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J Virol. 2003 March; 77(5): 3099–3118.
PMCID: PMC149756

Simian-Human Immunodeficiency Virus SHIV89.6-Induced Protection against Intravaginal Challenge with Pathogenic SIVmac239 Is Independent of the Route of Immunization and Is Associated with a Combination of Cytotoxic T-Lymphocyte and Alpha Interferon Responses

Abstract

Attenuated primate lentivirus vaccines provide the most consistent protection against challenge with pathogenic simian immunodeficiency virus (SIV). Thus, they provide an excellent model to examine the influence of the route of immunization on challenge outcome and to study vaccine-induced protective anti-SIV immune responses. In the present study, rhesus macaques were immunized with live nonpathogenic simian-human immunodeficiency virus (SHIV) 89.6 either intravenously or mucosally (intranasally or intravaginally) and then challenged intravaginally with pathogenic SIVmac239. The route of immunization did not affect mucosal challenge outcome after a prolonged period of systemic infection with the nonpathogenic vaccine virus. Further, protection from the SIV challenge was associated with the induction of multiple host immune effector mechanisms. A comparison of immune responses in vaccinated-protected and vaccinated-unprotected animals revealed that vaccinated-protected animals had higher frequencies of SIV Gag-specific cytotoxic T lymphocytes and gamma interferon (IFN-γ)-secreting cells during the acute phase postchallenge. Vaccinated-protected animals also had a more pronounced increase in peripheral blood mononuclear cell IFN-α mRNA levels than did the vaccinated-unprotected animals in the first few weeks after challenge. Thus, innate as well as cellular anti-SIV immune responses appeared to contribute to the SHIV89.6-induced protection against intravaginal challenge with pathogenic SIVmac239.

In the rhesus macaque model of simian immunodeficiency virus (SIV) infection, attenuated lentivirus vaccines have provided the most consistent protection against systemic and mucosal challenge with pathogenic SIV (6, 15, 17, 54, 98). Although live attenuated lentiviruses may never be used in humans due to safety concerns, understanding the immune mechanisms that confer protection in live attenuated vaccine primate models may be useful for developing other vaccine approaches. Further, the results of these studies will be useful in defining relevant immunological endpoints in clinical trials of human immunodeficiency virus (HIV) vaccines.

The results of prior studies suggest that multiple immune mechanisms contribute to attenuated vaccine-mediated protection against challenge with pathogenic SIV. There is evidence that CD8-mediated cytotoxicity (35, 36) as well as noncytolytic CD8-mediated responses (21) contribute to the control of virus replication. Other studies have shown an association between protection and neutralizing antibodies, the maturation of antibodies (12), or T helper 1 responses (Th1) (20, 90). However, the relative contribution of these various immune effector mechanisms to live attenuated vaccine-induced protective anti-SIV immunity is still unclear.

Chimeric lentiviruses, namely, simian-human immunodeficiency viruses (SHIVs) consisting of an SIVmac239 backbone and HIV type 1 (HIV-1) envelope (env) and regulatory genes, were produced with the goal of developing monkey challenge models that could be used to directly test the efficacy of HIV-1 env-based vaccines. SHIV HXBc2 (45) was constructed by using the vpu, tat, rev, and env genes of HIV-1 IIIB/LAI, which is a prototype of laboratory-adapted T-tropic viruses. A second virus, designated SHIV89.6 (81), was identical to SHIV HXBc2 except for the fragment from KpmI (nucleotide 5925) to BamHI (nucleotide 8053), which encodes the ectodomain of the gp120 and gp41 envelope glycoproteins derived from HIV-1 89.6, a highly cytopathic, M-tropic variant (13). SHIV89.6 can infect rhesus macaques after intravenous (i.v.) and intravaginal (i.vag.) inoculation (51, 81). However, we and other investigators found that insertion of the HIV-1 genes into SIVmac239 dramatically attenuated the highly pathogenic phenotype of SIVmac239. In fact, these SHIVs were attenuated for both pathogenicity and replication in monkeys compared to the parental SIVmac239 (45, 51, 80, 81). Subsequently it was shown that serial passage of these SHIVs in monkeys can be used to produce acutely pathogenic SHIV variants (50a, 80).

In early studies with SHIV89.6 and SHIV HXBc2 (51), the attenuated nature of the SHIVs was noted, and a decision was made to test whether prior infection with these viruses conferred protection from challenge with pathogenic SIVmac239. Compared to attenuated SIVmac deletion mutant vaccines, the SHIV immunization-SIV challenge system provides a unique opportunity to determine if immune responses to variable epitopes in the HIV envelope glycoproteins are a requirement for protection from challenge with SIVmac239. The completely heterologous nature of the envelope gene and some regulatory genes and the homologous nature of the rest of the genome in this vaccine/challenge system emphasize the importance of immune responses to nonenvelope antigens and test the relevance of concerns related to clade-specific vaccines in the setting of live attenuated vaccines.

In a previous study, it was shown that rhesus macaques i.vag. infected with nonpathogenic SHIV89.6 were protected from i.vag. challenge with pathogenic SIVmac239 (60). The main goal of the present study was to determine if the route of immunization was a factor in the observed protection from vaginal SIV challenge. Further, we sought to define the SHIV89.6-induced host immune responses that confer protection against challenge with pathogenic SIVmac239. Therefore, three groups of rhesus macaques were either i.v., intranasally (i.n.), or i.vag. immunized with nonpathogenic SHIV89.6 and then challenged i.vag. with SIVmac239.

After SIV challenge, the vaccinated animals were categorized as either protected or unprotected based on virological parameters, such as plasma viral RNA (vRNA) levels and the detection of the challenge virus envelope gene (SIV env) by PCR. The focus of the laboratory analysis was a comprehensive assessment of immune responses in the acute phase postchallenge (p.c.). We compared these responses in vaccinated-protected and vaccinated-unprotected animals, with the assumption that there should be qualitative and/or quantitative differences in immune responses between protected and unprotected animals. As it is not likely that a single, specific immune response is responsible for protective immunity, and to address the complexity of antiviral immune responses, we assessed innate (alpha/beta interferons [IFN-α/β], proinflammatory cytokines, chemokines, and CD8 T-cell-mediated noncytolytic antiviral activity) as well as adaptive cellular (antigen-specific cytotoxicity, IFN-γ secretion and proliferation, and Th1 and Th2 cytokines) immune responses in vaccinated and naïve animals p.c. In addition, we assessed mucosal and systemic antibody responses. This is the first attempt to integrate such a broad analysis of immune responses to identify the protective components of live attenuated vaccine-induced anti-SIV immunity. We found that compared to vaccinated-unprotected animals, vaccinated-protected animals had higher precursor frequencies of SIV-specific cytotoxic T lymphocytes (CTLs), higher numbers of SIV-specific IFN-γ-secreting cells, and a greater ability to increase IFN-α mRNA levels in peripheral blood mononuclear cells (PBMC) during the acute phase p.c. Thus, the nonpathogenic vaccine virus induced both cellular and innate antiviral immune responses that were associated with protection from SIV challenge.

MATERIALS AND METHODS

Animals.

Rhesus macaques (Macaca mulatta) were housed at the California Regional Primate Research Center in accordance with the regulations of the American Association for Accreditation of Laboratory Animal Care standards. All animals were negative for antibodies to HIV-2, SIV, type D retrovirus, and simian T-cell lymphotropic virus type 1 at the time the study was initiated.

Immunization.

Table Table11 lists all monkeys and the routes of immunization used in the present study. Two SHIVs were used for the animal inoculations (51). Both SHIVs contain functional HIV-1 vpu, tat, rev and env genes in the context of the SIVmac239 provirus and were grown in rhesus macaque PBMC. The first virus was designated SHIV HXBc2 (45) and was constructed by using the HIV-1 IIIB/LAI variant, which is the prototype of the T-tropic viruses. The second virus, designated SHIV89.6 (81), was identical to SHIV HXBc2 except for the fragment from KpmI (nucleotide 5925) to BamHI (nucleotide 8053), which encodes the ectodomain of the gp120 and gp41 envelope glycoproteins. This env fragment in the SHIV89.6 virus was derived from HIV-1 89.6, a highly cytopathic, M-tropic variant (13). The SHIV HXBc2 stock contained 4,800 50% tissue culture infective doses (TCID50)/ml, and the two SHIV89.6 stocks contained 1,800 and 104 TCID50/ml, as determined by titration on CEMx174 cells; they were produced as described previously (51). The low-titer stock was used for the initial animal inoculation studies and the higher-titer stock was used for later animal inoculations. The SHIV HXBc2 and 89.6 stocks infect rhesus macaques after i.v. inoculation of approximately 1 to 10 TCID50 (51).

