JOURNAL OF VIROLOGY, June 2007, p. 6175–6186
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 81, No. 12
Mucosal Innate Immune Response Associated with a Timely Humoral
Immune Response and Slower Disease Progression after Oral
Transmission of Simian Immunodeficiency Virus to
Jeffrey M. Milush,1Kelly Stefano-Cole,2Kimberli Schmidt,3Andre Durudas,1
Ivona Pandrea,4and Donald L. Sodora1*
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 753901; University of Pittsburgh,
Pittsburgh, Pennsylvania2; California National Primate Research Center, University of California, Davis, California3; and
Tulane National Primate Research Center, Covington, Louisiana, and Tulane Health Sciences Center,
Tulane University, New Orleans, Louisiana4
Received 7 January 2007/Accepted 27 March 2007
Mucosal transmission is the predominant mode of human immunodeficiency virus (HIV) infection world-
wide, and the mucosal innate interferon response represents an important component of the earliest host
response to the infection. Our goal here was to assess the changes in mRNA expression of innate mucosal genes
after oral simian immunodeficiency virus (SIV) inoculation of rhesus macaques (Macaca mulatta) that were
followed throughout their course of disease progression. The SIV plasma viral load was highest in the macaque
that progressed rapidly to simian AIDS (99 days) and lowest in the macaque that progressed more slowly
(>700 days). The mRNA levels of six innate/effector genes in the oral mucosa indicated that slower disease
progression was associated with increased expression of these genes. This distinction was most evident when
comparing the slowest-progressing macaque to the intermediate and rapid progressors. Expression levels of
alpha and gamma interferons, the antiviral interferon-stimulated gene product 2?-5? oligoadenylate synthetase
(OAS), and the chemokines CXCL9 and CXCL10 in the slow progressor were elevated at each of the three oral
mucosal biopsy time points examined (day 2 to 4, 14 to 21, and day 70 postinfection). In contrast, the more
rapidly progressing macaques demonstrated elevated levels of these cytokine/chemokine mRNA at lymph
nodes, coincident with decreased levels at the mucosal sites, and a decreased ability to elicit an effective
anti-SIV antibody response. These data provide evidence that a robust mucosal innate/effector immune
response is beneficial following lentiviral exposure; however, it is likely that the anatomical location and timing
of the response need to be coordinated to permit an effective immune response able to delay progression to
Initiating a human immunodeficiency virus (HIV) infection
generally requires that the virus traverse a mucosal barrier,
such as the vaginal, rectal, penile, or oral mucosa, in order to
establish an infection (39, 54). Mucosal transmission via the
oral route occurs in both newborns and adults (41, 42, 47, 48,
52, 56). Transmission to newborns can occur during the birth-
ing process, likely through contact between the vaginal fluids
and the oral cavity, or after birth via breast feeding, with the
virus present in the milk (41, 42, 51). Oral transmission in
adults can occur as a result of unprotected receptive oral
intercourse in which the virus is present in the semen (9, 47,
The simian immunodeficiency virus (SIV) infection of rhe-
sus macaques provides an excellent model system to assess the
innate and adaptive immune responses to viral infection (6, 7,
28, 38, 50). While the analysis of HIV immunity has historically
focused on HIV/SIV-specific T- and B-cell responses, more
recent studies have assessed the innate cytokine and chemo-
kine immune responses in the host. Within the blood and
lymph nodes of chronically SIV-infected macaques, an in-
crease in the expression of various innate cytokines/chemo-
kines, including Mip-1?, Mip-1?, alpha interferon (IFN-?),
and CXCL9 and CXCL10, has been observed. Interestingly,
this increase in mRNA expression correlated with higher levels
of viral replication (1, 31, 49), indicating that high levels of
these cytokines/chemokines are unable to control viral repli-
cation in blood and lymph tissues of SIV?monkeys. With
regard to the immune events occurring at the mucosa, the
reduced plasma viral loads observed for some macaques di-
rectly correlate with the markedly increased cytotoxic factors
(i.e., granzyme A, lysozyme, and perforin) and proinflamma-
tory gene transcript levels in the gut-associated lymphoid tissue
(GALT; jejunum), indicating a dichotomy between inflamma-
tory responses in the GALT and those in the lymph nodes (20).
In addition, expression of certain immune response gene prod-
ucts (i.e., interleukin 2 [IL-2], ?2 microglobulin, and SDF-1) in
the GALT involved in eliciting a cytotoxic T-cell response was
also upregulated in macaques with lower viral loads (20).
Therefore, the effectiveness of an inflammatory immune re-
sponse may depend on the timing of these innate/effector
* Corresponding author. Mailing address: University of Texas,
Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX
75390-9113. Phone: (214) 648-2438. Fax: (214) 648-0231. E-mail:
?Published ahead of print on 11 April 2007.
genes, as well as which genes are expressed, at different tissue
Innate/effector gene expression at the site of SIV mucosal
inoculation likely reflects some of the earliest host responses to
SIV infection (3, 4). Following vaginal transmission, the early
innate response at the vaginal mucosa is predominantly com-
prised of proinflammatory cytokines (4). The induction of cy-
tokines with antiviral activity (alpha/beta interferons) was de-
layed and consequently was too late to prevent virus
replication and dissemination. Therefore, the early cytokine
response favors immune activation potentially resulting in the
recruitment of additional target cells for SIV infection (4).
