIV RNA?cells were detected (green,
arrows), and some of these cells had dendritic processes (arrowhead). (B) Most cells express p55?(red), a marker for DC. (C) Viewed through a double band-pass
filter, all the SIV RNA?cells in this field are p55?DC (arrows). Panels D to F show a single, high-magnification field in a cytospin slide of vaginal epithelium from
animal 23319 (18 h PI). (D) SIV RNA?cells (green, arrows). (E) Most cells (red) express CD1a, a marker for LC. (F) Viewed through a double band-pass filter, all
the SIV RNA?cells in this field are CD1a?LC (arrows). Combined ISH (digoxigenin-labeled riboprobe, Tyramide-FITC detection system) and immunofluorescent
antibody labeling of cell markers (Texas red detection system) were used.
TABLE 3. Percentage of SIV RNA?cells in cytospin slides
of cell suspensions from the genital tract
% SIV RNA?cellsa
Unsorted Sorted Unsorted Sorted
aPercentage of SIV RNA?cells in the cytospin slides of cell suspensions from
the genital tract. To estimate the percentage of SIV RNA?cells, at least 200 cells
in each preparation were analyzed and the number of SIV RNA?cells was
determined by ISH.
bSample was mixed mononuclear cells.
cSample was HLA-DR (hi)-enriched cell suspension (HLA-ECS).
6092HU ET AL.J. VIROL.
FIG. 5. Overview of the experimental design used to determine the minimum amount of contact time required for SIV to pass from the vaginal lumen into the
vaginal mucosa. (A) In experiment A, the initial study, animals were exposed intravaginally to SIV for 15, 30, or 60 min and then the vagina was flushed with vinegar.
(B) In experiment B, the animals that did not become infected in experiment A were reexposed to two SIV inoculations and vinegar lavage procedures in a single day
with a 4-h resting interval between inoculations. The inoculum was completely inactivated by the vinegar lavage after the first exposure, so the two exposures in a single
day should be considered independent transmission opportunities. (C) In experiment C, control animals were lavaged with vinegar, allowed to rest for 4 h, and then
exposed intravaginally to SIV for 15 min. (D) In experiment D, control animals were exposed to SIV once. The inoculum was left undisturbed after deposition in the
vagina. (D) In experiment E, control animals were exposed to SIV once, allowed to rest for 4 h, and then exposed intravaginally to SIV again. The inoculum was left
undisturbed after deposition in the vagina. To assess the results of the experiment, the animals were monitored for 16 weeks for systemic SIV infection and the results
are shown in Table 4.
VOL. 74, 2000 DENDRITIC CELLS ARE TARGETS IN VAGINAL SIV TRANSMISSION6093
ples for DC, also maximized the probability that we would
detect DC infection. In addition, because cytoplasmic RNA is
more accessible to hybridization in cytospin slides than in for-
malin-fixed, paraffin-embedded tissues, we were able to sensi-
tively detect infected DC. Another advantage of the cytospin
preparations is that immunophenotypic characterization of in-
fected cells does not require antigen retrieval and a broader
range of antibodies is available to detect cell surface markers.
Our ISH protocol uses seven riboprobes, some of which detect
expression of regulatory genes that are expressed relatively
early in the viral life cycle. The sensitivity of the standard ISH
NBT/BCIP assay (without Tyramide amplification) was con-
firmed by our ability to detect SIV RNA?cells 12 h after in
vitro infection of T-cell lines or PBMC (data not shown).
The number of SIV RNA?cells in the vaginal mucosa can
be estimated by using the frequency of SIV RNA?cells in the
formalin-fixed histologic sections of vagina. On average, we
detected one SIV RNA?cell in each 6-?m-thick section of
vaginal mucosa. Once opened along the longitudinal axis, the
rhesus macaque vagina is approximately 4 by 7 cm. At least
11,600 histologic tissue sections (6 ?m thick) can be produced
from a tissue sample of that size. Assuming that the frequency
of one SIV RNA?cell per section of vagina is accurate, then
approximately 10,000 cells in the vaginal mucosa, mostly DC
and LC, became infected with SIV within 18 h of intravaginal
inoculation with 105TCID50of SIVmac251. This may be an
underestimate, considering that an infected cell must contain
at least 10 RNA copies for detection by ISH and that tran-
scriptionally inactive provirus cannot be detected.
