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*
Tulane Regional Primate Research Center, Covington, LA 70433; and
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent studies of mucosal immune responses in SIV-infected macaques have demonstrated that this system will be highly useful in identifying protective immunity at the mucosal surface. SIV-specific CTL have been identified in both the vaginal and intestinal mucosae of chronically infected macaques (3, 4). Moreover, macaques chronically infected with attenuated virus have been shown to be protected against rectal challenge (5). Transient infections, whereby monkeys have only sporadic evidence of SIV in the periphery but remain seronegative, have also been demonstrated following mucosal exposure to limiting doses of virus (6, 7, 8, 9). These animals may have immune responses that remain localized to the mucosa because several investigators have now observed that these animals are protected from mucosal challenge (7, 8). A direct correlation between mucosal immunity and protection, however, has not been established. Toward this end, we have performed a series of colonic exposures with limiting doses of two molecular clones (SIVmac239 and SIV/17E-Fr) and found an absolute correlation (4 of 4 vs 0 of 7 monkeys) between the selective induction of an MHC class I-restricted CTL response directed against the viral env in the jejunal lamina propria (LP)6 and protection following colonic challenge with the heterologous primary isolate SIV/DeltaB670.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Twenty-three adult Indian origin rhesus monkeys (Macaca mulatta) of either sex were involved in this study. Observation of activity, stool consistency, appetite, and general condition was performed daily. Physical examinations, performed weekly for the first four weeks postinoculation and monthly thereafter, consisted of body temperature and weight measurements, palpation and size grading of lymph nodes and spleen, abdominal palpation, and assessment of general condition. Whole blood was collected at the time of physical examination for diagnostic PCR, flow cytometry, viral coculture, ELISA, and p27 antigenemia assays. Appropriate treatment was instituted as necessary, and moribund animals were humanely sacrificed with an i.v. barbiturate overdose.
Viral isolates
Three virus stocks were used to infect the macaques in these studies: SIV/DeltaB670rh, SIV/17E-Fr, and SIVmac239. SIV/DeltaB670rh was obtained by a single passage of SIV/DeltaB670hu stock onto rhesus PHA-stimulated blasts. SIV/DeltaB670hu was obtained by coculturing lymph node tissue from SIV-infected monkey B670 with primary human PHA-stimulated PBMC (PHA blasts). SIVmac239 is an infectious molecular clone that is primarily T cell tropic (10). SIV/17E-Fr is an infectious, molecular congenic clone of SIVmac239 that is macrophage-tropic (kindly provided by J. Clements Johns Hopkins University, Baltimore, MD) (11, 12).
Animal inoculations
Animals were treated with warm water enemas and Golytely (Raintree Laboratories, Raintree, MA) the day prior inoculation. The day of inoculation the animals were chemically restrained with ketamine HCl (10 mg/kg) and treated with Torbutrol (0.05 mg/kg) to minimize pain. An Olympus flexible fiberoptic endoscope (model GIF type PQ20) was used to intracolonically inoculate the animals. The endoscope was introduced rectally and advanced approximately 50 cm in an oral direction to the transverse colon where inoculations of virus stock occurred. Following inoculation, each macaque underwent a series of three ventral abdominal midline celiotomies for the purpose of collecting jejunum and mesenteric lymph nodes. One day before surgery all animals were treated with warm water enemas and Golytely and started on the following treatments: Kefzol (25 mg/kg twice daily for 5 days intramuscularly), lactobacillus paste (1.0 ml per day, mouth), and Torbutrol (0.05 mg/kg three times daily for 3 days i.m.). For surgery, the animals were preanesthetized with glycopyrrolate and acepromazine, induced with Ketamine HCl, and anesthesia was maintained with isoflurane and O2. One or four months following the last survival surgery, each animal was challenged intracolonically with 1.0 ml containing 10,000 TCID50 of SIV/DeltaB670rh. The same routine described for the initial inoculations was followed for the challenge inoculations.
Isolation of lamina propria and mesenteric lymph node mononuclear cells
Mononuclear cells (MNC) from the jejunal LP were isolated using a neutral protease method (13). Briefly, minced 20-cm sections of tissue were treated with 1 mM DTT (Sigma, St. Louis, MO) for 20 min at 37°C followed by incubation in calcium/magnesium-free PBS containing 0.75 mM EDTA and 5% FCS four times for 30 min at 37°C to release intraepithelial lymphocytes (IEL), which were pooled and placed in Iscove’s modified Dulbecco’s media (Life Technologies, Grand Island, NY), 20% FCS plus Abs, and 0.25 µg/ml Fungizone (Life Technologies) overnight at room temperature. The tissues were sliced into smaller pieces and digested with 1.5 mg/ml Dispase, Grade II (Boehringer Mannheim, Indianapolis, IN) for 30 min. The supernatant containing dissociated cells was collected, and the treatment was repeated four more times. The pooled LP cells were resuspended in Iscove’s modified Dulbecco’s media overnight at 37°C to reexpress CD8 molecules. Mesenteric lymph nodes (MLN) were teased to release the MNC and placed in Iscove’s media (Life Technologies) overnight at room temperature. PBMC were isolated the day of surgery and held overnight as described for MLN. MNC from IEL were passed through glass wool columns followed by discontinuous gradient centrifugation on Percoll (14) (Pharmacia, Piscataway, NJ). Cells from the LP were also purified by Percoll centrifugation. PBMC and MNC from lymph nodes were purified by Ficoll-Paque (Pharmacia) density barrier centrifugation.