Assessment of neonatal macaques following multiple oral ex-
posures at 7 days postinfection has also identified a predomi-
nantly proinflammatory response and delayed interferon effec-
tor response (IFN-?, IFN-?, 2?-5? oligoadenylate synthetase
[OAS], and Mx) at the mucosal site of inoculation (3). The
ability of SIV to rapidly spread from the site of transmission at
the oral or vaginal mucosa to lymph nodes in as little as 1 to 2
days postinfection (3, 27, 28, 40) might indeed present a chal-
lenge to the innate immune system to respond in a timely
manner to benefit the host. To determine the importance of
timing of the innate immune response on disease outcome, we
reasoned that assessing the expression of innate/effector genes
at the mucosa would be more informative if the animals were
followed throughout their disease course. Following successful
SIV oral inoculation of the animals, biopsies were obtained
from oral mucosal tissue (gingiva adjacent to the molars) at
three time points (2 to 4, 14 to 21, and 70 days postinfection)
throughout the disease course. The levels of 13 different in-
nate/effector mRNA levels were quantified, and changes in the
expression of these genes in the SIV-infected macaques were
monitored. Interestingly, assessing these macaques throughout
their disease courses determined that the rate of disease pro-
gression was inversely associated with the ability to increase
the expression of a select group of innate/effector genes
(IFN-?, IFN-?, CXCL9, CXCL10, OAS, and IL-12) at the
mucosa. These data indicate that a robust innate/effector im-
mune response at the mucosa may be beneficial to a host
confronted with a lentivirus, particularly when the response is
initiated during the earliest time points and maintained
throughout the disease course.
MATERIALS AND METHODS
Animal inoculations and virus stock. The macaques used in these studies were
colony-bred rhesus macaques (Macaca mulatta) housed at the California Na-
tional Primate Research Center (CNPRC). Upon beginning these studies, all
animals were seronegative for SIV, simian T-cell leukemia virus (STLV), and
simian retrovirus (SRV), as determined by antibody enzyme immunoassay. In
addition, all animals were negative for SIV, STLV, and SRV proviral DNA as
determined by virus-specific PCR assays using DNA extracted from peripheral
blood mononuclear cells (PBMC) (Simian Retrovirus Laboratory, CNPRC) (5,
32, 33). Animals utilized in this study had the following CNPRC designations:
RM11 (33291), RM12 (32167), RM13 (32174), RM14 (32296), RM15 (33353),
and RM16 (32127). All animals were cared for in accordance with National
Institutes of Health guidelines, and appropriate approvals from the local Animal
Care and Use Committees were obtained. Each macaque was orally inoculated
with two 1 ? 10550% tissue culture infective doses (TCID50) of SIVmac251-5/98
(22, 35) 1 h apart to ensure infection. Macaques were followed throughout
infection and observed for signs of simian AIDS, as previously described (57), at
which time they were humanely euthanized by a pentobarbital overdose, in
accordance with California National Primate Research Center and federal guide-
Tissue collection, processing, and assessment of cellular infiltrates. Numer-
ous biopsies were obtained from the macaques while they were under ketamine
hydrochloride anesthesia (10 mg/kg). Mucosal biopsies were approximately 2
mm in diameter and 2 mm thick and consisted of squamous epithelium, as well
as underlying connective tissue. Therefore, these biopsies represent a mixed
population of cell types, including epithelial and lymphoid cells. Oral mucosal
biopsies were obtained from each macaque at three time points (2 to 4, 14 to 21,
and 70 days postinfection), lymph node biopsies were obtained at four time
points (7 to 15, 21 to 28, 45 to 56, and 85 days postinfection), and each biopsy was
placed in RNAlater (Ambion, Inc., Austin, TX) and then stored at ?20°C for
RNA isolation. In addition, rectal mucosal biopsies were placed in Streck tissue
fixative buffer (Streck Laboratories, Inc.) and were then paraffin embedded. Day
0 mucosal biopsies were not acquired from the six study animals due to concerns
that the biopsies would alter the mucosal integrity and affect the outcome of the
study. Instead, similar biopsies were obtained from four age-matched, uninfected
macaques to achieve baselines. Assessment of cellular infiltrates in the mucosa
was performed on standard hematoxylin-and-eosin-stained tissue sections by a
pathologist. Microscopy was performed using a Zeiss microscope and PASCAL
version 3.2 image software (512-by-512-pixel resolution) (Carl Zeiss, Oberkochen,
Quantification of plasma viral RNA. Viral RNA in the plasma was quantified
by a Chiron Corporation branch DNA (bDNA) signal amplification assay, ver-
sion 4.0, specific for SIV (57). Viral load in the plasma is reported as copies of
viral RNA per milliliter of plasma. The limit of detection of the bDNA assay is
125 copies of viral RNA per milliliter of plasma.