A detailed discussion of the relevance of the SIV-rhesus
macaque model to heterosexual HIV transmission is beyond
the scope of this study, and a number of reviews are available
(15–17, 19, 23). Briefly, it is widely accepted that the HIV
variants transmitted by sexual contact are macrophage-tropic
and use CCR5 as a coreceptor (reviewed in reference 16).
SIVmac251, used in our studies, replicates well in primary
macrophages and uses CCR5 as a coreceptor (3). The inocu-
lum contains high-titer virus (105TCID50and 109RNA copies/
ml). We have shown that, while inoculation with a low-titer
inoculum can produce systemic infection in rhesus macaques,
the efficiency of transmission with a particular virus stock is
directly related to the titer of infectious virus inoculum (20). A
similar relationship between the virus load in an HIV-infected
person and transmission to an uninfected partner is well es-
tablished (5, 29a). Use of the high-titer SIV inoculum increases
the probability of interactions between infectious virions and
susceptible target cells in the genital tract, but it is unlikely to
alter the basic biology of the virus-target cell relationship. In
fact, the frequencies and types of virus-infected cells in the
genital tracts of chronically SIV-infected female rhesus ma-
caques and HIV-infected women are similar (15). Thus, in
both species, lentivirus-infected macrophages, T cells, and DC
can be routinely detected in the female genital tract during the
chronic stage of the infection (7, 25, 26, 29). Studies using
human tissues collected in the first few hours after HIV expo-
sure can never be conducted to verify the findings reported
here. However, the similarities between tissue-based studies in
chronic SIV and HIV infection in the female genital tract
suggest that the findings in the SIV model are relevant to HIV
sexual transmission. It is worth noting that in chronic SIV
infection, there may be regional differences in the types of cells
that are infected at different mucosal surfaces. Numerous SIV-
infected DC are found in the genital tract of an animal, but
they are difficult to detect in the tonsils of the same animal (6,
7). Thus, other mucosal tissues, such as tonsils, cannot be used
as surrogates for studying genital tract HIV infection. In fact,
these regional differences may exist between the endocervix
and the rest of the cervicovaginal mucosa, and findings in one
tissue cannot be extrapolated to the other.
The results of the experiments described here are consistent
with the hypothesis (23, 31; L. R. Braathen, G. Ramirez, R. O.
Kunze, and H. Gelderblom, Letter, Lancet 2:1094, 1987) that
intraepithelial DC are the initial target cells of HIV infection
in the genital tract. We also provide evidence that these in-
fected DC then migrate to draining lymph nodes, where the
infection is passed to CD4?T lymphocytes that disseminate
the virus systemically as they recirculate through the body. It
was recently demonstrated that following intravaginal inocula-
tion of mice with HIV, DC take up and transport virus to
genital lymph nodes in less than 24 h (14). Thus, in vivo ex-
periments in both mice and monkeys now support the hypoth-
esis (23, 31; Braathen, et al., Letter) that DC play a critical role
in disseminating HIV from the genital tract to lymphoid tissues
in the first 24 h after virus exposure.
The results also suggest that a second pathway of dissemi-
nation may be involved in HIV sexual transmission. We found
SIV RNA?T lymphocytes in the genital tract of one animal at
18 h PI. The ISH technique used for these studies could not
detect SIV RNA expression until 12 h after in vitro infection of
T-cell lines; thus, it is unlikely that the T-cell infection repre-
sents passage of the infection from infected DC to the T cells.