Flow cytometry
Lymphocyte subsets and monocyte/macrophages were directly stained per manufacturer’s instructions with FITC- or phycoerythrin (PE or RD-1)-conjugated mAbs specific for human subsets, including CD20 (B1-FITC), CD2 (T11-RD1), CD29 (4B4-RD1) (Coulter Immunology, Hialeah, FL); CD4 (OKT4-FITC) (Ortho Diagnostics, Raritan, NJ); CD8 (Leu-2a-FITC or -PE), CD14 (Leu-M3-PE), and CD16 (Lea 11a-FITC) (Becton Dickinson, San Jose, CA). MNC subset compositions of various stained cell preparations were evaluated by flow cytometry using either EPICS 541 (Coulter, Irvine, TX) or FACSCalibur (Becton Dickinson) instruments. Lymphocytes and/or monocyte/macrophages were gated based on light scatter characteristics, and percentages of stained cells were determined relative to labeled isotype controls (Becton Dickinson). For two-color evaluations, the electronic compensation for spectral overlap was set using lymphocyte preparations stained with mutually exclusive OKT4-FITC and Leu-2a-PE.
Quantitation of CTL
Effector cells. Potential effector MNC from blood and mesenteric lymph nodes were purified by Ficoll-Paque (Pharmacia) barrier density centrifugation. MNC from jejunal LP and IEL were purified on discontinuous gradients of 20, 44, and 67% Percoll (Pharmacia) and washed twice with RPMI 1640 (Life Technologies) supplemented with antibiotics, 2 mM L-glutamine, and 2% human AB serum (Irvine Scientific, Santa Anna, CA) before resuspending them at 2 x 106/ml in the RPMI 1640:Click’s (Life Technologies) (1:1) medium with 0.04 mM 2-ME (Sigma), 10 ng/ml IL-7 (R&D Systems, Minneapolis, MN), and 2 x 105 PFU/ml (0.1 PFU/cell) of each of the following recombinant vaccinia viruses encoding sequences for SIVmac251 gp160 (env-vaccinia), gag and protease (gag-vaccinia), and pol (pol-vaccinia) (kindly provided by G. P. Mazzara and D. Panicali, Therion Biologic Corp., Cambridge, MA). The cells were incubated in culture tubes or 75-cm2 flasks at 37°C in 5% CO2. After 3 days, half of the medium was replaced with the RPMI 1640:Click’s medium without IL-7 and the recombinant viruses but including 4 U/ml recombinant IL-2 (Hoffmann-La Roche, Nutley, NJ) for the remaining 4 days of culture.
Target cells. PBMC were isolated by Ficoll-Paque density gradient centrifugation and transformed by exposure to rhesus EBV (15) from a persistently infected cell line. Transformed B cells were maintained on RPMI 1640 supplemented with 2 mM L-glutamine, antibiotics, and 15% FCS. The day before the CTL assay, autologous and MHC class I mismatched B cell lines, as determined by isoelectric focusing (16), were infected with either wild-type vaccinia virus or recombinant viruses env-vaccinia, gag-vaccinia, or pol-vaccinia in 200 µl 2.5 x 108 PFU/ml for each 2 x 106 cells (25 PFU/cell) for 2 h at 37°C, adjusted to 1 x 106/ml, and incubated at 37°C in 5% CO2 for 16 h. Except for the portion of wild-type vaccinia-infected cells to be used for "cold" targets, aliquots of all infected cells were labeled with Na2CrO4 (50 µCi/106 cells) (DuPont NEN Research Products, Boston, MA) for 1 h at 37°C. The cell suspensions were then centrifuged through FCS barriers, washed two times, and resuspended to 5 x 105/ml.
CTL assay.
Cultured effector cells were pooled, subjected to positive selection of
CD8+ cells using Dynabeads M450 with Detachabead (Dynal,
Great Neck, NY) as per manufacturer’s instructions. Various
concentrations of CD8+ effector cells were added to 5
x 103 51Cr-labeled target cells to achieve E:T
ratios of 20:1, 10:1, or 5:1 along with 1 x 105
"cold" targets in 96-well U-bottom plates to a total of 100 µl.
Wild-type vaccinia-infected cold targets were used to reduce the effect
of any existing anti-EBV or -vaccinia effector cells. However,
since fixed recombinant vaccinia-infected EBV-transformed cell lines
were not used to stimulate cultures, this effect was dependent on the
individual animal and was minimal. Cold targets were also added to the
spontaneous 51Cr-release controls. Medium and 0.5% sodium
desoxycholate were added in place of effector cells to spontaneous and
maximum 51Cr-release controls, respectively. After 1 h
incubation at 37°C, an additional 100 µl of medium was added, and
the plates were incubated another 4 h. Supernatants were collected
with the Skatron Supernatant Collecting System (Skatron, Sterling, VA)
and counted on a Packard Cobra II Auto
solid crystal scintillation
system (Packard Instrument, Downers Grove, IL). Spontaneous
51Cr release was always less than 20% of the maximum
51Cr release.