Quantitative real-time PCR analysis of immune effector genes. Total RNA
was extracted from the mucosal and lymph node biopsies as previously described,
utilizing mechanical homogenization, followed by Trizol extraction (2). Real-
time PCRs utilizing gene-specific primer/probe were performed on an ABI 7700
or ABI 7300 (Applied Biosystems) sequencer, utilizing the default settings as
described previously (1, 2). Changes in expression of 13 innate immune genes
(IFN-?, IFN-?, IFN-?, IL-4, IL-6, IL-10, IL-12, CXCL9, CXCL10, tumor ne-
crosis factor alpha [TNF-?], Mip1?, Mx, and OAS) and the glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) housekeeping gene were calculated as pre-
viously described, utilizing delta cycle threshold (?CT) values (2). Briefly, the
GAPDH CTvalue was subtracted from the CTvalue of the target gene, thereby
generating a ?CTvalue. For the four uninfected macaques, an average of the
?CTvalues was derived, and this average ?CTvalue was then subtracted from the
?CTvalue of a target gene to achieve the ??CTvalue. Change (n-fold) was then
determined by the following formula: 2???CT(User Bulletin no. 2; ABI Prism
7700 Sequence Detection System; Applied Biosystems). In the event that the
??CTvalue was positive, indicating that the change was ?1-fold (a negative fold
change), the negative fold-change value was calculated by the following formula:
?1/fold-change value. For example, a ??CTvalue of 3 would result in a fold
change of 2?3, equal to 0.125 or a negative fold change of ?1/(0.125) or ?8-fold
change. An average fold change and a standard deviation of the target gene were
calculated for the uninfected macaques. Changes in mRNA expression of a
target gene in an infected macaque were deemed either increased or decreased
if its fold change was greater than 2 standard deviations of the average of the four
SIV envelope-specific antibody endpoint titer and avidity. Antibody responses
to native SIV envelope were measured as previously described utilizing a con-
canavalin A (ConA) enzyme-linked immunosorbent assay (ELISA) (17). Briefly,
detergent-disrupted SIV envelope proteins from SIVsmB7 captured on the
ConA plate were exposed for 1 h at room temperature to plasma antibodies,
monoclonal antibodies, or plasma from SIV-negative control macaques. To de-
termine endpoint titers, the plates were washed with phosphate-buffered saline
(PBS) and developed using peroxidase-labeled goat anti-monkey immunoglob-
ulin G antibody and TM blue (Serologicals Corp., Gaithersburg, Md.) as the
substrate. Endpoint titers represent the last twofold dilution with an optical
density at 450 nm (OD450) that is twice that of the SIV-negative control animals.
The avidity of antibody binding was determined by measuring the stability of
antibody-antigen binding in the presence of 8 M urea. The percentage of anti-
body avidity was calculated as follows: (OD450of urea-treated wells/OD450of
PBS-treated wells) ? 100. The results are averages of at least two independent
experiments, with variation in individual antibody avidity values of less than 10%.
Statistical analysis. A Spearman nonparametric correlation test was per-
formed to determine whether mRNA gene expression correlated with viral load,
antibody titers, or disease progression. To compare the number of upregulated
genes in the oral gingiva to those in the rectal mucosa, an adjusted chi-square test
was performed. All calculations were performed utilizing Prism statistical soft-
ware, version 4.0c (GraphPad Software, Inc.), and a P value of less than 0.05 was
considered to be significant.
6176MILUSH ET AL.J. VIROL.
Oral inoculation of SIV: plasma viral load and innate/effec-
tor gene levels. These studies were initiated via a nontraumatic
oral inoculation of SIVmac251 to the cheek pouch of six ma-
caques, and each macaque became infected and developed
peak viremia at 1 to 2 weeks postinfection (Fig. 1). As is
commonly observed following an SIV infection, there was a
variable rate in disease progression, including one rapid pro-
gressor (RM11 developed simian AIDS in 14 weeks), four
intermediate progressors (RM12 and RM15 developed simian
AIDS in 21 and 36 weeks, respectively), and one slow progres-
sor (RM16 developed signs of simian AIDS after 106 weeks of
infection) (Table 1). Similar to results from previous studies, a
slower rate of disease progression was associated with lower
plasma viral loads (P ? 0.0538) (13, 14, 25, 37, 44, 55). The
decrease in viral load following the acute peak was most dra-
matic in the slow progressor, in which the set point viral load
(weeks 2 to 4 postinfection) was 37-fold lower (106to 106.5
copies of viral RNA per milliliter of plasma) than the average
5.8-fold decrease in the other five macaques (107to 108copies
of viral RNA) (Fig. 1). Over the course of this study, five of the
macaques developed opportunistic infections of the respiratory
(i.e., Klebsiella pneumoniae, Moraxella spp., and Cryptospo-
ridium spp.) and/or intestinal (i.e., Candida albicans, Blastocys-
tis hominis, and Blantidium coli) tract, representing the onset
of simian AIDS (Table 1).
Throughout SIV infection, pinch biopsies were obtained
from oral mucosa, where the virus was administered, and from
rectal mucosa to determine how innate/effector gene mRNA
levels compared at these different mucosal sites. Quantitative
real-time PCR analysis of 13 immune response genes and one
housekeeping gene (GAPDH) for the purpose of normaliza-
tion between samples was undertaken with each of the biopsies
obtained. In general, within the 13 innate/effector gene mRNA
levels assessed (listed in Materials and Methods), a signifi-
cantly higher percentage (34%) of the genes assessed in the
oral mucosa (gingiva adjacent to teeth) were increased beyond
the standard deviation range of ?2 from four uninfected ma-
caques than that of rectal mucosa (27%) during acute infection
(Table 2) (adjusted chi-square test, P ? 0.026). The increased
numbers of upregulated innate/effector genes at the oral mu-
cosa could possibly be due to the oral route of inoculation
utilized here. Progression to simian AIDS at different rates was
associated with distinctions in the innate/effector gene levels
when these were compared to levels from four uninfected
FIG. 1. Plasma viral RNA copies per milliliter of plasma were
quantified by the bDNA signal amplification assay specific for SIV
(limit of detection is 125 copies per milliliter of plasma). Peak viral
loads occurred at 1 to 2 weeks postinfection, and set point viral loads
were established by 8 weeks postinfection. Results for the rapid pro-
gressor RM11 are shown with open boxes, for the four intermediate
progressors in gray lines, and for the slow progressor RM16 in the solid
TABLE 1. Clinical and pathological findings following SIV infection
Clinical findingsNecropsy findings
Diarrhea, Campylobacter coli
Diarrhea, esophageal Candida albicans
Diarrhea, wt loss
Diarrhea, oral Candida albicans
Gastritis, enteritis, colitis, cystitis, glomerulonephritis
Encephalitis, focal cerebral hematoma
Lymphoid depletion: mucosa-associated lymphoid
tissue, lymph nodes, thymus
99Oral candidiasis, diarrhea, wt loss, euthanasia
Increased heart respiratory rates, labored breathing RM12224 Colitis, multifocal pneumonia, generalized
lymphadenopathy, severe urinary bladder
Pneumonia, scrotal edema, euthanasia
Diarrhea, Campylobacter coli, trichomonas,
Blastocystis hominis, cryptosporidium
Diarrhea, Iodamoeba butschlii, trichomonas
Diarrhea, Balantidium coli, trichomonas
Diarrhea, colitis, wt loss, euthanasia
Weak, unsteady in cage, wt loss, euthanasia
Nasal discharge: coagulase and Staphylococcus spp.