TABLE 1.
Virological assessment of SHIV89.6-vaccinated animals at the time of challenge

With the goal of testing the ability of SHIV HXBc2 to protect against vaginal challenge with SIVmac239, five animals were initially inoculated i.v. and became infected with SHIV HXBc2 (45, 51) (Table (Table1).1). However, we found that the SHIV HXBc2 was severely attenuated for in vivo replication, as judged by the inconsistency of virus isolation, compared to other attenuated forms of SIVmac239 (SIVmac239Δnef, SIVmac239Δ3) that had proven efficacy as vaccines (98). Thus, we concluded that this SHIV was unlikely to be an effective vaccine. This conclusion was supported when other investigators demonstrated that SHIV HXBc2 could not protect monkeys from i.v. challenge with SIVmac32H (43). Based on these considerations, the five monkeys were reinoculated at 12 weeks post-SHIV HXBc2 inoculation with nonpathogenic SHIV89.6.

Three monkeys received cholera toxin (CT) at the time of i.n. immunization with SHIV89.6 (Table (Table1).1). The addition of CT to mucosally administered protein vaccines has been shown to enhance the induction of antibodies, especially immunoglobulin A (IgA), at the cervico-vaginal mucosa and can also induce anti-SIV cellular immune responses (34).

In a small preliminary study, rhesus macaques were infected i.vag. with SHIV89.6 and subsequently challenged i.vag. with pathogenic SIVmac239 (60). Two monkeys infected with SHIV89.6 for a shorter period of time (6 months), but not three monkeys challenged after more than 12 months postimmunization, became infected with SIVmac239 and tested positive by PCR for the SIV envelope gene. Hence, the temporal relationship between the time of immunization and the time of challenge seemed to be important for challenge outcome. This was consistent with data from other studies showing that the time between the initial immunization with the attenuated virus and challenge with the pathogenic virus can influence the vaccine efficacy (10, 98). In contrast, after immunization with an SIVmac nef deletion mutant, SIVmacC8, protection was achieved after only 10 weeks (70). Thus, we also sought to test if the length of immunization affected challenge outcome.

Thus, to streamline the experimental design and to achieve statistically meaningful data sets, the monkeys were assigned to one of three groups that were immunized with live, nonpathogenic SHIV89.6 either i.v. (n = 16), i.n. (n = 11), or i.vag. (n = 16), as indicated in Table Table1.1. At weeks −1, 0, 1, 2, 4, 6, and 8 postimmunization and monthly thereafter, blood was collected and analyzed for vRNA levels and antiviral immune responses. Twelve monkeys were challenged at 6 months post-SHIV89.6 immunization, and 31 monkeys were challenged between 9 and 15 months post-SHIV89.6 inoculation (Table (Table11).

In addition, 18 vaccine-naïve, SIV-infected animals were included in the study as challenge controls (see Tables Tables11 and and2).2). These animals were age matched to the SHIV89.6-vaccinated animals (range, 5 to 13 years) and included a percentage of rhesus macaques of Chinese origin similar to that included in the vaccine groups (4 of 18, or 22%, of the control animals were of Chinese origin, compared to 11 of 43, or 25%, of the vaccinated animals).

TABLE 2.
Postchallenge evaluation of virological and immune response parameters

Challenge.

The pathogenic virus challenge of the SHIV89.6-immunized and naïve monkeys consisted of two i.vag. inoculations with 1 ml of SIVmac239 at 105 TCID50/ml. This virus stock was produced in rhesus PBMC as previously described (60). Blood samples were collected at weeks 1, 2, and 5 postinfection, monthly thereafter, and at necropsy. Six months p.c., the monkeys were killed by phenobarbital sedation, and blood and tissues were collected.

Virus load measurement.

Plasma samples were analyzed for vRNA by a quantitative branched DNA assay (16). Virus load in plasma samples is reported as vRNA copy numbers per ml of plasma. The detection limit of this assay is 500 vRNA copies/ml of plasma.

Virus isolation.

Virus isolation from rhesus PBMC was performed as previously described (60).

Nested PCR.

Nested PCRs for SIV gag, SIV env, and HIV env were performed as previously described (56, 60). As previously reported (60), the SIV env PCR assay is less sensitive than the SIV gag PCR assay. Serial dilution of appropriate plasmid DNA into PBMC lysates from uninfected animals demonstrated that this assay could consistently detect 100 SIV gag copies/105 PBMC, 100 to 1,000 SIV env copies/105 PBMC, and 10,000 HIV env copies/105 PBMC (data not shown). Importantly in the evaluation of challenge outcome, it should be noted that the sensitivity of the nested PCR for the detection of the challenge virus (SIV env PCR) was higher than that for the detection of the vaccine virus (HIV env PCR).

PBMC isolation.

PBMC were isolated from heparinized blood by using lymphocyte separation medium (ICN Biomedicals, Aurora, Ohio). Freshly isolated PBMC were used for limiting dilution analysis (LDA) of SIV-specific CTL precursor frequencies and T-cell proliferative responses. Additional PBMC samples were frozen in 10% dimethyl sulfoxide (Sigma, St. Louis, Mo.)-90% fetal bovine serum (Gemini BioProducts, Calabasas, Calif.) and stored in liquid nitrogen until future analysis in immunological and virological assays.

Measurement of anti-SIV antibody titers.

Anti-SIV binding antibody titers in serum and cervico-vaginal secretions (CVS) were measured as previously described (50). To confirm that IgG was present in a CVS sample, the total IgG concentration in all samples was determined by enzyme-linked immunosorbent assay (ELISA) as described previously (49). The results of the anti-SIV antibody ELISAs are reported as the dilution of a sample that produced optical density values above the cutoff value. The serum of vaccinated animals was tested for neutralizing antibodies at the day of challenge and on weeks 4 and 13 p.c., as described previously (63).

Proliferation assay.

SIV-specific T-cell proliferative responses were measured as previously described (56). The SIV antigen used for this assay, whole AT-2 inactivated SIVmac239, was kindly provided by J. Lifson (Laboratory of Retroviral Pathogenesis, SAIC Frederick, Bethesda, Md.). Due to batch-to-batch variations in the level of cellular protein contaminants in the virus preparations, the antigen concentrations used to stimulate PBMC were based not on total protein concentration but on SIV p28CA concentration as determined by ELISA (Coulter Corporation, Miami, Fla.). In most batches of the AT-2 SIV, 10 ng of p28CA antigen/ml corresponds to about 1 μg of total protein/ml. The following p28CA antigen concentrations were used: 0.1, 1.0, and 10.0 ng of p28CA/well. For each sample, only the highest stimulation index (SI) in the dilution series is reported. An SI of >2.5 was considered positive. This cutoff was established by testing PBMC from eight healthy, SIV-uninfected rhesus macaques. In every assay, PBMC from an uninfected animal are included as control.

IFN-γ ELISPOT assay.

The number of IFN-γ-secreting cells in PBMC was determined by using an IFN-γ monkey cytokine ELISPOT kit (U-CyTech, Utrecht University, Utrecht, The Netherlands). Frozen PBMC samples were thawed, washed with AIM V media (Invitrogen, Grand Island, N.Y.) supplemented with 20% fetal bovine serum (Invitrogen) (complete medium), and cultured overnight in 24-well tissue culture plates in complete medium. After overnight culture, 2 million cells/ml were stimulated with an SIVmac239 Gag p28CA peptide pool at a concentration of 1 μg of each peptide/ml in a 96-well flat-bottom tissue culture plate and incubated for 18 h at 37°C. The SIV Gag p55 peptide pool containing 25 overlapping 20-mers spanning SIV Gag p28CA was obtained through the National Institutes of Health AIDS Research and Reference Reagent Program, Division of AIDS. Negative controls consisted of cells that were cultured in medium only and cells from uninfected monkeys. Positive control wells were stimulated with phorbol myristate acetate-ionomycin (Sigma), as suggested in the U-CyTech protocol. The next day, cells were transferred directly to an anti-IFN-γ-coated ELISPOT plate and incubated for 5 h. After the incubation, cells were washed off and all remaining steps were performed in accordance with the manufacturer's protocol. The developed plates were read by using the ZEISS ELISPOT reader (Carl Zeiss Inc., Jena, Germany) and KS ELISPOT software (Zeiss). A sample was considered positive only if the number of IFN-γ-secreting cells/well exceeded 50 cells per 1 × 106 PBMC and if the number of positive IFN-γ spot-forming cells (SFC) was greater than the mean number of SFC found in the medium-only wells ± 2 standard deviations. Data are reported as the number of IFN-γ SFC per 1 × 106 PBMC. For reporting purposes, the background IFN-γ spot numbers observed in medium-only wells were subtracted from the IFN-γ spot numbers of SIV peptide-stimulated wells. By these criteria, PBMC samples taken from study animals before the initial immunization were consistently negative for SIV p28CA-specific IFN-γ secretion (data not shown). In every assay, PBMC from SIV-negative monkeys and SIV-positive responder animals are included as controls.

LDA of CTL precursor frequencies.