Percent specific 51Cr release (Fig. 1
A for PBMC and 1C
for LP) was calculated using the following formula:
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B
for PBMC and 1D for LP). A difference >10% at the 20:1 E:T
ratio was considered to be positive. As a means of quantitating
relative responses, LUs were obtained by first extrapolating to an
estimated E:T ratio that would give 15% SIV Ag-specific lysis (Fig. 1, B and D). Using the estimated 15% E:T ratio,
15% LUs per 106 purified CD8+ effector cells
after culture were calculated as follows:
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or
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Therefore:
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SIV-specific serum Ab
SIV-specific gp120 Ab responses were identified in serum by an ELISA using Con A-captured baculovirus-produced SIV/DeltaB670 gp120 as described (17). Recombinant SIV gp120 was kindly provided by Dr. Ronald Montelaro at the University of Pittsburgh School of Medicine.
SIV p27 assay
Antigenemia was determined by measuring the levels of SIV p27 Ag in serum using a commercially available enzyme-linked immunoassay kit specific for SIV (Coulter, Hialeah, FL).
SIV-specific IgA ELISPOT assay
Nitrocellulose HA 96-well plates (Millipore, Bedford, MA) were coated with 0.2 µg SIV envelope recombinant gp140 (kindly provided by Drs. K. Javaherian and G. LaRosa, Repligen, Cambridge, MA) in 100 µl PBS and placed at 4°C overnight. After washing five times with PBS, the wells were blocked with a milk solution (Kirkegaard & Perry Laboratories, Gaithersburg, MD) and incubated for 4 h at 4°C. Following further washing with PBS, various numbers of MNC from jejunum in RPMI 1640 with 10% human AB serum were added to wells and incubated overnight at 37°C in 5% CO2. Detection of B lymphocytes secreting SIV envelope-specific IgA Abs was accomplished by incubating the plates 4 h at 37°C with goat anti-monkey IgA conjugated with peroxidase (Nordic Immunological Labs, Capistrano Beach, CA) after first washing with PBS and PBS with 0.05% Tween 20 (Sigma). Following extensive washing with PBS-Tween (10 times), development was accomplished with TMB membrane peroxidase substrate (Kirkegaard & Perry) for about 30 min at room temperature. The plates were then washed with water and dried overnight at 4°C. Spots representing gp140-specific IgA-secreting B lymphocytes were enumerated with a binocular dissecting scope.
Detection of SIV
Identification of SIV in PBMC was performed by nested PCR utilizing conserved primers specific for the viral long terminal repeat (LTR) as described (18). When identification of specific SIV genomes was desired, the first hypervariable region of the viral gp120 was PCR amplified using conserved sequences flanking this region, cloned in a TA cloning vector (Inbitrogen, San Diego, CA), and sequenced as described (18).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peripheral blood was sampled at weekly intervals postinoculation (p.i.) to monitor the appearance of SIV and SIV-specific Ab. At 3 and/or 8 wk p.i., CTL responses were also measured in both the peripheral blood and in the gut-associated tissues. In some cases, the presence of SIV-specific IgA-producing B cells was also enumerated in the gut-associated tissues. For analysis of gut-specific immunity, 20-cm sections of jejunum and colonic lymph node(s) were obtained for analysis. Jejunal tissue was selected because it can be surgically excised without adversely affecting the health of the animal. This procedure allowed serial collection of tissue so that a causal relationship between gut-specific immunity and protection from experimental challenge could be obtained. The jejunum is appropriate for analysis of effector responses because, once Ag-specific effectors become primed and pass through the regional draining lymph nodes, they are redistributed to the epithelium and lamina propria that line the entire intestine (19, 20, 21).
Infection outcome of colonic exposure to limiting doses of SIVmac239 and SIV/17E-Fr
Two doses of each virus preparation were utilized in two separate
experiments so that a spectrum of no infection 
transient infection
disseminated infection could be achieved with each of the clones.
The first experiment evaluated the outcome of animals receiving either
10, 100, or 1000 TCID50 of either SIVmac239 or SIV/17E-Fr.
A second experiment was later initiated to expand the number of monkeys
exposed to the higher dose (1000 TCID50) of SIV/17E-Fr.
Assisted by an endoscope, 1 ml of appropriately diluted cell-free
culture supernatant was atraumatically inoculated into the lumen of the
transverse colon. This site was chosen to assure accurate delivery of
the virus dose, to avoid self-inoculation of leaked virus into other
mucosal sites, and to minimize inadvertent i.v. inoculation of virus by
exposure of existing rectal abrasions to the inoculum.
One monkey died of causes unrelated to virus exposure; infection
outcome in the remaining 11 monkeys is shown in Table I. One of four monkeys exposed to
SIVmac239 (monkey M224) and two of seven animals exposed to SIV/17E-Fr
(monkeys N385 and N138) developed a disseminated infection as
determined by repeated identification of viral sequences in PBMC by PCR
amplification. PCR was used to identify virus in the periphery because,
in our hands, it is the most sensitive measurement of SIV in the
infected animal. Three of the remaining monkeys exposed to SIVmac239
(monkeys N255, M029, and L870) were sporadically PCR positive. No virus
was detected by PCR in five of the monkeys exposed to SIV/17E-Fr (L987,
M259, M577, N041, and M155).