Weight loss, nasal discharge: Moraxella spp.
Dehydration, wt loss, euthanasia
RM13Cholecystitis and choledochitis, colitis, enlarged
VOL. 81, 2007 INNATE MUCOSAL IMMUNITY FOLLOWING ORAL SIV INOCULATION6177
control macaques. Of the six macaques, the slow progressor,
RM16, had the largest number of genes (8 of 12) upregulated
at the earliest (2 to 4 days postinfection) time point assessed
postinfection (Table 2). In contrast, the rapid progressor,
RM11, had the largest number of genes (6 of 13) downregu-
lated at this earliest (2 to 4 days postinfection) time point
(Table 2). Assessment of the mRNA levels of these genes was
complicated by the fact that some genes did not achieve effi-
cient PCR amplification with every biopsy obtained and re-
mained at levels below detection (Table 2). Therefore, we have
focused on a careful assessment of six immune response genes
that were efficiently amplified in the majority of samples, in-
cluding three cytokines (IFN-?, IFN-?, and IL-12), two che-
mokines (CXCL9 and CXCL10), and one interferon-stimu-
lated intracellular antiviral gene (OAS) product for the in-
depth analysis presented here.
Assessment of the changes in mucosal cytokine/chemokine
mRNA levels during acute SIV infection (days 2 to 21). Early
events postinfection were assessed at two distinct phases of
acute infection: 2 or 4 days postinfection (d.p.i.) represents
time points prior to the initiation of the adaptive immune
response, whereas 14 or 21 d.p.i. represents time points during
the initiation of the adaptive immune response. As the virus
was administered orally, the innate/effector gene mRNA levels
at the oral mucosa were of particular interest. Of the six
mRNA levels that we focused on in detail, type I interferons
(represented by IFN-?) are known to be induced at very early
times following a viral infection (reviewed in reference 8).
TABLE 2. Fold change in expression of 13 innate/effector genes following oral SIV inoculationa
Fold change in the expression of the following innate/effector gene products relative to the indicated control value
Control9.36 126.96.36.1995.63 7.993.821.82 1.782.682.76 3.81.91
?4.55 ?2.44 ?200
16.67 ?5.26 ?500
2.07 ?1.45 ?100
aSummary of the normalized fold change in expression of 13 innate/effector genes assessed in the oral and rectal mucosa following oral SIV inoculation. The six genes
discussed in detail in the text are in bold. Values of increased expression are shown in bold with underlining, while values of reduced expression are shown in bold italics.
6178MILUSH ET AL.J. VIROL.
Indeed, an increase in IFN-? expression in the gingiva was
observed at the 2-to-4 and 14-to-21 d.p.i. time points in three
macaques that progressed relatively more slowly to disease
(RM14, RM15, and the slow progressor RM16) compared to
the uninfected controls (Fig. 2A). Interestingly, in the rapid
progressor (RM11) as well as RM13, expression of IFN-? was
not elevated at either the 2-to-4 or the 14-to-21 d.p.i. time
points (Fig. 2A). The type I interferon-stimulated gene prod-
uct OAS degrades viral and cellular mRNA, thereby limiting
viral replication and spread to other cells. RM13 and the slow
progressor RM16 had elevated levels (5-fold and 34-fold, re-
spectively) of OAS mRNA expression in the gingiva at 2 to
4 d.p.i. (Fig. 2B). These levels dropped to within normal ranges
in the gingiva at 14 to 21 d.p.i. in the slow progressor RM16.
FIG. 2. The changes (n-fold) in mRNA expression of immune response genes in the gingiva of six orally infected macaques at 2 to 4, 14 to 21,
and 70 days postinfection are shown. The rapid progressor (RM11) is shown as an open bar, the slow progressor (RM16) is shown in solid black,
and the intermediate progressors are shown in gray. The mRNA levels shown are reported as n-fold changes with regard to mRNA levels in
matched gingival samples of four uninfected macaques. The shaded area represents the averages ? 2 standard deviations of expression in
uninfected macaques. Bars extending beyond the gray shaded area represent samples that are increased or decreased with regard to the uninfected
VOL. 81, 2007INNATE MUCOSAL IMMUNITY FOLLOWING ORAL SIV INOCULATION 6179
Levels of OAS expression in RM12, RM14, and RM15 were
delayed until 14 to 21 d.p.i. or never increased as in the rapid
progressor RM11. Interestingly, there appears to be a trend
between a delay in OAS expression and higher acute and set
point plasma viral loads with these macaques (Fig. 1). The
mRNA level of the proinflammatory cytokine IL-12 was in-
creased in the gingiva of RM14 and the slow progressor RM16
at 2 to 4 d.p.i. (Fig. 2E), while the rapid progressor RM11
exhibited decreased gingival IL-12 expression at 2 to 4 d.p.i.
that remained decreased at 14 to 21 d.p.i. (Fig. 2E). IFN-?
expression at 2 or 4 d.p.i. in the gingiva of RM13, RM14, RM15,
and the slow progressor RM16, however, was within normal
ranges compared to that of SIV-negative macaques and re-
duced in RM12 and the rapid progressor RM11 (Fig. 2F). This
trend was maintained at 14 to 21 d.p.i. in all six macaques (Fig.