SIV Gag-specific CTL frequencies were determined as previously described (47), with the following modifications. Effector cells were plated in 24 replicates at 7,000, 5,000, 3,000, 2,000, 1,000, 700, 500, 300, 200, 100, 50, and 0 cells per well. Before the serial dilutions were set up, the effector cells were CD4 depleted by using DYNAbeads (human anti-CD4) in accordance with the manufacturer's instructions (DYNAL, Oslo, Norway), and at the day of the CTL assay (day 14 of culture), the effector cells consisted mostly of CD8+ T cells (95% purity), as determined by flow cytometry.

In vitro inhibition of viral replication by CD8+ T cells.

The noncytolytic inhibition of viral replication by CD8 T cells in autologous CD4 T cells was assessed as previously described (46, 53), with minor modifications. Because this assay has repeatedly been shown to measure noncytolytic suppression of viral replication and not lysis of infected cells (95, 97), it can be assumed that we were measuring noncytolytic suppression in our assays, too. In fact, we found that SIV-naïve animals in our colony exhibited in vitro CD8+-T-cell-mediated suppression of SIV replication in autologous CD4+ T cells ranging from 20 to 70%. Thus, this in vitro suppression was not due to an adaptive anti-SIV CD8+- cytolytic-T-cell response.

Briefly, CD4+ T cells and CD8+ T cells were prepared from PBMC by positive selection by using DYNAbeads (human anti-CD4/8) in accordance with the manufacturer's instructions (DYNAL). CD4+ T cells were stimulated for 3 days with 5.0 μg of concanavalin A (Sigma)/ml and 20 U of human interleukin 2 (IL-2)/ml (Biotest Diagnostics Corporation, Denville, N.J.). After 3 days, CD4+ T cells were treated with Polybrene (Sigma) (10 μg/ml) for 30 min at 37°C and then infected for 3 h with SIVmac239 (100 TCID50/106 cells). CD4+ T cells were washed and resuspended in complete RPMI medium containing 20 U of IL-2/ml. CD8+ T cells were added to the CD4+ T cells at different ratios (2:1, 1:1, 0.5:1, 0.25:1, 0.125:1, 0.062:1, and 0:1). On days 4, 7, 10, and 14 of the cultures, 100 μl of culture supernatant was collected and replaced with fresh medium. Supernatants were frozen and subsequently analyzed for SIV p27 antigen by using the Coulter ELISA kit (Coulter). Percent suppression of viral replication by CD8 T cells was calculated relative to replication in CD4 SIV-infected cultures without CD8 T cells. Only cultures with >90% inhibition of viral replication were considered positive for CD8-mediated noncytolytic antiviral activity.

RNA isolation and cDNA preparation.

Total PBMC RNA was isolated by using the Ambion RNAqueous kit (Ambion, Austin, Tex.) in accordance with the manufacturer's instructions. All samples were DNase treated with DNA-free (Ambion) for 1 h at 37°C. cDNA was prepared by using random hexamer primers (Amersham Pharmacia Biotech, Inc., Piscataway, N.J.) and Moloney murine leukemia virus reverse transcriptase (Invitrogen).

Cytokine mRNA analysis by reverse transcriptase real-time PCR.

Real-time PCR was performed as previously described (2). Briefly, samples were tested in duplicate, and the PCRs for the housekeeping gene GAPDH and the target (cytokine) gene from each sample were run in parallel on the same plate. The reaction was carried out on a 96-well optical plate (Applied Biosystems, Foster City, Calif.) in a 25-μl reaction volume containing 5 μl of cDNA and 20 μl of Mastermix (Applied Biosystems). All sequences were amplified by using the 7700 default amplification program, namely, 2 min at 50°C and 10 min at 95°C, followed by 40 to 45 cycles of 15 s at 95°C and 1 min at 60°C. Results were analyzed with the SDS 7700 system software, version 1.6.3 (Applied Biosystems) by using a G4 Macintosh computer (Apple Computer, Cupertino, Calif.). Cytokine mRNA expression levels were calculated from delta Ct (dCt) values and are reported as increase of cytokine mRNA levels in SIV/SHIV-infected PBMC compared to levels in PBMC from control samples (see below). Ct (for cycle threshold) values correspond to the cycle number at which the fluorescence due to enrichment of the PCR product reaches significant levels above the background fluorescence (threshold). In this analysis, the Ct value for the housekeeping gene (GAPDH) is subtracted from the Ct value of the target (cytokine) gene. The dCt value for the SIV/SHIV-infected sample is then subtracted from the dCt value of the corresponding control sample to yield ddCt. Thus, the increase in cytokine mRNA levels in SIV/SHIV-infected samples compared to those in control samples is then calculated as 2−ddCt (User Bulletin No. 2, ABI Prism 7700 Sequence Detection System [Applied Biosystems]).

Cytokine mRNA data generated by real-time reverse transcriptase PCR were analyzed by using two strategies. In the first approach (strategy A), the p.c. PBMC cytokine mRNA levels of each animal were directly compared to that animal's prechallenge cytokine mRNA levels. This approach accounts for the fact that increased cytokine mRNA levels due to the SHIV89.6 infection were present in PBMC of the vaccinated animals at the day of challenge. We report increased cytokine mRNA levels only if the p.c. mRNA levels were at least twofold higher than the same cytokine mRNA levels in the same monkey before challenge.

In a second approach (strategy B), PBMC cytokine mRNA levels in naïve and vaccinated monkeys p.c. were compared to cytokine mRNA levels in uninfected rhesus PBMC. PBMC from six uninfected monkeys (age matched to the experimental animals) were sampled at two different time points and analyzed for their cytokine mRNA levels (expressed as dCt value). For all of these samples, the mean dCt value for each cytokine was calculated, and then the increase in mRNA levels for each individual sample relative to the average mRNA level was determined. Next, to control for variation in cytokine mRNA levels among individuals in the study, the increases for a particular cytokine in all the samples from the six uninfected monkeys were averaged and then used as a calibrator value to determine if an experimental sample had normal or increased cytokine mRNA levels relative to this control population. We concluded that a PBMC sample from an experimental animal had increased cytokine mRNA levels if the increase in the mRNA level was equal to or higher than the average increase for the same cytokine in uninfected PBMC plus 2 standard deviations. By comparing PBMC cytokine mRNA levels in naïve, unvaccinated, and vaccinated animals after SIV challenge with the levels in PBMC from uninfected monkeys, we were able to evaluate the changes in cytokine mRNA levels of each study group relative to a single population of uninfected monkeys.

To report the data, which was collected at multiple time points (weeks 1, 2, and 5 p.c.) and generated from a large number of animals, in a reasonable and understandable manner, the results are given givenasas the percentages of all animals within each group that had increased PBMC cytokine mRNA levels at least once during weeks 1 to 5 p.c.

Statistical analysis.

For statistical analysis, data were log10 transformed and analyzed by a one-way analysis of variance with post hoc Tukey comparisons by using InStat software (Graph Pad Software Inc., San Diego, Calif.).

RESULTS

Immunization with nonpathogenic SHIV89.6.

Three groups of rhesus macaques were immunized i.v. (n = 16), i.n. (n = 11), or i.vag. (n = 16) with nonpathogenic SHIV89.6 (Table (Table1).1). Six of the 16 i.v. SHIV89.6-immunized monkeys had previously been immunized i.v. with nonpathogenic SHIV HXBc2 (Table (Table1).1). Following SHIV HXBc2 immunization, these animals developed peak SHIV HXBc2 viremia at week 2 postinoculation (p.i.) (3.9 to 6.2 log10 vRNA copies/ml of plasma) and had undetectable plasma SHIV HXBc2 vRNA levels by week 12 p.i. (data not shown). At no point during this time could virus be isolated from PBMC. Therefore, these monkeys were reimmunized at week 14 with SHIV89.6 by i.v. inoculation.

In agreement with earlier reports (51, 60, 81), all SHIV89.6-immunized monkeys had detectable plasma vRNA levels during the first 8 to 12 weeks p.i. (Fig. (Fig.1A).1A). In most animals, plasma vRNA levels peaked between weeks 1 and 2, although some i.vag. immunized animals had the highest vRNA levels at week 4 p.i. (Fig. (Fig.1A).1A). One i.vag. immunized monkey did not develop acute viremia until week 12 p.i. (animal 30445). The six SHIV HXBc2 i.v. immunized animals had lower peak vRNA levels (2.7 to 4.5 log10 vRNA copies/ml) post SHIV89.6 inoculation than all other i.v. immunized monkeys (5.7 to 6.9 log10 vRNA copies/ml of plasma). It is worth noting that the dose of SHIV89.6 used to inoculate these monkeys was 10-fold lower than that used for the other monkeys (Table (Table1).1). Peak vRNA levels (week 2 p.i.) were 1 to 2 log10 higher in i.v. and i.n. immunized monkeys than in i.vag. immunized monkeys (Fig. (Fig.1)1) (P < 0.001). Virus was consistently isolated from PBMC of the SHIV89.6-infected animals during the first 8 to 12 weeks p.i. but only sporadically thereafter. Viral DNA (SIV gag) could be detected in PBMC of SHIV89.6-immunized monkeys at all monthly time points tested until the day of challenge (data not shown).