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MNC were purified from the peripheral blood (PBMC), jejunal LP,
and MLN draining the large intestine. Flow cytometric evaluation showed
that the composition of MNC found in these tissues was similar, with
several minor exceptions (Fig. 2). All
three organs had significant populations of T and B lymphocytes and
macrophages as measured by surface expression of CD2, CD20, and CD14,
respectively, with the exception that a higher percentage of B cells
was found in MLN than in the other two organs. All organs had
significant populations of both CD4+ (helper) and
CD8+ (suppressor-cytotoxic) subsets of T lymphocytes. The
ratio of CD4+ to CD8+ lymphocytes, however, was
higher in MLN and LP than that found in PBMC. These findings,
consistent with previously published data (4), assured us we were not
deleteriously affecting these populations during the purification
process.
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) (22). Conversely, none of the monkeys
that were either sporadically PCR positive or PCR negative had
detectable Ab in the serum by conventional ELISA.
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). Interestingly, SIV
was identified in the PBMC of this animal at only one time point (4 wk
p.i.), and it remained seronegative. Ag-specific T cell proliferative
responses were uniformly negative among all LP samples tested
(data not shown); these results were not unexpected given the apparent
inability of T effectors from the LP to proliferate in response to Ag
(23).
Purified CD8+ T lymphocytes from PBMC, LP, and MLN were
also evaluated for cytolytic activity using EBV-transformed autologous
and allogeneic mismatched targets infected with vaccinia recombinant
viruses containing SIV env, gag, and pol at 3 and/or 8 wk postexposure.
These data are presented as LU in Table II. Graphic presentation of SIV
Ag-specific 51Chromium release at E:T ratios of 20:1, 10:1,
and 5:1 are shown for representative animals in Fig. 3.
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12 LU) of pol- or gag -specific CTL were
observed in monkeys M029 and M259, respectively. Neither of these
animals was persistently infected. A higher level (
33 LU) of CTL
activity specific for both env and gag was observed in the PBMC of
monkey N385. This result was not surprising, given that this animal was
persistently infected with SIV/17E-Fr, an attenuated strain that is a
potent inducer of CTL in the periphery (our unpublished
observations). CTL activity was not detected in the PBMC of the
other two persistently infected monkeys (monkeys N138 and M224). The
failure to identify CTL responses in all monkeys infected with SIV is
consistent with the observations of others (24) and may be explained by
an MHC nonresponder haplotype in these animals.
With respect to the mesenteric lymph nodes, only monkey M577 had
demonstrable CTL in this organ among the seven monkeys evaluated (Table II). In contrast, CTL activity was frequently detected in the jejunal
LP that, with one exception (monkey N385), was directed solely against
viral env determinants. These responses could be divided into two
categories: a low level of activity (8 LU), as observed in monkeys
N255 and M029, and a high level of activity (17 LU), as observed in
monkeys L870 (Fig. 3, A and B), M577 (Fig. 3, C and D), N041 (Fig. 3E), and N385
(Fig. 3F). This response was a striking finding because
three of these animals (monkeys L870, M577, and N041) had little or no
evidence of virus in the periphery.
MHC class I restriction of CTL
We routinely employ CD8+ purified effector cells that,
by flow cytometric analysis, are >90% CD2+
CD8+ and CD16- (data not shown). In our hands,
cytolysis by PBMC effectors is always MHC class I restricted. The
CD8+ population in the intestinal mucosa may differ from
that in the blood, however, since mucosal tissue is known to be
enriched for TCR 
, CD8+ T cells that do not require
MHC class I recognition for cytolysis (25). To address this issue, CTL
activity was assessed in LP taken 3 wk before exposure and compared
with the results obtained 8 wk postexposure against autologous and
allogeneic-mismatched targets expressing either SIV gag or env. CTL
activity detected in PBMC and LP before infection is shown for the
representative monkey N385 in Fig. 4. As
expected, no cytolysis of either autologous or mismatched targets was
observed in the PBMC of the naive animal (Fig. 4A). By 8 wk
p.i., significant activity against both env and gag targets was
observed, but these responses were restricted to autologous targets, a
finding that confirms a requirement for MHC class I recognition (Table II). In contrast, a low level of CTL activity against env, but not gag,
was detected in the jejunal LP before SIV exposure (Fig. 4B). This activity was not MHC class I restricted, however,
a finding suggesting the presence of functionally active TCR ,
CD8+ T lymphocytes in the LP of this animal. At 8 wk
postexposure, CTL activity against env was significantly higher in the
LP, with activity against gag also apparent (Fig. 3F and
Table II). In contrast to that observed before virus exposure, this
activity required MHC class I recognition. A similar pattern was
observed in monkey N041 (data not shown). CTL activity was also
analyzed in IEL. One animal (monkey N041) had env-specific CTL in IEL,
which mirrored that seen in LPL. The remaining monkeys had no
detectable response (data not shown).