2F). The mRNA expression levels of two interferon-inducible
chemokines that are genetically and functionally similar,
CXCL9 and CXCL10, were also assessed. Increases at 2 to
4 d.p.i. in the expression of both CXCL9 and CXCL10 in the
gingiva occurred in two macaques (RM12 and the slow pro-
gressor RM16), while CXCL9 expression alone was increased
in RM13 (Fig. 2C and D). By 14 to 21 d.p.i., five of the six
macaques exhibited elevated expression levels of at least one of
the chemokines above the levels observed for SIV-negative
macaques (Fig. 2C and D). The observation that the expression
of interferon-independent genes, such as IL-4 and IL-10 (Ta-
ble 2), did not appear to follow the same patterns of expression
as the interferon-related genes provides further evidence that
the mucosal immune response during acute SIV infection is
primarily driven by interferon and interferon-responsive genes.
To determine if the innate/effector gene changes at the oral
mucosa were reflected at other mucosal sites, we also assessed
the rectal mucosa and observed both similarities and differ-
ences compared to those of the oral mucosa. Similarities in-
cluded higher levels of OAS, CXCL9, and CXCL10 at the
rectal mucosa during the 2-to-4-day and 14-to-21-day time
points for the slow progressor RM16 and decreased levels of
these genes in the rapidly progressing macaque (RM11) (Fig.
2 and Fig. 3). Differences included IFN-? expression that was
increased in the slow progressor RM16 and intermediate pro-
gressors RM14 and RM15 in the gingiva but remained within
normal ranges or decreased in the rectal tissues of all six
macaques (Fig. 2F and Fig. 3F). Additionally, IFN-? expres-
sion levels differed among mucosal sites as none of the ma-
caques exhibited increased IFN-? mRNA levels in the gingiva;
however, RM11, RM13, RM14, and the slow progressor RM16
each exhibited an increased level at the 2-to-4-day or 14-to-21-
day time points in the rectal mucosa. These differences are
understandable as these sites represent distinct regions of the
digestive tract. However, identifying similar mRNA levels for
some of the genes analyzed (encoding OAS, CXCL9, and
CXCL10) is interesting as the oral and rectal mucosal sites may
be reacting to SIV infection of the host in a similar manner,
even at these earliest time points.
Assessment of the mucosal cytokine/chemokine mRNA lev-
els at 70 days after SIV infection. Following acute infection,
the mucosal immune system is impacted by the depletion of
CD4?T cells (reviewed in references 10 and 23) that provides
an opportunity for opportunistic infections to establish them-
selves, resulting in many of the diseases commonly associated
with the onset of AIDS (e.g., thrush, pneumocystis, etc.). The
day 70 time point was chosen to represent the chronic stage of
the disease, as by this time, macaques have undergone the
depletion of CD4 cells at the mucosa (11, 24, 30, 34, 36, 45, 58)
and have established their viral set point (Fig. 1). Furthermore,
the presence of opportunistic infections at these various mu-
cosal sites was monitored at this later time point (Table 1) and
assessed with regard to changes in innate/effector gene mRNA
levels. The oral gingival biopsies obtained at 70 d.p.i. exhibited
a pattern of expression that was similar to the acute time points
for the genes encoding IFN-?, OAS, IL-12, and IFN-? (Fig. 2).
In general, the rapid- and intermediate-progressing macaques
exhibited declines or similar levels of these genes compared to
those of uninfected macaques; however, the slowest-progress-
ing macaque, RM16, exhibited elevated levels (similar to those
at the acute time points). The most remarkable finding at day
70 in the oral mucosa was the increased expression levels of
interferon-inducible chemokines CXCL9 and CXCL10 in the
slow progressor RM16 (730- and 145-fold for CXCL9 and
CXCL10 expression, respectively [Fig. 2C and D]).
In general, the assessment of rectal mucosal biopsies re-
sulted in similar patterns of gene expression as those observed
for the gingiva of the slowest progressor, with increases in five
of the six gene products (OAS, IFN-?, IL-12, CXCL9, and
CXCL10), and neither RM12 nor the rapid progressor RM11
showed increased levels of expression of any of the six genes at
70 days postinfection (Fig. 3). Also of interest was the obser-
vation that IFN-? expression in the rectal tissue was either
decreased (RM11, RM12, and RM15) or within normal ranges
(RM13, RM14, and RM16) of SIV-negative macaques, which
contrasted with observations for the gingiva (Fig. 2A and Fig.
3A). Similar to levels observed for the gingiva, increased levels
of the chemokines CXCL9 and CXCL10 were observed for the
rectal tissue of three of the macaques, including the slow pro-
gressor RM16 (Fig. 3C and D).