FIG. 1.
Mean plasma vRNA levels and mean anti-SIV antibody titers in SHIV89.6-immunized rhesus macaques. (A) Plasma vRNA levels of SHIV89.6-infected monkeys, expressed as log10 vRNA copies per ml of plasma. (B) Serum anti-SIV IgG antibody titers are reported ...

Consistent with the delayed and lower plasma vRNA levels in i.vag. immunized monkeys, i.v. and i.n. immunized monkeys seroconverted within the first 2 to 4 weeks p.i., whereas some i.vag. immunized monkeys did not have detectable serum anti-SIV binding antibodies before weeks 4 to 8 p.i. (Fig. (Fig.1B).1B). One i.vag. immunized monkey (no. 30445) did not seroconvert before week 12 p.i. (data not shown), and this animal also had no detectable plasma vRNA levels until this time point (see above). Once elicited, high antibody titers persisted throughout the immunization period (Fig. (Fig.1B).1B). At the time of challenge, vaccinated-protected monkeys and vaccinated-unprotected animals had similar endpoint binding antibody titers (ranging from 200 to 800,000 and from 400 to 1,600,000, respectively) (data not shown).

Anti-SIV binding antibodies in CVS could be detected in 26 of 31 vaccinated animals tested (data not shown). The highest anti-SIV binding antibody titers in CVS were found in mucosally immunized monkeys (with endpoint titers of 160 in monkeys 25979, 26154, and 28288 and endpoint titers of 1,600 in monkey 28288). Eight additional monkeys of the 21 mucosally immunized and tested animals had anti-SIV endpoint titers of ≤10, and 11 of the 21 animals had anti-SIV endpoint titers of >10 but <100 (data not shown). Only two of the 21 mucosally immunized monkeys had no detectable anti-SIV antibodies in CVS, despite the consistent detection of total IgG in the same CVS samples. The anti-SIV antibody titers in CVS of mucosally immunized monkeys were similar to anti-SIV antibody titers observed in the CVS samples of 10 i.v. immunized and tested monkeys (undetectable titer, n = 3; titer of ≤10, n = 5; titer of >10 but <100, n = 2). No difference in anti-SIV binding antibody titers was detectable in CVS between vaccinated-protected and vaccinated-unprotected monkeys (data not shown). In fact, one i.n. immunized, vaccinated-unprotected monkey (no. 26154) had one of the highest CVS anti-SIV antibody titers (endpoint dilution, 160). Thus, mucosal immunization did not generally result in higher anti-SIV antibody titers in mucosal secretions, and anti-SIV antibodies present in CVS at the time of challenge did not appear to influence challenge outcome.

All animals were challenged i.vag. with pathogenic SIVmac239 at 6 months p.i. (n = 12) (short-term immunized monkeys) or between 9 and 15 months p.i. (n = 31) (long-term immunized monkeys) (Table (Table1).1). At the time of challenge, vRNA was detectable in the plasma of 6 of the 43 SHIV89.6-immunized monkeys (<3.0 log10 vRNA copies/ml in monkeys 21349, 26249, and 26509; 3.0 to 4.0 log10 vRNA copies/ml in monkeys 23744, 26012, and 31413), and 3 of 43 monkeys (21349, 26154, and 31413) were virus isolation positive (Table (Table1).1). All of the monkeys tested positive by PCR for viral DNA (SIV gag) in their PBMC at the time of challenge (Table (Table11).

Outcome of intravaginal challenge with SIVmac239. (i) Assessment of virological parameters.

Challenge outcome was determined by assessing the level of vRNA in plasma and the ability to detect the SIVmac239 envelope (SIV env) gene in DNA from PBMC. Plasma vRNA levels were measured at weeks 1, 2, and 5 p.c. and monthly thereafter until 6 months p.c. In naïve monkeys, the plasma vRNA levels peaked at week 2 p.c., viral set point was reached by week 8 (Fig. (Fig.2),2), and no further significant change (P > 0.05) in plasma vRNA levels occurred during the next 6 months (Fig. (Fig.2).2). All naïve monkeys challenged with SIVmac239 had peak plasma vRNA levels above 104 copies/ml, and the vast majority (16 of 18, or 88%) of these naïve monkeys continued to have vRNA levels above 104 copies/ml at all time points tested throughout the 6-month follow-up period. vRNA levels reflect the extent of virus replication; therefore, we judged the relative level of vaccine-mediated protection based on vRNA levels, in analogy to the prediction of distinct clinical outcomes by plasma vRNA levels in chronically SIV-infected macaques (30). We define vaccinated-protected animals as those having plasma vRNA levels of less than 104 copies/ml at all times during the 6-month period p.c., whereas vaccinated but unprotected animals had plasma vRNA levels above 104 copies/ml at least once during this time period.

FIG. 2.
Plasma vRNA levels in vaccinated and naïve monkeys p.c. with SIVmac239. Each panel shows the plasma vRNA levels for individual monkeys in the vaccine-naïve (A) and in the i.v. (B), i.n. (C), and i.vag. (D) SHIV89.6-immunized monkeys p.c. ...

For the 6-month p.c. period, 15 of 43 vaccinated monkeys had undetectable plasma vRNA levels and were negative by PCR for SIV env in PBMC (Table (Table2,2, protected monkeys no. 1 to 15). Thus, these monkeys appeared to be completely protected from the SIVmac239 challenge. Another 12 of the 43 vaccinated monkeys had plasma vRNA levels below 104 copies/ml and were also considered protected (Table (Table2,2, protected monkeys no. 16 to 27). By the above criteria, 62% of i.v. (10 of 16), 73% (8 of 11) of i.n., and 56% (9 of 16) of i.vag. immunized monkeys were protected. The similar levels of protection achieved in i.v. and mucosally (i.n. and i.vag.) immunized animals indicated that mucosal immunization did not improve the mucosal SIV challenge outcome. It should be noted that the challenge occurred after a prolonged period of systemic infection with the vaccine virus. The conclusion that the route of immunization did not affect challenge outcome was confirmed by statistical analysis comparing vRNA levels of i.v., i.n., and i.vag. immunized monkeys at the time of peak viremia (week 2) and viral set point (week 8) and during chronic infection (week 20). Plasma vRNA levels were significantly higher in naïve animals than in i.v., i.n., or i.vag. vaccinated animals at all time points tested (P < 0.01), but the analysis failed to demonstrate any differences in vRNA levels between the three vaccinated groups (P > 0.05). Thus, for all subsequent analyses, monkeys were grouped into vaccine-naïve, SIVmac239-infected control (n = 18), vaccinated-protected (n = 27, or 62.8%), and vaccinated-unprotected (n = 16, or 37.2%) monkeys.

One interesting difference in challenge outcome among the three vaccinated groups was noted. Longitudinal analysis of vRNA levels in immunized monkeys showed that in i.vag. immunized monkeys, the vRNA levels at 2 weeks p.c. were significantly lower than at week 20 p.c. This trend was not observed in i.v. or i.n. immunized monkeys. Rising vRNA levels in the later stages of the p.c. observation period are consistent with late escape from vaccine-mediated protection. This late escape accounts for the slightly higher percentage of unprotected animals in the i.vag. immunized monkeys.

Differences in p.c. plasma vRNA levels between short- and long-term immunized animals did not reach the level of statistical significance (data not shown). The fact that protection was observed in a similar percentage of short- (6 months) and long-term (>12 months) SHIV89.6-immunized monkeys indicates that 6 months was sufficient time for protective immune responses to develop.

Further, no difference in challenge outcome was observed between the long-term i.v. immunized monkeys that had received a prior immunization with SHIV HXBc2 before SHIV89.6 immunization (3 of 6 were protected) and the animals that were immunized with SHIV89.6 only (4 of 6 were protected) (Tables (Tables11 and and2).2). Coadministration of CT at the time of i.n. SHIV89.6 immunization did not affect the challenge outcome in the small number of animals tested (2 of 3 were protected) (Tables (Tables11 and and22).

It should also be noted that positive virus isolation results and/or the detection of vRNA in the plasma at the time of challenge were not predictive of challenge outcome. Further, there was no correlation between peak plasma vRNA levels postimmunization with nonpathogenic SHIV89.6 and peak vRNA levels p.c. with pathogenic SIVmac239 (r2 = 0.001; P = 0.812). Thus, the relative ability to control the vaccine virus was not predictive of challenge outcome.