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The relationship of the immune responses observed in animals
mucosally exposed to the two SIV clones, particularly those identified
in the intestinal LP, with protection against mucosal challenge was
further assessed by colonic challenge with a 100% infectious dose
(10,000 TCID50) of the heterologous primary isolate,
SIV/DeltaB670. Monkeys in experiment I were challenged 6 mo
postexposure, whereas monkeys in experiment II were challenged 3 mo
postexposure (4 and 1 mo, respectively, from the last evaluation of
immune responses in the gut-associated tissues) (Table III). Four naive animals were included as
infection controls; all four of these animals became persistently PCR
positive by 7 days postchallenge. Three of the infected control animals
responded to virus infection with the production of virus-specific Ab
in the periphery. One monkey (animal L821) failed to seroconvert, a
finding consistent with the rapid and severe disease noted soon after
infection (see below).
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Three monkeys were PCR positive before challenge (monkeys M224, N385, and N138). Two of these animals subsequently developed AIDS in a manner similar to that observed in the challenge controls. One animal (monkey N385), however, failed to develop disease (see below); this animal had significant levels of CTL activity in the LP before challenge. This finding prompted us to analyze the virus found in this animal postchallenge at the sequence level. For this analysis, the V1 region of the viral gp120 from PBMC obtained at 2, 4, and 8 wk postchallenge was PCR amplified, cloned, and sequenced. At 2 wk postchallenge, both the immunizing strain SIV/17E-Fr (seven clones) and the challenge strain SIV/DeltaB670 (three clones) were found. By 8 wk, however, only SIV/17E-Fr could be detected among 17 clones sequenced, a finding that suggests transient infection with the challenge strain (data not shown).
Analysis of SIV-specific CTL activity in the jejunal LP and/or PBMC was
repeated at 3 wk postchallenge to determine the persistence of these
responses and to relate these responses to the outcome of mucosal
challenge. CTL activity was identified in only one control animal
(monkey M558). This activity was env specific and detected only in PBMC
(Table III). In the two monkeys where LP was analyzed postchallenge
(animals L870 and M577), the CTL activity observed before challenge was
again observed after challenge. The inclusion of allogeneic-mismatched
targets in these assays demonstrated that the responses were MHC class
I restricted. Lymphocytes from the LP taken from the other two monkeys
that had CTL in LP before challenge (animals N041 and N385) were
unavailable for analysis; however, both animals had detectable CTL in
PBMC. Like that observed before challenge, analysis of CTL in the LP
for MHC class I restriction revealed activity of two types: a larger,
MHC class I-restricted component, and a smaller, MHC class
I-unrestricted component. This latter activity, however, was apparently
localized to the LP because it was not found in the PBMC of these
animals, nor has it been seen in other studies (data not shown). In
monkeys M577 and N041, CTL activity was also seen for the first time in
the peripheral blood, a finding that may suggest boosting of these
responses by challenge, even though the challenge virus was never
detected in the periphery.
To confirm our virological assessment of protection following
challenge, monkeys were also monitored clinically for signs of disease
progression. Flow cytometric enumeration of lymphocyte subsets and
SIV-specific p26 antigenemia are shown over time postchallenge for
these animals in Figs. 5-7. All 4 control
monkeys developed progressive disease characterized by a selective
decline in CD4+CD29+ helper/inducer T
lymphocytes (monkeys L821 and M558; Fig. 5, A and
C) or total CD4+ lymphocytes (monkeys L632 and
M079; Fig. 5, B and D) and died of
AIDS-associated illness by 450 days postinfection. Like that observed
previously (26, 27), the selective decrease in
CD4+CD29+ T lymphocytes observed in these
animals was an early indicator of rapid disease progression.
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. Monkey M224 (Fig. 6A),
which became persistently infected after exposure to SIVmac239, had a
small peak in antigenemia 14 days postexposure, which coincided with a
decline in CD4+ lymphocytes and an increase in
CD8+ T lymphocytes. The decline in CD4+ T
lymphocytes persisted, with the increase in CD8+ T
lymphocytes subsequently declining to preinoculation levels by the time
the animal was challenged with SIV/DeltaB670 6 mo later. As expected,
monkeys N255 and M029 showed no change in T lymphocytes until
challenge. Following challenge, monkey N255 developed rapid disease
characterized by an SIV p26 antigenemic peak of over 47 ng/ml, a
selective decline in CD4+CD29+ helper/inducer T
lymphocytes and an increase in CD8+ T suppressor/cytotoxic
lymphocytes, and died 119 days later (Fig. 6B). Monkey M029
developed disease more slowly and displayed a slow decline in
CD4+ T lymphocytes and elevated levels of CD8+
lymphocytes (Fig. 6C). This animal was sacrificed at 481
days postinfection for reasons unrelated to disease. In striking
contrast to the other animals, the only change noted in monkey L870,
which was never virus positive, was a transient decline in
CD4+CD29+ helper-inducer lymphocytes during the
time of challenge, but these numbers returned to normal levels
thereafter (Fig. 6D).
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), only three
monkeys showed no significant changes in T cell subsets in response to
either immunization or challenge. These results are in agreement with
the virological assessment of infection. Taken together, these data
further demonstrate a correlation of the induction of env-specific, MHC
class I-restricted CTL in the LP with protection from disease
progression following colonic challenge.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transient infection of the mucosal tissues induced SIV-specific responses that remained localized to the jejunal lamina propria. A persistent level of SIV-specific IgA-secreting B cells was detected in the lamina propria of one animal (monkey N255) and a strong CTL response against viral env determinants in three others (monkeys L870, M577, and N041). A CTL response to both gag and env determinants was also detected in the lamina propria of one persistently infected monkey (animal N385). The incorporation of allogeneic-mismatched targets into the assay further demonstrated that the CTL activity observed in the lamina propria was MHC class I restricted. Although CTL responses have been identified in both the intestinal (4) and vaginal (3) mucosa of chronically infected monkeys, this is the first report to our knowledge of SIV-specific mucosal immunity induced by transient infection. This finding may be analogous to HIV-exposed humans who remain seronegative despite repeated sexual exposure (1, 2).