Apparently the T cells were directly, and rapidly, infected by
the inoculum. The mechanism of T-cell infection is unclear, as
the vaginal epithelium provides a barrier to the entry of water-
soluble dyes and presumably larger particles, such as lentivi-
ruses, from the vaginal lumen into the mucosa (10). A few
CD4?T cells are present in the cervicovaginal epithelium of
rhesus macaques (21), and these cells could be directly infected
if they entered the superficial layers of the epithelium. It is also
possible that there were breaks in the vaginal epithelium which
provided the virus direct access to CD4?T cells in the lamina
propria, but we did not see such features in the histologic slides
examined. The early infection of T cells after mucosal inocu-
lation is consistent with the results of other SIV mucosal trans-
mission studies (33) and may explain the presence of the SIV
provirus detected in lymphoid tissues beyond the lymph nodes
that drain the genital tract by PCR. If T cells were directly
infected in the genital tract, then they could enter the periph-
eral vasculature and recirculate widely, disseminating the in-
fection. Further study is required to determine the relative
TABLE 4. Minimum time of exposure to vaginal SIV that
results in systemic infection
Duration of each
No. of SIV
Outcome of SIV
C2 151 0/2
D6 NA1 4/6
E8 NA1 8/8
aSee Fig. 5 for an explanation of the interventions used for each experimental
group. NA, not applicable (no intervention).
6094HU ET AL. J. VIROL.
significance of these two pathways of viral dissemination from Download full-text
the genital tract.
We have documented the rapid penetration of SIV into the
genital mucosa, infection of intraepithelial DC, and dissemi-
nation of SIV-infected cells to the draining lymph node within
hours of vaginal exposure to the virus. These findings may have
practical implications for developing strategies to block HIV
sexual transmission. If our findings related to vaginal SIV
transmission accurately reflect HIV biology, then HIV infects
DC and begins to disseminate very rapidly after sexual contact.
It would appear that, in order to stop systemic spread of HIV
infection after genital exposure, a vaccine will need to elicit
potent immunologic memory cell populations that rapidly ex-
pand in response to the presence of HIV recall antigens in the
This work was supported in part by grants PHS NCRR00169, PHS
AI40877, and PHS AI39435 and by the Rockefeller Foundation.
We thank Steve Joye, Paul Brosio, Ding Lu, Yichuan Wang, Zhong-
min Ma, and Judy Torten for technical assistance.
1. Barratt-Boyes, S. M., S. C. Watkins, and O. J. Finn. 1997. In vivo migration
of dendritic cells differentiated in vitro: a chimpanzee model. J. Immunol.
2. Brahic, M., L. Stowring, P. Ventura, and A. T. Haase. 1981. Gene expression
in visna virus infection in sheep. Nature 292:240–242.
3. Chen, Z., P. Zhou, D. D. Ho, N. R. Landau, and P. A. Marx. 1997. Genetically
divergent strains of simian immunodeficiency virus use CCR5 as a corecep-
tor for entry. J. Virol. 71:2705–2714.
4. Edwards, J. N. T., and H. B. Morris. 1985. Langerhans cells and lymphocyte
subsets in the female genital tract. Br. J. Obstet. Gynecol. 92:974–982.
5. Garcia, P. M., L. A. Kalish, J. Pitt, H. Minkoff, T. C. Quinn, S. K. Burchett,
J. Kornegay, B. Jackson, J. Moye, C. Hanson, C. Zorrilla, and J. F. Lew.
1999. Maternal levels of plasma human immunodeficiency virus type 1 RNA
and the risk of perinatal transmission. N. Engl. J. Med. 341:394–402.
6. Hu, J., C. J. Miller, U. O’Doherty, P. A. Marx, and M. Pope. 1999. The
dendritic cell-T cell milieu of the lymphoid tissues of the tonsil provides a
locale in which SIV resides and propagate at chronic stages of infection.
AIDS Res. Hum. Retrovir. 15:1305–1314.
7. Hu, J., M. Pope, C. Brown, U. O’Doherty, and C. J. Miller. 1998. Immuno-
phenotypic characterization of SIV-infected dendritic cells in the cervix,
vagina and draining lymph nodes of rhesus macaques. Lab. Invest. 78:435–
8. Kimber, I., and M. Cumberbatch. 1992. Stimulation of Langerhans cell
migration by tumor necrosis factor alpha (TNF-alpha). J. Invest. Dermatol.