Although the gingival biopsies did not yield sufficient tissue
to assess for cellular infiltrates, we were able to assess the levels
of lymphocyte infiltration at the rectal mucosa at some time
points. Indeed, hematoxylin and eosin staining indicated that
the elevated levels of cells were likely lymphocytes and mac-
rophages in the rectal biopsies of the slow progressor RM16 at
70 d.p.i. (data not shown). In summary, elevated IL-12 and
IFN-? expression levels in the gingival and rectal tissues of the
slow progressor RM16 were associated with high CXCL9 and
CXCL10 expression levels as well as more immune cells at the
mucosa, which may indicate a heightened mucosal immune
defense that might aid in preventing opportunistic infections.
Assessment of the interferon-inducible chemokine re-
sponses in lymph nodes. A number of studies have been un-
dertaken that establish a direct correlation between elevated
cytokine/chemokine levels in lymph nodes of SIV-infected ma-
caques and disease progression (1, 31, 49). Here, we focused
on the chemokines CXCL9 and CXCL10, as these were highly
elevated in the mucosae of the slow-progressing macaques and
have been observed to correlate with a poor prognosis during
SIV infection (1, 31, 49). Assessment of the chemokines
CXCL9 and CXCL10 in the macaques in this study revealed
strikingly different expression patterns when lymph node sam-
ples (Fig. 5) were compared to mucosal samples (Fig. 2 and
Fig. 3). Similar to the assessment of mucosal biopsies, lymph
6180MILUSH ET AL.J. VIROL.
node biopsies were assessed at multiple time points postinfec-
tion, including day 7 to 15, 21 to 28, 45 to 56, and at day 85.
CXCL9 and CXCL10 were highly expressed in the lymph node
of the rapid progressor (RM11) at all time points analyzed
(Fig. 4A and B). The intermediate progressors exhibited in-
creased expression of both CXCL9 and CXCL10; however,
these levels were generally lower than those observed for the
rapid progressor (Fig. 4A and B). In the slow-progressing ma-
FIG. 3. The changes (n-fold) in mRNA expression of immune response genes in the rectal tissue of six orally infected macaques at 2 to 4, 14
to 21, and 70 days postinfection are shown. The rapid progressor (RM11) is shown as an open bar, the slow progressor (RM16) is shown in solid
black, and the intermediate progressors are shown in gray. The mRNA levels shown are reported as n-fold changes with regard to mRNA levels
in matched rectal samples of four uninfected macaques. The shaded area represents the averages ? 2 standard deviations of expression in
uninfected macaques. Bars extending beyond the gray shaded area represent samples that are increased or decreased with regard to the uninfected
VOL. 81, 2007INNATE MUCOSAL IMMUNITY FOLLOWING ORAL SIV INOCULATION 6181
caque (RM16), the levels of CXCL10 generally remained
within levels normally observed in healthy macaques (Fig. 4A);
however, CXCL9 expression was increased during the acute
infection before dropping to within normal levels of expression
at 45 to 56 d.p.i. (Fig. 4B). These data indicate that increased
immune/effector gene expression at mucosal sites is associated
with slower disease progression, whereas increased effector
gene expression (CXCL9/CXCL10) at secondary lymphoid
sites is associated with increased rates of disease progression.
SIV Env-specific antibody response following oral inocula-
tion. To investigate the association between increased innate
immune responses and the induction or maturation of adaptive
immune responses, we assessed anti-SIV Env-specific antibody
titers and avidity. We observed very low levels and poor mat-
uration of SIV-specific antibodies in the rapid progressor
RM11 (Fig. 5A), consistent with other studies assessing rapidly
progressing SIV?macaques (18, 26, 43, 61). In contrast, the
slow progressor RM16 exhibited Env-specific antibodies as
early as 2 weeks postinfection and achieved maximal, steady-
state endpoint titers by 6 weeks postinfection (Fig. 5A). Fur-
thermore, these levels were maintained throughout the study
period of 50 weeks in the slow progressor. Interestingly, the
intermediate progressors (RM12, RM13, RM14, and RM15)
developed SIV Env-specific antibody responses, but their lev-
els were delayed by 2 to 3 weeks compared to that of the slow
progressor (Fig. 5A). Antibody avidity assesses the strength of
an antibody-antigen interaction and can be utilized as a qual-
itative assessment of antibody maturation. Based on previous
studies (15, 16), avidity indexes greater than 35% have been
determined to be consistent with a mature antibody response
of the host to the SIV Env protein. The very low levels of
SIV-specific antibodies precluded our abilities to assess SIV
antibody avidity indexes in the most rapidly progressing ma-
caque, RM11 (Fig. 5A). In contrast, the normal progressor
RM15 and the slowest progressing macaque RM16 had in-
creasing avidity indexes that reached levels indicative of a
mature SIV-specific antibody response at 28 and 36 weeks
FIG. 4. The changes (n-fold) in mRNA expression of the chemokines CXCL9 (B) and CXCL10 (A) in the lymph nodes of six orally infected
macaques at 7 to 15, 21 to 28, 45 to 56, and 85 days postinfection are shown. The rapid progressor (RM11) is shown as an open bar, the slow
progressor (RM16) is shown in solid black, and the intermediate progressors are gray. The mRNA levels shown are reported as n-fold changes
with regard to mRNA levels in matched lymph node samples of four uninfected macaques. The shaded area represents the averages ? 2 standard
deviations of expression in uninfected macaques. Bars extending beyond the gray shaded area represent samples that are increased or decreased
with regard to the uninfected controls.