Colonic challenge with 100% infectious dose of SIV/DeltaB670 resulted in infection and disease in all four naive controls and all of the exposed animals (7 of 11 monkeys tested) that had little or no detectable CTL in the lamina propria before or following infection. Conversely, none of the monkeys (4 of 11 animals tested) that had strong, env-specific CTL in the lamina propria became persistently infected with the challenge virus or developed disease. In three of the protected animals, no other detectable SIV-specific immune responses were identified in either the periphery or the gut-associated tissues. Taken together, these data provide compelling evidence for the requirement of virus-specific MHC class I CTL in mucosal protection.
Although most of the CTL responses observed in the lamina propria
in protected animals was MHC class I restricted, a portion of this
activity did not require MHC class I recognition. Since this activity
was identified only in this tissue, it is possible that
CD8+ effectors with a , rather than an 
ß, TCR
phenotype were responsible. Confirmation of this hypothesis will
require a more detailed analysis of these populations using mAbs
specific for each phenotype. This finding is noteworthy, however, in
that, where this activity was detected before SIV exposure, its
presence correlated with the subsequent induction of higher levels of
MHC class I-restricted CTL after exposure. Whether this is a
prerequisite for the induction of TCR ß effector cells required
for protection is a topic for further investigation.
The common denominator for mucosal protection observed in this study was CTL recognition of viral env determinants. The preferential induction of env-specific responses following colonic exposure to limiting virus doses is intriguing. Perhaps the preferential uptake of virus by dendritic cells in the mucosa, coupled with their superior Ag-presenting capabilities, permitted the induction of this response even though only small amounts of Ag were produced. Infection of these cells may not initially be productive, a condition that might promote the preferential expression of env proteins. This finding may be important because, unlike the systemic protection observed following infection with attenuated SIV (17, 28), protection was induced by low dose, transient expression of virus. Furthermore, a correlation with protection and a response to a single virus protein was observed. Taken together, these observations should rapidly facilitate the development and implementation of safer, noninfectious vaccine approaches such as that described in a recent report of mucosal protection in mice by induction of env-specific CTL following immunization with a synthetic multideterminant HIV gp160 peptide (29).
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
3 Address correspondence and reprint requests to Dr. Michael Murphey-Corb, University of Pittsburgh School of Medicine, Department of Molecular Genetics and Biochemistry, BST Room 1240, Pittsburgh, PA 15261. E-mail address:
4 Indicated authors shared equally in this project and should be considered co-first authors.
5 Current address: Columbia University College of Surgeons and Physicians, Department of Anesthesiology, St. Luke’s-Roosevelt Hospital, 1111 Amsterdam Avenue, New York, NY 10025.
6 Abbreviations used in this paper: LP, lamina propria; MNC, mononuclear cells; IEL, intraepithelial lymphocytes; MLN, mesenteric lymph nodes; p.i., postinoculation; ELISPOT, enzyme-linked immunospot; PE, phycoerythrin; PFU, plaque-forming unit; TCID50, tissue culture infectious dose 50.
Received for publication March 25, 1998. Accepted for publication September 1, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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  M. D. George, D. Verhoeven, S. Sankaran, T. Glavan, E. Reay, and S. Dandekar Heightened Cytotoxic Responses and Impaired Biogenesis Contribute to Early Pathogenesis in the Oral Mucosa of Simian Immunodeficiency Virus-Infected Rhesus Macaques Clin. Vaccine Immunol., February 1, 2009; 16(2): 277 - 281. [Abstract] [Full Text] [PDF]
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 I. M. Belyakov, D. Isakov, Q. Zhu, A. Dzutsev, D. Klinman, and J. A. Berzofsky Enhancement of CD8+ T Cell Immunity in the Lung by CpG Oligodeoxynucleotides Increases Protective Efficacy of a Modified Vaccinia Ankara Vaccine against Lethal Poxvirus Infection Even in a CD4-Deficient Host J. Immunol., November 1, 2006; 177(9): 6336 - 6343. [Abstract] [Full Text] [PDF]
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 I. M. Belyakov, V. A. Kuznetsov, B. Kelsall, D. Klinman, M. Moniuszko, M. Lemon, P. D. Markham, R. Pal, J. D. Clements, M. G. Lewis, et al. Impact of vaccine-induced mucosal high-avidity CD8+CTLs in delay of AIDS viral dissemination from mucosa Blood, April 15, 2006; 107(8): 3258 - 3264. [Abstract] [Full Text] [PDF]