To confirm that the high p.c. vRNA levels in unprotected animals were due to the presence of the challenge virus (SIVmac239) and to detect low levels of SIV infection in the absence of detectable plasma vRNA, nested PCR for the envelope (env) genes of HIV89.6 and SIVmac239 was performed. In PBMC of unvaccinated monkeys, SIV gag and SIV env were detected during the acute phase of infection. During the chronic phase of infection, SIV gag was detectable at every time point, and SIV env was occasionally detected (Table (Table2).2). All of the vaccinated-unprotected animals tested PCR positive for SIV env in PBMC (Table (Table2).2). Overall, SIV env could be detected in PBMC of 24 of 43 vaccinated animals (namely, protected monkeys 20 to 27 and unprotected monkeys 1 to 16) within 6 months p.c. (Table (Table2).2). Eight of the 24 SIV env-positive monkeys (protected monkeys 20 to 27) had vRNA levels below 104 copies/ml and were defined as protected (Table (Table2).2). In fact, two of the 8 vaccinated-protected and SIV env PCR positive monkeys (nos. 22131 and 31420) had plasma vRNA levels below the detection limit throughout the p.c. period. In the majority of the vaccinated-protected monkeys (19 of 27) (protected monkeys 1 to 19), viral DNA for SIV env could not be detected (Table (Table2).2). Among the 19 vaccinated-protected, PCR SIV env-negative animals, 15 animals (protected monkeys 1 to 15) had vRNA levels below the detection limit (<2.7 log10 copies/ml) throughout the p.c. period, whereas 4 of 19 animals (protected monkeys 16 to 19) had low but detectable plasma vRNA levels (Table (Table2).2). However, even in the absence of detectable plasma vRNA levels and the detection of the challenge virus by PCR, these monkeys had increases in cytokine mRNA levels that were consistent with exposure to the challenge virus (see below and Table Table33).

TABLE 3.
Cytokine mRNA induction in PBMC of naïve and vaccinated monkeys during the acute phase p.c.a

Most of the vaccinated animals (42 of 43), including the unprotected animals, did not exhibit the typical pattern of primary lentiviral replication that was seen in all the naïve animals after i.vag. challenge with pathogenic SIVmac239 (Fig. (Fig.2).2). For the first 8 weeks p.c., more than 80% of the vaccinated animals (n = 36) had plasma vRNA levels that remained below 104 copies/ml. Thus, these animals were able to effectively control SIVmac239 replication in the acute p.c. stage. However, 16 of these animals had elevated plasma vRNA levels (>104 copies/ml) after week 8 p.c., and thus, these monkeys were considered unprotected (Table (Table2,2, unprotected monkeys 1 to 16). Only 1 of 43 vaccinated monkeys (no. 26154) had a plasma vRNA pattern that was similar to that of the naïve monkeys. In this animal, peak plasma viremia (5.7 log10 vRNA copies/ml) was detected between weeks 1 and 2 p.c., and vRNA levels were consistently high (>5.5 log10 vRNA copies/ml) up to 20 weeks p.c. (time of euthanasia). Three other vaccinated monkeys (monkeys 28408, 23804, and 26011) developed an acute viremia, with plasma vRNA levels above 104 copies/ml, but their plasma vRNA levels dropped below this threshold by week 5 p.c. Based on the previously defined criteria, all four of the vaccinated animals described above (monkeys 26154, 28408, 23804, and 26011) were considered unprotected.

(ii) CD4+-T-cell levels in PBMC.

In naïve and vaccinated-unprotected animals, mean CD4+-T-cell counts dropped within the first 2 weeks p.c. and increased slightly by week 5 but remained low for the duration of the observation period (Fig. (Fig.3).3). At weeks 9 and 21 p.c., CD4+-T-cell counts in naïve and vaccinated-unprotected animals were significantly lower than in vaccinated-protected animals (P < 0.05), but counts in naïve and vaccinated-unprotected monkeys were indistinguishable. Consistent with lower CD4+-T-cell numbers, the CD4:CD8 T-cell ratio was reduced in naïve and vaccinated-unprotected monkeys (Table (Table2).2). Vaccinated-protected monkeys had a significantly higher CD4:CD8 T-cell ratio than naïve animals at weeks 5 and 21 p.c. (P < 0.05). In addition, the CD4:CD8 T-cell ratio in naïve animals was significantly lower at weeks 5 and 21 p.c. than at week 1 p.c. (P < 0.05). This level of CD4+-T-cell decline was not observed in either vaccinated-unprotected or vaccinated-protected animals. Thus, the changes observed in CD4+-T-cell levels were consistent with our categorization of vaccinated animals as protected or unprotected based on plasma vRNA levels (Table (Table22).

FIG. 3.
Percent change in absolute CD4 T-cell numbers p.c. with SIVmac239. p.c. CD4 T-cell numbers for monkeys categorized as vaccinated-protected and vaccinated-unprotected and for SIVmac239 control monkeys are shown as the average percent change ± standard ...

Assessment of immune responses in naive and SHIV89.6-vaccinated monkeys challenged with pathogenic SIVmac239.

Although two virological outcomes can be distinguished among the monkeys classified as vaccinated-protected (see above), we could not detect major differences in immune responses between solidly protected (animals 1 to 15) and partially protected (animals 16 to 27) animals (Table (Table2).2). Thus, we have made no effort to distinguish immune responses among these two vaccinated-protected groups (Table (Table22 and data not shown).

(i) Anti-SIV antibody responses.

All vaccine-naïve control animals, except one rapid progressor monkey (no. 23756), had detectable anti-SIV antibodies by week 5 p.c., and maximum antibody titers were reached between weeks 9 and 13 p.c. (data not shown). Although the vaccinated monkeys had serum anti-SIV antibodies on the day of challenge, p.c. antibody responses were induced with kinetics similar to that of the primary response seen in the vaccine-naïve animals. In general, p.c. binding antibody titers were higher in the vaccinated-unprotected monkeys than in the vaccinated-protected monkeys. During the acute phase p.c., 55% of vaccinated-protected monkeys had anti-SIVmac251 binding antibody endpoint titers of ≥104 (range, 2,000 to 800,000), compared to 100% of vaccinated-unprotected monkeys (range, 25,000 to 1,600,000) and 75% of naïve monkeys (range, 4,000 to 32,000) (Table (Table2).2). It should be noted that among the vaccinated-protected animals, the animals with plasma vRNA levels of <2.7 log10 copies/ml and undetectable SIV env DNA (animals 1 to 15) had lower anti-SIV binding antibody titers (200 to 200,000) than vaccinated-protected animals with plasma vRNA levels of >2.7 log10 but <4.0 log10 copies/ml and/or detectable SIV env DNA (animals 16 to 27; anti-SIV binding antibody titers, 16,000 to 800,000) (Table (Table2).2). These antibody responses persisted throughout the p.c. period, and at 6 months p.c. antibody titers of >104 were detected in 55% of vaccinated-protected monkeys, 100% of vaccinated-unprotected monkeys, and 90% of naïve monkeys (data not shown).

Neutralizing antibodies to SHIV89.6 were detectable in the serum of 23 of 37 animals tested at the time of challenge (data not shown), and 15 of these 23 monkeys were subsequently categorized as vaccinated-protected. However, p.c., neutralizing antibodies to the challenge virus SIVmac239 were detectable in only 2 of the 37 tested SHIV89.6-vaccinated monkeys, and they were detected at very low titers (data not shown). Based on p.c. plasma vRNA levels, both of these monkeys (nos. 26154 and 26011) were considered vaccinated-unprotected (data not shown). Thus, strong serum anti-SIV antibody responses and anti-SIVmac239 neutralizing antibodies did not play a role in the challenge outcome.

(ii) SIV-specific T-cell proliferative responses.

Most of the vaccinated monkeys tested had SIV-specific proliferative responses before challenge (26 of 31) (data not shown) and in the first 5 weeks p.c. (14 of 17) (Table (Table2).2). No significant differences in the strength of T-cell proliferative responses (SI) were detected between vaccinated-protected (n = 11) and vaccinated-unprotected (n = 6) animals. During the acute phase p.c., positive responses were detected in 9 of 11 (82%) vaccinated-protected animals (SIs ranging from 2.7 to 23.1), in 5 of 6 (83%) vaccinated-unprotected monkeys (SIs ranging from 4.4 to 16.5), and in 4 of 9 (44%) naïve monkeys (ranging from 2.3 to 4.8) (data not shown and Table Table2).2). Thus, p.c. anti-SIV proliferative responses were not predictive of challenge outcome. At the time of necropsy (6 months p.c.), 8 of 9 naïve monkeys (88%) had SIs from 2.1 to 22.6, whereas only 8 of 17 vaccinated animals (47%) had a proliferative response to SIV antigens (data not shown).

CD8+-T-cell responses. (i) CTL activity.

In vaccinated animals, vaccine-induced anti-SIV Gag CTL activity was examined 1 month prior to challenge and 5 weeks p.c. (Fig. (Fig.4).4). At the time of challenge, the vaccinated-protected and vaccinated-unprotected animals had similar frequencies of SIV Gag-specific precursor CTLs (pCTLs) (mean, 481 and 497 pCTLs per 106 CD8+ T cells, respectively) (Fig. (Fig.4).4). However, vaccinated-protected animals had significantly higher pCTL frequencies (mean pCTL count, 1,125 per 106 CD8+ T cells) p.c. (P < 0.05) than vaccinated-unprotected animals (mean pCTL count, 370 per 106 CD8+ T cells) (Fig. (Fig.44 and Table Table2).2). Thus, higher pCTL frequencies in the first few weeks after challenge, but not prechallenge, were associated with protection from SIVmac239 challenge.