FIG. 5. (A) Serum antibody endpoint titers were analyzed for re-
activity to SIVsmB7 envelope proteins, using the ConA ELISA. End-
point titers were determined to be the last twofold dilution with an
OD450of twice that of normal monkey serum and are reported as the
log10of the reciprocal endpoint titer. (B) Maturation of SIV envelope-
specific antibody avidity following oral inoculation. Antibody avidity
was determined by measuring the stability of the antigen-antibody
complexes to 8 M urea and is expressed as the (OD of wells washed
with 8 M urea/OD of wells washed with PBS) ? 100. The rapid
progressor RM11 is shown with open boxes, the intermediate progres-
sors with gray lines, and the slow progressor RM16 with solid black
lines. Avidity indexes of ?35% are mature SIV Env-specific antibodies
6182MILUSH ET AL. J. VIROL.
postinfection, respectively. Delayed or insufficient maturation
of Env-specific antibodies was observed in the intermediate-
progressing macaques, RM12, RM13, and RM14 (Fig. 5B).
These data are in agreement with previous studies indicating
that slower disease progression is associated with a more ro-
bust SIV-specific antibody response (18, 26, 43, 61); however,
our findings also identify a significant correlation between the
induction of certain innate/effector genes at the mucosa and a
more rapid triggering (by 3 weeks postinfection) of a mature
SIV-specific antibody response (P ? 0.0333).
The ability of HIV to replicate in and kill CD4?T cells has
made the understanding of HIV pathogenesis a difficult and
complex task. While CD4?T cells are required for initiating a
successful adaptive immune response that could potentially
clear the HIV infection, activation of CD4?T cells also creates
optimum virus replication factories, thereby perpetuating the
HIV infection. The innate immune response plays a key role in
inhibiting HIV infection, although the exact cytokines, chemo-
kines, and immune cells needed to impact HIV replication are
not known. The innate immune system responds to a variety of
pathogen-associated molecular patterns (PAMPs), as exempli-
fied in the Toll-like receptors, which are present on different
immune cells (dendritic cells, macrophages, gamma-delta T
cells, NK cells, etc.). To date, the data from a number of
laboratories, including ours, support a model of mucosal trans-
mission in which CD4?T cells, macrophages, and dendritic
cells are the first cells infected, followed by rapid dissemination
of SIV to draining and peripheral lymph nodes (27, 28, 40, 46).
Studies designed to assess the innate immune response to this
initial infection have assessed fixed time points following nec-
ropsy of the monkeys in order to acquire the necessary tissues
for analysis (1, 3, 4, 20, 53). A recent study assessed innate
immune responses at 7 d.p.i. in a model that utilized multiple
low-dose SIV exposures (to reflect infection following breast-
feeding) in neonatal rhesus macaques (3). Although immune
responses varied in the innate/effector genes expressed (gingi-
val mucosa was less prone to inducing type I interferon re-
sponses than lymphoid tissues), an overall increased proin-
flammatory innate/effector response was observed following
infection for all tissues examined (3). Similarly increased levels
of innate/effector genes were previously obtained from vaginal
mucosa following vaginal transmission (4). The study results
shown here are distinct from those of previous studies because
mucosal tissues were acquired for analysis while following the
macaques until they progressed to simian AIDS. Unfortu-
nately, this study design limited the number of macaques that
could be assessed; however, the diverse range of outcomes
permitted an analysis of macaques that progressed to disease
rapidly or at a relatively normal rate (intermediate progres-
sors), as well as one slow progressor. This is the first study, to
our knowledge, that combines an assessment of mucosal and
lymphatic innate/effector genes, SIV-specific adaptive immu-
nity, and disease outcome such that associations between these
factors can be identified.
Comparing cytokine/chemokine expression patterns with
SIV-specific antibody responses and rates of disease progres-
sion permitted assessment of multiple events occurring in the
orally inoculated SIV macaques throughout the acute and
chronic phases of infection. The rapid progressor RM11 up-
regulated only TNF-? expression 2 days postinfection in the
oral mucosa, while the slow progressor RM16 upregulated
eight genes (encoding IFN-?, OAS, CXCL9, CXCL10, IL-12,
IL-10, TNF-?, and MIP-1a) (Table 2). The intermediate pro-
gressors showed an increase in two to four of the gene mRNA
levels assessed in the oral mucosa at 2 to 4 days postinfection
and were associated with intermediate set point viral loads
(Fig. 1). These intermediate progressors contained a pheno-
type that was intermediate between those of the contrasting
rapid and slow progressors, and therefore provided additional
confirmation that these findings reflect a phenomenon that will
be observed in future SIV/macaque studies. The lower gastro-
intestinal tract, including the rectal mucosa, has been a site of
intense investigation due to the rapid, extensive depletion of
CD4?T cells from this mucosal site (11, 24, 30, 34, 36, 45, 58).