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P. M. Sheth, A. Danesh, K. Shahabi, A. Rebbapragada, C. Kovacs, R. Dimayuga, R. Halpenny, K. S. MacDonald, T. Mazzulli, D. Kelvin, et al. HIV-Specific CD8+ Lymphocytes in Semen Are Not Associated with Reduced HIV Shedding J. Immunol., October 1, 2005; 175(7): 4789 - 4796. [Abstract] [Full Text] [PDF]
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 F. J. Ibarrondo, P. A. Anton, M. Fuerst, H. L. Ng, J. T. Wong, J. Matud, J. Elliott, R. Shih, M. A. Hausner, C. Price, et al. Parallel Human Immunodeficiency Virus Type 1-Specific CD8+ T-Lymphocyte Responses in Blood and Mucosa during Chronic Infection J. Virol., April 1, 2005; 79(7): 4289 - 4297. [Abstract] [Full Text] [PDF]
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C. Devito, B. Zuber, U. Schroder, R. Benthin, K. Okuda, K. Broliden, B. Wahren, and J. Hinkula Intranasal HIV-1-gp160-DNA/gp41 Peptide Prime-Boost Immunization Regimen in Mice Results in Long-Term HIV-1 Neutralizing Humoral Mucosal and Systemic Immunity J. Immunol., December 1, 2004; 173(11): 7078 - 7089. [Abstract] [Full Text] [PDF]
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H. Zhang, R. Fayad, X. Wang, D. Quinn, and L. Qiao Human Immunodeficiency Virus Type 1 Gag-Specific Mucosal Immunity after Oral Immunization with Papillomavirus Pseudoviruses Encoding Gag J. Virol., October 1, 2004; 78(19): 10249 - 10257. [Abstract] [Full Text] [PDF]
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R. Stevens, K. E. Howard, S. Nordone, M. Burkhard, and G. A. Dean Oral Immunization with Recombinant Listeria monocytogenes Controls Virus Load after Vaginal Challenge with Feline Immunodeficiency Virus J. Virol., August 1, 2004; 78(15): 8210 - 8218. [Abstract] [Full Text] [PDF]
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M. M. Gherardi, E. Perez-Jimenez, J. L. Najera, and M. Esteban Induction of HIV Immunity in the Genital Tract After Intranasal Delivery of a MVA Vector: Enhanced Immunogenicity After DNA Prime-Modified Vaccinia Virus Ankara Boost Immunization Schedule J. Immunol., May 15, 2004; 172(10): 6209 - 6220. [Abstract] [Full Text] [PDF]
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L. Musey, Y. Ding, M. Elizaga, R. Ha, C. Celum, and M. J. McElrath HIV-1 Vaccination Administered Intramuscularly Can Induce Both Systemic and Mucosal T Cell Immunity in HIV-1-Uninfected Individuals J. Immunol., July 15, 2003; 171(2): 1094 - 1101. [Abstract] [Full Text] [PDF]
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B. L. Shacklett, C. A. Cox, J. K. Sandberg, N. H. Stollman, M. A. Jacobson, and D. F. Nixon Trafficking of Human Immunodeficiency Virus Type 1-Specific CD8+ T Cells to Gut-Associated Lymphoid Tissue during Chronic Infection J. Virol., May 15, 2003; 77(10): 5621 - 5631. [Abstract] [Full Text] [PDF]
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C. Peters, X. Peng, D. Douven, Z.-K. Pan, and Y. Paterson The Induction of HIV Gag-Specific CD8+ T Cells in the Spleen and Gut-Associated Lymphoid Tissue by Parenteral or Mucosal Immunization with Recombinant Listeria monocytogenes HIV Gag J. Immunol., May 15, 2003; 170(10): 5176 - 5187. [Abstract] [Full Text] [PDF]
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T. U. Vogel, H. Horton, D. H. Fuller, D. K. Carter, K. Vielhuber, D. H. O'Connor, T. Shipley, J. Fuller, G. Sutter, V. Erfle, et al. Differences Between T Cell Epitopes Recognized After Immunization and After Infection J. Immunol., October 15, 2002; 169(8): 4511 - 4521. [Abstract] [Full Text] [PDF]
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J. Baig, D. B. Levy, P. F. McKay, J. E. Schmitz, S. Santra, R. A. Subbramanian, M. J. Kuroda, M. A. Lifton, D. A. Gorgone, L. S. Wyatt, et al. Elicitation of Simian Immunodeficiency Virus-Specific Cytotoxic T Lymphocytes in Mucosal Compartments of Rhesus Monkeys by Systemic Vaccination J. Virol., October 11, 2002; 76(22): 11484 - 11490. [Abstract] [Full Text] [PDF]
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L. Stevceva, X. Alvarez, A. A. Lackner, E. Tryniszewska, B. Kelsall, J. Nacsa, J. Tartaglia, W. Strober, and G. Franchini Both Mucosal and Systemic Routes of Immunization with the Live, Attenuated NYVAC/Simian Immunodeficiency Virus SIVgpe Recombinant Vaccine Result in Gag-Specific CD8+ T-Cell Responses in Mucosal Tissues of Macaques J. Virol., October 11, 2002; 76(22): 11659 - 11676. [Abstract] [Full Text] [PDF]