9. Kimber, I., S. Hill, J. A. Mitchell, S. W. Peters, and S. C. Knight. 1990.
Antigenic competition in contact sensitivity. Evidence for changes in den-
dritic cell migration and antigen handling. Immunology 71:271–276.
10. King, B. F. 1983. The permeability of nonhuman primate vaginal epithelium:
a freeze-fracture and tracer-perfusion study. J. Ultrastruct. Res. 83:99–110.
11. Macatonia, S. E., A. J. Edwards, and S. C. Knight. 1986. Dendritic cells and
the initiation of contact sensitivity to fluorescein isothiocyanate. Immunology
12. Macatonia, S. E., S. C. Knight, A. J. Edwards, S. Griffiths, and P. Fryer.
1987. Localization of antigen on lymph node dendritic cells after exposure to
the contact sensitizer fluorescein isothiocyanate. Functional and morpholog-
ical studies. J. Exp. Med. 166:1654–1667.
13. Martin, L. S., J. S. McDougal, and S. L. Loskoski. 1985. Disinfection and
inactivation of the human T lymphotropic virus type III/lymphadenopathy-
associated virus. J. Infect. Dis. 152:400–403.
14. Masurier, C., B. Salomon, N. Guettari, C. Pioche, F. Lachapelle, M. Guigon,
and D. Klatzmann. 1998. Dendritic cells route human immunodeficiency
virus to lymph nodes after vaginal or intravenous administration to mice.
J. Virol. 72:7822–7829.
15. Miller, C. J. 1998. Correspondence re: Immunophenotypic characterization
of SIV-infected dendritic cells in the cervix, vagina, and draining lymph
nodes of rhesus monkeys, by Hu J, Pope M, Brown C, O’Doherty U, and
Miller CJ (Lab Invest 1998;78:435–451). Lab. Invest. 78:1343–1344.
16. Miller, C. J. 1998. Host and viral factors influencing heterosexual HIV
transmission. Rev. Reprod. 3:42–51.
17. Miller, C. J. 1994. Mucosal transmission of SIV. Curr. Top. Microbiol.
18. Miller, C. J., N. J. Alexander, P. Vogel, J. Anderson, and P. A. Marx. 1992.
Mechanism of genital transmission of SIV: a hypothesis based on transmis-
sion studies and the location of SIV in the genital tract of chronically
infected female rhesus macaques. J. Med. Primatol. 21:64–68.
19. Miller, C. J., M. Marthas, J. Greenier, D. Lu, P. J. Dailey, and Y. Lu. 1998.
In vivo replication capacity rather than in vitro macrophage tropism predicts
efficiency of vaginal transmission of simian immunodeficiency virus or sim-
ian/human immunodeficiency virus in rhesus macaques. J. Virol. 72:3248–
20. Miller, C. J., M. Marthas, J. Torten, N. J. Alexander, J. P. Moore, G. F.
Doncel, and A. G. Hendrickx. 1994. Intravaginal inoculation of rhesus ma-
caques with cell-free simian immunodeficiency virus results in persistent or
transient viremia. J. Virol. 68:6391–6400.
21. Miller, C. J., M. McChesney, and P. F. Moore. 1992. Langerhans cells,
macrophages and lymphocyte subsets in the cervix and vagina of rhesus
macaques. Lab. Invest. 67:628–634.
22. Miller, C. J., M. B. McChesney, X. Lu ¨, P. J. Dailey, C. Chutkowski, D. Lu,
P. Brosio, B. Roberts, and Y. Lu. 1997. Rhesus macaques previously infected
with simian/human immunodeficiency virus are protected from vaginal chal-
lenge with pathogenic SIVmac239. J. Virol. 71:1911–1921.
23. Miller, C. J., J. R. McGhee, and M. B. Gardner. 1992. Mucosal immunity,
HIV transmission and AIDS. Lab. Invest. 68:129–145.
24. Miller, C. J., P. Vogel, N. J. Alexander, S. Dandekar, A. G. Hendrickx, and
P. A. Marx. 1994. Pathology and localization of SIV in the reproductive tract
of chronically infected male rhesus macaques. Lab. Invest. 70:255–262.