It is interesting to note in this study that different mucosal sites
within the same macaque can respond similarly to infection
with regard to a subset of innate/effector genes. Similar in-
creases in CXCL9 and CXCL10 observed here at the oral and
rectal mucosa have also been observed in the lung tissues of
acutely and chronically SIV-infected macaques (53). In con-
trast, a disparate regulation of IFN-? expression was observed
at these two sites as increased expression at the oral gingiva of
the slower-progressing macaques occurred at times when the
rectal biopsies exhibited similar or decreased IFN-? levels
compared to those of the uninfected macaques. The reduced
expression of IFN-? in the rectal tissue may be indicative of an
early innate immune dysfunction possibly contributing to the
rapid depletion of CD4?T cells from the gut or may simply
indicate an inherent distinction between the different mucosal
sites. In contrast, studies assessing lymph nodes and PBMC
innate immune responses in SIV-infected macaques deter-
mined that increased levels of cytokine/chemokine expression
correlate with a poor disease prognosis (typically measured by
increased viral loads) (1, 31, 49). High levels of both CXCL9
and CXCL10 in the lymph nodes were generally associated
with higher viral loads, a poor SIV Env-specific antibody re-
sponse, and a faster rate of disease progression. These data
indicate that the rapid progressor was likely responding to the
infection by producing proinflammatory chemokines in the
lymph nodes; however, this response was not effective. Among
the cell types potentially recruited by CXCL9 and CXCL10 are
activated CD4?T cells that might serve as additional target
cells for the virus, providing an explanation for the higher
plasma viral load in these macaques (RM11, RM12, RM13,
RM14, and RM15). In contrast, high levels of CXCL9/
CXCL10 expression at the mucosal sites may help to slow the
spread of the virus from the portal of entry to secondary
lymphoid tissues, allowing the host time to mount a more
effective SIV-specific immune response, as was observed in the
slow progressor RM16. These data indicate that the timing and
the anatomical location of proinflammatory cytokine/chemo-
kine expression during acute infection may impact the levels of
viral replication, SIV-specific immune responses, and rate of
During chronic HIV/SIV infection, the onset of AIDS is
generally associated with opportunistic infections (e.g., oral
candidiasis, Pneumocystis pneumonia, and enteric cryptospo-
VOL. 81, 2007INNATE MUCOSAL IMMUNITY FOLLOWING ORAL SIV INOCULATION6183
ridiosis) of mucosal tissues. We hypothesized that maintaining
mucosal expression of cytokines and chemokines during
chronic infection would be beneficial by preventing the onset
of opportunistic infections. Indeed, our findings indicate that
the rapid progressor RM11 developed an opportunistic infec-
tion at 70 d.p.i. (Table 1), at which time levels of many of the
cytokines/chemokines, including CXCL9 and CXCL10, were
similar to or lower than levels observed for uninfected ma-
caques. The potential benefit of elevated cytokine/chemokine
levels at mucosal sites was determined to be statistically sig-
nificant due to a correlation of higher gene expression in gin-
gival (P ? 0.0333) and rectal (P ? 0.0167) mucosae during
chronic infection (70 d.p.i.) and delayed onset of AIDS-related
death. In contrast, these and other studies have observed a
poor prognosis associated with high levels of proinflammatory
cytokine/chemokine expression in the lymph nodes during
chronic infection (1, 31, 49). These data indicate that if the site
where high levels of CXCL9 and CXCL10 are expressed is a
replication site for HIV/SIV, then recruitment of additional
activated CD4?cells may be detrimental; however, if the site
is important for inhibiting opportunistic infections (mucosal
tissues), then the recruitment of activated CD4?T cells, as well
as of other effector cells such as NK cells, may be beneficial.
This dichotomy of elevated chemokine levels between the dif-
ferent anatomical sites provides a rationale for assessing these
chemokines in future studies.
These data highlight a potentially important role for innate/
effector molecules during the first few days postexposure and
may explain why mucosal transmission of HIV is a relatively
rare event in humans (19, 21). Our findings suggest that the
timing and magnitude of the innate immune response at the
site of inoculation (oral gingiva) play potentially important
roles in eliciting the anti-SIV immune response and slowing
progression to simian AIDS. In this light, these data provide a
rationale for upregulating interferons and interferon-stimu-
lated innate/effector gene expression to increase mucosal effi-
cacy of HIV vaccines. Indeed, providing PAMPs, such as CpG
motifs, during a vaccine administration has been demonstrated
in some studies to boost both innate and adaptive mucosal
immune responses and prevent infection following SIV chal-
lenge (12, 29). However, mucosal immune activation would
likely need to be undertaken in a careful manner, as the ap-
plication of imiquimod (Toll-like receptor 9 agonist) or CpGs
(Toll-like receptor 7 agonist) to the vaginal mucosa 30 min
prior to SIV administration resulted in increased plasma viral
loads, indicating that the immune activation favored viral rep-
lication (60). Our studies also suggest that disease progression
may be inhibited during the chronic stages of SIV infection due
to sustained expression of mucosal cytokines/chemokines, par-
ticularly the chemokines CXCL9 and CXCL10. Additional
studies in the SIV?macaques and HIV?humans are needed
to more definitively address whether the cytokines/chemokines
assessed here (e.g., CXCL9 and CXCL10) are important for
maintaining the proper threshold of CD4?effector T cells at
the mucosa as described by Picker et al. (45), potentially illu-
minating new approaches to recruit T cells back to the severely
depleted mucosal sites. In addition, our data suggest that as-
sessing mucosal sites by assessing expression levels of the in-
CXCL10, OAS, IL-12, and IFN-? may be useful as an indicator
of immunologic health. As such, these studies provide insights
as to the direction of future studies to further assess the role of
mucosal and lymphoid cytokine/chemokine responses in dis-
ease progression following SIV/HIV infections.
These studies were supported by NIH grant P51 RR00169 to the
Immunology Core of the California National Primate Research Center
(CNPRC), by base operating grant RR00169 to CNPRC, and by NIH
grants DE12926 and AI35522 (to D.L.S.).
We thank Marta Marthas for help with the animal study design and
undertaking of these experiments. Also, we acknowledge the excellent
animal care and veterinary staff at the California National Primate
Research Center, where the macaque experiments were performed.
We also acknowledge Kristina Abel and the Immunology Core of the
California National Primate Research Center for undertaking the in-
nate/effector mRNA expression analysis. We also thank Alagar
Muthukumar, David Kosub, Amanda Leone, and Kiran Mir for careful
readings of the manuscript.
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