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A. Biragyn, I. M. Belyakov, Y.-H. Chow, D. S. Dimitrov, J. A. Berzofsky, and L. W. Kwak DNA vaccines encoding human immunodeficiency virus-1 glycoprotein 120 fusions with proinflammatory chemoattractants induce systemic and mucosal immune responses Blood, July 30, 2002; 100(4): 1153 - 1159. [Abstract] [Full Text] [PDF]
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S. Sabbaj, B. H. Edwards, M. K. Ghosh, K. Semrau, S. Cheelo, D. M. Thea, L. Kuhn, G. D. Ritter, M. J. Mulligan, P. A. Goepfert, et al. Human Immunodeficiency Virus-Specific CD8+ T Cells in Human Breast Milk J. Virol., June 27, 2002; 76(15): 7365 - 7373. [Abstract] [Full Text] [PDF]
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D. H. Fuller, P. A. Rajakumar, L. A. Wilson, A. M. Trichel, J. T. Fuller, T. Shipley, M. S. Wu, K. Weis, C. R. Rinaldo, J. R. Haynes, et al. Induction of Mucosal Protection against Primary, Heterologous Simian Immunodeficiency Virus by a DNA Vaccine J. Virol., March 7, 2002; 76(7): 3309 - 3317. [Abstract] [Full Text] [PDF]
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L. Stevceva, B. Kelsall, J. Nacsa, M. Moniuszko, Z. Hel, E. Tryniszewska, and G. Franchini Cervicovaginal Lamina Propria Lymphocytes: Phenotypic Characterization and Their Importance in Cytotoxic T-Lymphocyte Responses to Simian Immunodeficiency Virus SIVmac251 J. Virol., January 1, 2002; 76(1): 9 - 18. [Abstract] [Full Text] [PDF]
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J. E. Schmitz, R. S. Veazey, M. J. Kuroda, D. B. Levy, A. Seth, K. G. Mansfield, C. E. Nickerson, M. A. Lifton, X. Alvarez, A. A. Lackner, et al. Simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocytes in gastrointestinal tissues of chronically SIV-infected rhesus monkeys Blood, December 15, 2001; 98(13): 3757 - 3761. [Abstract] [Full Text] [PDF]
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Z. Hel, J. Nacsa, B. Kelsall, W.-P. Tsai, N. Letvin, R. W. Parks, E. Tryniszewska, L. Picker, M. G. Lewis, Y. Edghill-Smith, et al. Impairment of Gag-Specific CD8+ T-Cell Function in Mucosal and Systemic Compartments of Simian Immunodeficiency Virus mac251- and Simian-Human Immunodeficiency Virus KU2-Infected Macaques J. Virol., December 1, 2001; 75(23): 11483 - 11495. [Abstract] [Full Text]
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R. S. Veazey, M.-C. Gauduin, K. G. Mansfield, I. C. Tham, J. D. Altman, J. D. Lifson, A. A. Lackner, and R. P. Johnson Emergence and Kinetics of Simian Immunodeficiency Virus-Specific CD8+ T Cells in the Intestines of Macaques during Primary Infection J. Virol., November 1, 2001; 75(21): 10515 - 10519. [Abstract] [Full Text] [PDF]
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M. V. Rayevskaya and F. R. Frankel Systemic Immunity and Mucosal Immunity Are Induced against Human Immunodeficiency Virus Gag Protein in Mice by a New Hyperattenuated Strain of Listeria monocytogenes J. Virol., March 15, 2001; 75(6): 2786 - 2791. [Abstract] [Full Text]
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D. Masopust, J. Jiang, H. Shen, and L. Lefrancois Direct Analysis of the Dynamics of the Intestinal Mucosa CD8 T Cell Response to Systemic Virus Infection J. Immunol., February 15, 2001; 166(4): 2348 - 2356. [Abstract] [Full Text] [PDF]
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J. E. Wigginton and D. Kirschner A Model to Predict Cell-Mediated Immune Regulatory Mechanisms During Human Infection with Mycobacterium tuberculosis J. Immunol., February 1, 2001; 166(3): 1951 - 1967. [Abstract] [Full Text] [PDF]
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I. M. Belyakov, J. D. Ahlers, J. D. Clements, W. Strober, and J. A. Berzofsky Interplay of Cytokines and Adjuvants in the Regulation of Mucosal and Systemic HIV-Specific CTL J. Immunol., December 1, 2000; 165(11): 6454 - 6462. [Abstract] [Full Text] [PDF]
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M. A. Cromwell, R. S. Veazey, J. D. Altman, K. G. Mansfield, R. Glickman, T. M. Allen, D. I. Watkins, A. A. Lackner, and R. P. Johnson Induction of Mucosal Homing Virus-Specific CD8+ T Lymphocytes by Attenuated Simian Immunodeficiency Virus J. Virol., September 15, 2000; 74(18): 8762 - 8766. [Abstract] [Full Text]
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R. Kaul, F. A. Plummer, J. Kimani, T. Dong, P. Kiama, T. Rostron, E. Njagi, K. S. MacDonald, J. J. Bwayo, A. J. McMichael, et al. HIV-1-Specific Mucosal CD8+ Lymphocyte Responses in the Cervix of HIV-1-Resistant Prostitutes in Nairobi J. Immunol., February 1, 2000; 164(3): 1602 - 1611. [Abstract] [Full Text] [PDF]
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, Negative antigenemia values.
Percent T lymphocyte subsets over time postinfection are indicated as
follows: 
, CD4+; x, CD8+; 
,
CD4+CD29+.