FIG. 4.
SIV Gag-specific pCTL frequencies in vaccinated-protected and vaccinated-unprotected animals. SIV Gag-specific pCTLs per 106 CD8+ T cells of vaccinated-protected and vaccinated-unprotected animals were measured at the time of challenge and compared ...

(ii) CD8+-T-cell-mediated noncytolytic antiviral activity.

To assess the potential role of noncytolytic inhibition of viral replication by CD8+ T cells in SHIV89.6-mediated protection, we analyzed the ability of CD8+ T cells from 14 of the SHIV89.6-vaccinated monkeys to suppress virus replication in autologous CD4+ T cells in vitro. At the time of SIV challenge, CD8-mediated inhibition of SIV replication in vitro was detected in approximately 60% of all vaccinated monkeys (data not shown). In addition, we found that SIV-naïve animals in our colony exhibited in vitro CD8+-T-cell-mediated suppression of SIV replication in autologous CD4+ T cells ranging from 20 to 70%. Further, the ability of CD8+ T cells to inhibit virus replication was not predictive of challenge outcome. p.c. CD8-mediated inhibition of virus replication was detectable in 80% of all naïve animals and 100% of vaccinated-unprotected monkeys (data not shown). In comparison, only 45% of vaccinated-protected animals had detectable CD8-mediated noncytolytic suppressor activity p.c. (data not shown). Thus, SHIV89.6-induced control of p.c. viremia was not associated with noncytolytic CD8+-T-cell-mediated control of virus replication (data not shown).

(iii) IFN-γ ELISPOT.

To determine if IFN-γ secretion by antigen-specific T cells was associated with protective immunity in vaccinated monkeys, the number of IFN-γ-secreting cells in PBMC was determined in vaccinated and naïve monkeys after incubation with an SIV Gag p28CA peptide pool (Fig. (Fig.5).5). At the time of challenge, SIV-specific IFN-γ-secreting cells were detectable in PBMC of 7 of 17 vaccinated animals tested (data not shown). At weeks 1 and 2 p.c., a higher number of vaccinated animals than naïve monkeys responded to SIV Gag peptide stimulation with detectable IFN-γ secretion (Fig. (Fig.55 and Table Table2).2). The faster response of vaccinated animals than naïve animals p.c. was indicative of an anamnestic T-cell response. Although some of the vaccinated monkeys showed no detectable IFN-γ response at week 2 p.c., the average number of IFN-γ-secreting cells in all vaccinated animals compared to that in all naïve animals was significantly higher (P < 0.05, Student t test). To determine if the vaccinated-protected or the vaccinated-unprotected animals were primarily responsible for the observed difference in the IFN-γ responses between vaccinated and naïve monkeys, a one-way analysis of variance of the number of IFN-γ-secreting cells in vaccinated-protected, vaccinated-unprotected, and naïve monkeys at week 2 p.c. was performed. Vaccinated-protected, but not vaccinated-unprotected, animals had statistically higher numbers of IFN-γ-secreting cells than naïve monkeys (P < 0.05) (Fig. (Fig.5).5). It is noteworthy that in one of the two vaccine-naïve animals (monkey 31423) (Table (Table3)3) with postpeak vRNA levels of <104 copies/ml of plasma, IFN-γ secretion was detectable at week 1 p.c. Antigen-specific IFN-γ secretion persisted in PBMC of vaccinated and naïve monkeys and was detectable in the majority of animals (>80%) at 6 months p.c. There was no correlation between the number of IFN-γ-secreting cells and plasma vRNA levels in either the acute or chronic phase p.c. (r2 = 0.03 and P > 0.05, and r2 = 0.06 and P > 0.05, respectively).

FIG. 5.
Frequency of SIV Gag-specific IFN-γ-secreting cells in PBMC of naïve and vaccinated monkeys p.c. with SIVmac239. The number of SIV Gag-specific IFN-γ-secreting cells during the acute and chronic phases p.c. is reported as the number ...

Cytokine mRNA analysis.

To clarify the role of cytokines in vaccine-induced protective immunity, PBMC mRNA levels of 11 cytokines were determined by real-time RT-PCR. The results were analyzed by using two different strategies (Table (Table3)3) (see Materials and Methods). To characterize differences in p.c. cytokine mRNA levels between vaccinated-protected and vaccinated-unprotected monkeys, the p.c. PBMC cytokine mRNA levels of an individual monkey were compared to the prechallenge PBMC cytokine mRNA levels of the same monkey (Table (Table3,3, strategy A). To characterize the differences in PBMC mRNA levels induced by the vaccine prior to challenge and to characterize challenge virus-induced changes in naïve animals, PBMC cytokine mRNA levels of all monkeys were compared to PBMC cytokine mRNA levels in uninfected animals (Table (Table3,3, strategy B). This approach determined relative changes in the vaccinated and naïve study populations compared to the normal monkey population.

By using strategy A, we found that of all cytokines examined, only IFN-α differed significantly in p.c. PBMC mRNA levels between vaccinated-protected and vaccinated-unprotected monkeys. A comparison of PBMC mRNA levels in individual monkeys before and after challenge demonstrated that 87% of vaccinated-protected compared to only 30% of vaccinated-unprotected monkeys had increased IFN-α PBMC mRNA levels in the acute phase p.c. (Tables (Tables22 and and3).3). At week 5 p.c., the increases in IFN-α PBMC mRNA levels were significantly higher in vaccinated-protected than in vaccinated-unprotected animals (P < 0.05) (Fig. (Fig.6).6). However, at the time of challenge, IFN-α PBMC mRNA levels were already elevated in 50% of vaccinated-unprotected monkeys and 20% of vaccinated-protected monkeys compared to uninfected animals (data not shown). Elevated IFN-α PBMC mRNA levels were detectable only in vaccinated-protected monkeys during the first few weeks p.c. and were no longer detected at 6 months p.c.

FIG. 6.
p.c. IFN-α mRNA levels in PBMC of vaccinated and naïve animals. The increase in PBMC IFN-α mRNA levels p.c. relative to prechallenge IFN-α PBMC mRNA levels of the same animal are shown for naïve ([filled triangle]), vaccinated-protected ...

IFN-β PBMC mRNA levels were undistinguishable from those in matched uninfected PBMC samples for the majority of the PBMC samples collected from naïve and vaccinated monkeys at the time of challenge and p.c. (Table (Table33).

PBMC mRNA levels of Mx, an antiviral effector molecule induced by IFN-α/β (89, 94), were persistently increased in naïve monkeys (80%) throughout the acute phase p.c. (Tables (Tables22 and and3).3). At weeks 1, 2, and 5 p.c., the increase in Mx PBMC mRNA levels relative to prechallenge levels in the naïve animals was significantly higher than the increase in Mx PBMC mRNA levels in vaccinated-protected animals (Fig. (Fig.7)7) (P < 0.05, P < 0.01, and P < 0.001, respectively). At the time of necropsy (6 months p.c.), vaccinated-protected animals had PBMC Mx mRNA levels undistinguishable from prechallenge mRNA levels and also undistinguishable from those of uninfected monkeys. In contrast, naïve and vaccinated-unprotected monkeys still had elevated Mx PBMC mRNA levels (Fig. (Fig.7).7). The strong induction of Mx PBMC mRNA in naïve monkeys and the persistence of elevated Mx PBMC mRNA levels in naïve animals throughout the p.c. period compared to vaccinated-protected monkeys were consistent with the ongoing high level of virus replication in naïve monkeys p.c. (see Fig. Fig.2).2). This conclusion is further confirmed by the observed increase in PBMC Mx mRNA levels in vaccinated-unprotected animals after viral escape.

FIG. 7.
p.c. Mx mRNA levels in PBMC of vaccinated and naïve animals. The symbols are as described in the legend to Fig. Fig.66.

During the acute phase p.c., 50% of the vaccinated monkeys had increased IFN-γ PBMC mRNA levels (Tables (Tables22 and and3),3), and 30% had increased IL-2 PBMC mRNA levels compared to their own prechallenge mRNA levels (Table (Table3).3). In contrast, during the acute phase p.c., only a few of the SIV-infected vaccine-naïve monkeys had increased PBMC mRNA levels of IFN-γ, IL-2, and IL-4 compared to prechallenge PBMC mRNA levels (Table (Table3).3). However, if an increase in IFN-γ and IL-2 PBMC mRNA levels was found, the magnitude of the increase was similar in monkeys from all three groups. Thus, a distinct Th1/Th2 polarization was not detectable in the acute phase p.c., and a specific pattern of increased Th1 or Th2 cytokine mRNA levels was not associated with a better challenge outcome. Consistent with the results of the IFN-γ ELISPOT assay, in which IFN-γ secretion was observed in most animals at the time of euthanasia (24 weeks p.c.), IFN-γ mRNA levels at 6 months p.c. were increased in about 50% of all monkeys independent of challenge outcome (data not shown). Thus, the increased IFN-γ mRNA levels and IFN-γ ELISPOT responses in PBMC were most consistent with an ongoing immune response to a productive infection but were not associated with control of virus replication.