25. Miller, C. J., P. Vogel, N. J. Alexander, S. Sutjipto, A. G. Hendrickx, and
P. A. Marx. 1992. Localization of SIV in the genital tract of chronically
infected female rhesus macaques. Am. J. Pathol. 141:655–660.
26. Nuovo, G. J., A. Forde, P. MacConnell, and R. Fahrenwald. 1993. In situ
detection of PCR-amplified HIV-1 nucleic acids and tumor necrosis factor
cDNA in cervical tissues. Am. J. Pathol. 143:40–48.
27. Parr, M., and E. Parr. 1990. Antigen recognition in the female reproductive
tract. I. Uptake of intraluminal protein tracers in the mouse vagina. J.
Reprod. Immunol. 17:101–114.
28. Parr, M. B., L. Kepple, and E. L. Parr. 1991. Antigen recognition in the
female reproductive tract. II. Endocytosis of horseradish peroxidase by
Langerhans cells in murine vaginal epithelium. Biol. Reprod. 45:261–265.
29. Pomerantz, R. J., S. M. de la Monte, C. E. Donegan, T. R. Rota, M. W. Vogt,
D. E. Craven, and M. S. Hirsch. 1988. Human immunodeficiency virus
(HIV) infection of the uterine cervix. Ann. Intern. Med. 108:321–327.
29a.Quinn, T. C., M. J. Wawer, N. Sewankambo, D. Serwadda, C. Li, F. Wabwire-
Mangen, M. O. Meehan, T. Lutalo, and R. H. Gray. 2000. Viral load and
heterosexual transmission of human immunodeficiency virus type 1. New
Engl. J. Med. 342:921–929.
30. Silberberg, I., R. L. Baer, S. A. Rosenthal, G. J. Thorbecke, and V. Ber-
ezowsky. 1975. Dermal and intravascular Langerhans cells at sites of pas-
sively induced allergic contact sensitivity. Cell. Immunol. 18:435–453.
31. Soto-Ramirez, L. E., B. Renjifo, M. F. McLane, R. Marlink, C. O’Hara, R.
Sutthent, C. Wasi, P. Vithayasi, V. Vithayasi, C. Apichartpiyakul, P.
Auewarakul, V. Pena Cruz, D.-S. Chui, R. Osathanondh, K. Mayer, T.-H.
Lee, and M. Essex. 1996. HIV-1 Langerhans cell tropism associated with
heterosexual transmission of HIV. Science 271:1291–1293.
32. Spira, A. I., P. A. Marx, B. K. Patterson, J. Mahoney, R. A. Koup, S. M.
Wolinsky, and D. D. Ho. 1996. Cellular targets of infection and route of viral
dissemination after an intravaginal inoculation of simian immunodeficiency
virus into rhesus macaques. J. Exp. Med. 183:215–225.
33. Stahl-Hennig, C., R. M. Steinman, K. Tenner-Racz, M. Pope, N. Stolte, K.
Matz-Rensing, G. Grobschupff, B. Raschdoff, G. Hunsmann, and P. Racz.
1999. Rapid infection of oral mucosal-associated lymphoid tissue with simian
immunodeficiency virus. Science 285:1261–1265.
34. Yamashita, K., and A. Yano. 1994. Migration of murine epidermal Langer-
hans cells to regional lymph nodes: engagement of major histocompatibility
complex class II antigens induces migration of Langerhans cells. Microbiol.
35. Zhang, Z.-Q., T. Schuler, M. Zupancic, S. Wietgrefe, K. A. Staskus, K. A.
Reimann, T. A. Reinhart, M. Rogan, W. Cavert, C. J. Miller, R. S. Veazey, D.
Notermans, S. Little, S. A. Danner, D. D. Richman, D. Havlir, J. Wong, H. L.
Jordan, T. W. Schacker, P. Racz, K. Tenner-Racz, N. L. Letvin, S. Wolinsky,
and A. T. Haase. 1999. Sexual transmission and propagation of simian and
human immunodeficiency viruses in resting and activated CD4?T cells.
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