The p.c. mRNA levels for the β-chemokines MIP-1α, MIP-1β (Fig. (Fig.8),8), and macrophage-derived chemokine were increased in similar percentages of vaccinated-protected and vaccinated-unprotected monkeys, relative to prechallenge PBMC mRNA levels (Tables (Tables22 and and33).

FIG. 8.
p.c. MIP-1β mRNA levels in PBMC of vaccinated and naïve animals. The symbols are as described in the legend to Fig. Fig.66.

Increased cytokine mRNA levels as marker of challenge virus exposure.

Compared to PBMC mRNA levels in unexposed animals and consistent with exposure to pathogenic SIVmac239, the majority of monkeys in all three groups had increased proinflammatory cytokine mRNA levels within the first 5 weeks p.c. At week 2 p.c., tumor necrosis factor alpha (TNF-α) and IL-6 PBMC mRNA levels were significantly higher in approximately 80% of naïve (P < 0.001), vaccinated-protected (P < 0.001), and vaccinated-unprotected (P < 0.05) monkeys (Tables (Tables22 and and33 and Fig. Fig.9).9). A comparison of p.c. PBMC mRNA levels of proinflammatory cytokines to prechallenge PBMC mRNA levels in an individual animal showed that 80 to 90% of the naïve monkeys responded to the challenge with an increase in TNF-α and IL-6 PBMC mRNA levels, compared to 50% of the vaccinated animals (Table (Table3).3). This is consistent with the fact that about 60% of all vaccinated animals already had elevated TNF-α and IL-6 PBMC mRNA levels (compared to levels in uninfected monkeys) at the time of challenge. In addition, about 60% of naïve and 50% of vaccinated monkeys had elevated IL-12 PBMC mRNA levels compared to levels in uninfected animals (Table (Table3).3). At the time of challenge, PBMC IL-12 mRNA levels of the vaccinated animals had been indistinguishable from IL-12 mRNA levels in matched PBMC of uninfected monkeys (data not shown).

FIG. 9.
TNF-α mRNA levels p.c. in PBMC of vaccinated and naïve animals. The increase in PBMC TNF-α mRNA levels p.c. compared to TNF-α PBMC mRNA levels of uninfected control animals are shown for naïve ([filled triangle]), vaccinated-protected ...

Also consistent with an innate immune response to virus exposure, p.c. PBMC mRNA levels for the chemokines MIP-1α, MIP-1β, and macrophage-derived chemokine were higher in approximately 60 to 70% of naïve and 40 to 60% of vaccinated monkeys compared to chemokine PBMC mRNA levels from uninfected animals (Table (Table3).3). A statistical analysis of proinflammatory cytokine and chemokine PBMC mRNA levels did not reveal any differences in the magnitude of the response between vaccinated-protected, vaccinated-unprotected, and naive animals (data not shown).

Importantly, consistent with exposure to the challenge virus, in 9 of 12 vaccinated-protected animals (Table (Table2,2, protected animals 1 to 12) in which there was no evidence for infection with the challenge virus (p.c. plasma vRNA level of <2.7 log10 copies/ml and PCR SIV env negative), there were increases in PBMC mRNA levels of proinflammatory cytokines in the acute phase p.c. (see Table Table22).

At the time of necropsy, 6 months p.c., the PBMC mRNA levels of most of the cytokines (except IFN-γ and Mx; see above) analyzed in naïve and vaccinated animals had returned to levels that were indistinguishable from PBMC mRNA levels in uninfected monkeys (data not shown).

DISCUSSION

It has been suggested that an HIV vaccine needs to elicit genital immune responses to effectively prevent heterosexual transmission (40, 42, 58, 59). Thus, the first goal of the present study was to directly compare the effect of the route of immunization on i.vag. challenge outcome by immunizing three large groups of monkeys with the same live attenuated vaccine, SHIV89.6, by three different routes (i.v., i.n., and i.vag.) and challenging all three vaccinated groups with SIVmac239 after an extended immunization period. We found that the SHIV89.6-induced protection against i.vag. challenge with pathogenic SIVmac239 was achieved independently of the route of immunization. It has been shown that i.n. immunization effectively induces female genital tract immunity (34, 48), and we did find that the most consistent protection was achieved by i.n. immunization with SHIV89.6. However, the plasma vRNA levels among monkeys immunized i.v. or mucosally were not statistically different from each other at any time p.c. Protection against mucosal challenge has also been achieved by systemic immunization with other live attenuated viruses (36, 69, 78). It is possible that the route of immunization is irrelevant in the challenge outcome of live attenuated vaccine studies because animals were infected systemically with the vaccine virus for a prolonged period of time (6 months or longer). The route of immunization may be more important for nonreplicating vaccines that do not propagate and disseminate in a manner analogous to an attenuated lentiviral vaccine.

The second goal of the study was to determine which of the immune responses that were present at the time of challenge and in the acute phase p.c. contributed to the observed protection in vaccinated animals (Table (Table2).2). Previous studies have provided conflicting data about the role that CTLs play in the protective immunity induced by live attenuated vaccines (15, 33, 36). We compared pCTL frequencies, the frequency of IFN-γ-secreting cells, and the levels of IFN-γ mRNA in PBMC of vaccinated-protected and vaccinated-unprotected animals at the time of challenge and during the acute phase p.c. At the time of challenge, more than 80% of all SHIV89.6-vaccinated monkeys had detectable SIV-specific CTL activity, but only the vaccinated-protected monkeys showed a significant increase in pCTL frequencies in the first 5 weeks p.c. (Table (Table2).2). Thus, as was previously reported (60), CTL responses were apparently an important part of the protective immune response in SHIV89.6-vaccinated-protected monkeys. Considering that vaccinated-protected and vaccinated-unprotected monkeys had comparable pCTL frequencies at the time of challenge, future studies need to determine which factors influence the induction of an anamnestic CTL response in the vaccinated-protected animals but not in the vaccinated-unprotected animals. The induction of strong CTL responses has also been observed after immunization with attenuated SIVmac deletion mutants (36). The observed role of CTL in vaccine-mediated protection is consistent with the well-documented role of CD8 T cells in the control of virus replication in HIV (55) and SIV infection (71, 72, 87).

In addition to increased pCTL frequencies in vaccinated-protected animals, we observed a more rapid induction of SIV Gag-specific IFN-γ-secreting cells in PBMC of vaccinated animals than in naïve monkeys during the acute phase p.c. Consistent with this, CD8+-T-cell-mediated cytolytic activity in viral infections has been associated with increased IFN-γ secretion (5, 32, 66). Further, treatment with exogenous IL-12 during the acute phase of SIVmac251 infection induced stronger CTL and IFN-γ responses, and this resulted in prolonged survival relative to that of untreated SIV-infected monkeys (8). In the present study, vaccinated-protected monkeys, but not the vaccinated-unprotected monkeys, had higher numbers of IFN-γ-secreting PBMC than naïve monkeys at week 2 p.c. (Table (Table2).2). Thus, the data suggested that during the acute phase p.c., SIV-specific IFN-γ responses contributed to the observed protection against mucosal challenge with SIVmac239. However, during the chronic phase p.c., antigen-specific IFN-γ-secreting cells were detectable in similar numbers in PBMC of naïve and vaccinated monkeys. Thus, during the chronic phase p.c., the frequency of IFN-γ-secreting PBMC did not correlate with vRNA levels, and therefore the frequency of IFN-γ-secreting PBMC cannot be used alone to predict disease progression.

In the present study, not every sample with detectable anti-SIV CTL activity also had detectable SIV-specific IFN-γ-secreting cells and vice versa. In addition, anti-SIV CTL activity and/or IFN-γ secretion was not always associated with increased IFN-γ mRNA levels in PBMC. Among 22 vaccinated monkeys for which anti-SIV CTL activity, IFN-γ secretion, and IFN-γ mRNA levels were determined in PBMC during the acute phase p.c., 2 monkeys (28408 and 30470) had increased PBMC IFN-γ mRNA levels but no detectable CTL activity or IFN-γ secretion and 6 monkeys (23478, 25409, 24251, 31420, 30474, and 31411) had detectable anti-SIV CTL activity and IFN-γ secretion, but no increase in IFN-γ mRNA levels was detected (Table (Table2).2). Similar discrepancies between CTL activity and IFN-γ secretion (as measured by ELISPOT assay) have been observed in SIV infection in response to various SIV peptides (4). It is estimated that LDA-based pCTL frequencies are 1 to 2 log10 lower than the actual in vivo precursor frequencies (24, 39, 71). Direct measurement of antigen-specific CD8+ T cells by using major histocompatibility complex peptide tetramers (7, 66, 72) or by measuring CTL effector molecules like granzyme and IFN-γ (86, 92) often reveals higher numbers of specific T cells than are detected in CTL assays. However, the relevance of these measures to the in vivo lytic function is not clear (4). The CTL precursor frequencies achieved by immunization with attenuated SHIV89.6 are comparable to or slightly lower than the frequencies in wild-type SIV-infected monkeys (up to 0.02% of PBMC) and HIV-infected humans (0.001 to 0.1% of PBMC), as determined by LDA (22, 39, 44, 84). Similarly, immunization with attenuated SIVmacΔnef or SIVmacΔ3 leads to the induction of strong CTL responses (35). Thus, there is a need for caution in comparing results obtained by different in vitro assays measuring antigen-specific recall responses (i.e., CTL and ELISPOT). To an even greater extent, a meaningful comparison between IFN-γ mRNA levels in freshly isolated PBMC and the in vitro induction of antigen-specific effector functions (i.e., CTL and ELISPOT test results) is problematic.

The assessment of CTL precursor frequencies directly in the cervico-vaginal mucosa or in the draining genital lymph nodes after i.vag. challenge in future studies should provide more conclusive information about the relative importance of the strength versus location of vaccine-induced CTL in providing effective immunity against i.vag. SIV challenge. This analysis will be particularly important in light of the findings that in HIV-1-infected patients, CTL activity measured in PBMC did not always correlate with CTL activity detectable in the cervix (67, 96).

It has been demonstrated in the mouse system that cytokines, especially IFN-γ and TNF-α, contribute to the clearance of viral infections via noncytolytic mechanisms (25, 26, 38, 52, 75). More recently, it has also been shown that IFN-γ is involved in the noncytolytic clearance of hepatitis B virus in the chimpanzee (27). Although CD8-mediated inhibition of SIV replication was detected in approximately 60% of all SHIV89.6-vaccinated monkeys at the time of challenge, the ability of CD8+ T cells of an individual monkey to inhibit virus replication was not predictive of challenge outcome in that monkey. Thus, in the present study, SHIV89.6-induced control of p.c. viremia was associated with CD8+-T-cell-mediated cytolytic activity but not with noncytolytic CD8+-T-cell-mediated suppression of viral replication.

In addition to more rapid and robust CD8+-T-cell responses, vaccinated-protected monkeys had increased IFN-α PBMC mRNA levels during the acute phase p.c. compared to vaccinated-unprotected monkeys (Table (Table3).3). Almost 90% of vaccinated-protected animals had higher IFN-α PBMC mRNA levels during the acute phase p.c. than at the time of challenge. This suggests that the ability to increase IFN-α mRNA levels p.c. contributed to the observed protection against challenge with pathogenic SIVmac239 in these animals.

The role of alpha/beta interferons in vaccine-mediated protection is unknown. We have recently shown that IFN-α mRNA levels in PBMC and lymphoid tissues of vaccine-naïve acute and chronically SIVmac-infected macaques reflect virus replication and do not correlate with control of virus replication (1). Further, increased serum IFN-α protein levels precede the detection of vRNA levels in SIV infection (23). Thus, it is possible that increased IFN-α PBMC mRNA levels in vaccinated-protected monkeys are a response to low levels of virus replication after exposure to the challenge virus. Consistent with the conclusion that IFN-α mRNA levels increase in response to viral replication, IFN-α PBMC mRNA levels in some naïve animals were increased at weeks 1 and 2 p.c. but reduced to baseline levels at week 5 p.c., relative to levels in uninfected animals.

Clearly, more studies are needed to determine the role of alpha/beta interferon in vaccine-induced anti-SIV immunity. It should also be emphasized that IFN-α has been shown to influence innate and adaptive antiviral immune responses through its coordinated action with other cytokines (68, 76). It has been shown that the timing of IFN-α/β responses is critical for the induction of NK cell cytotoxicity and to induce NK cell proliferation via induction of IL-15 (68). Further, in the mouse model of lymphocytic choriomeningitis virus infection, an early CD8+-T-cell-dependent induction of IFN-γ in vivo was associated with the prior expression of IFN-α/β and IL-18 (76). Thus, in vaccine-mediated protection, the absolute amount of IFN-α mRNA levels expressed by an individual may be less critical than the expression at the appropriate time and anatomic location after exposure to the challenge virus.

As recently reported, PBMC Mx mRNA levels were increased in the majority (80%) of unvaccinated SIV-infected animals, but only few PBMC samples had concomitant increases in IFN-α PBMC mRNA levels (1). In the present study, the PBMC Mx mRNA levels of the vaccine-naïve animals were significantly higher than PBMC Mx mRNA levels in vaccinated-protected monkeys during the acute (Table (Table3)3) and chronic phases p.c. Thus, increased Mx mRNA levels reflected virus replication and were not associated with protection from SIV challenge. In fact, by week 24 p.c., Mx PBMC mRNA levels of vaccinated-protected monkeys had decreased to levels that were indistinguishable from Mx PBMC mRNA levels of uninfected animals. In contrast, vaccinated-unprotected animals and naïve animals had increased Mx mRNA levels, consistent with the high plasma vRNA levels and ongoing virus replication in these animals. Importantly, it was shown in a group of HIV-infected patients that the level of PBMC-associated Mx protein correlated with the clinical stage of the patient (94). Patients with more advanced disease had higher levels of Mx (93). This is consistent with our data showing persistently increased Mx PBMC mRNA levels in the naïve and vaccinated-unprotected animals p.c. Thus, the early induction of IFN-α mRNA levels is associated with control of challenge virus replication in vaccinated-protected monkeys. However, the persistence of increased Mx mRNA levels in PBMC is a marker of ongoing virus replication and does not reflect a protective antiviral immune response.

Anti-HIV/SIV cellular immune responses in disease and vaccine-mediated protection have been the focus of intense study in recent years. Immunization with nonpathogenic SHIV89.6 led to the induction of persistent anti-SIV proliferative responses in the majority of animals, as has been observed after immunization with live attenuated viruses (19). In the present study, SHIV89.6-vaccinated animals were able to maintain SIV-specific proliferative responses during the acute p.c. period. The strength of this response was similar in vaccinated, protected, and unprotected monkeys. In contrast, only 45% (4 of 9 monkeys tested) of naïve monkeys showed SIV-specific proliferative activity during the acute phase p.c., compared to 82% (14 of 17 tested) of all vaccinated monkeys. This was consistent with the early control of challenge virus replication in the majority of the vaccinated animals. However, during the acute phase p.c., the T-cell proliferative response was similar in vaccinated-protected and vaccinated-unprotected monkeys, and thus, the presence or strength of lymphoproliferative responses did not correlate with challenge outcome (Table (Table2).2). At the time of chronic infection (20 to 24 weeks p.c.), the two naïve monkeys (31423 and 25402) with the lowest plasma vRNA levels showed higher proliferative responses to SIV antigen than the remaining naïve animals with intermediate plasma vRNA levels (data not shown). The naïve animal (23756) with the highest plasma vRNA levels had the lowest T-cell numbers at the time of euthanasia, and the proliferative response could not be tested due to the lack of available cells (data not shown). These data were consistent with the results obtained in HIV-infected patients, where an inverse correlation between plasma vRNA levels and proliferative responses was seen (83). However, T-cell proliferative responses were tested only at a single time point during the chronic phase of infection and therefore might not be representative of a general trend.

To further assess the role of CD4-T-helper cytokine responses in SHIV89.6-mediated protection, Th1 and Th2 cytokine mRNA levels were determined. However, we did not detect polarized Th1 or Th2 responses in any monkeys, and a specific pattern of Th1/Th2 cytokine expression was not associated with challenge outcome. Similarly, simultaneous production of IFN-γ, IL-2, and IL-4 was observed in PBMC of vaccinated rhesus macaques after challenge with a pathogenic SHIV (28).

The role of other cytokines in the observed vaccine-mediated protection was also unclear. PBMC mRNA levels for various proinflammatory cytokines (TNF-α, IL-6, IL-12) were increased p.c., and no difference in the magnitude or the kinetics of the cytokine mRNA induction was observed between naïve and vaccinated animals (Table (Table2).2). Consistent with this finding, mRNA levels for IL-6 and TNF-α are induced with similar kinetics and strength in lymph nodes from rhesus macaques acutely infected with either attenuated or pathogenic SIV (99).

In the present study, only a few SHIV89.6-immunized animals had elevated β-chemokine (MIP-1α and MIP-1β) PBMC mRNA levels at the time of challenge. During the first 5 weeks p.c., a similar percentage of vaccinated and naïve monkeys had elevated β-chemokine PBMC mRNA levels (Table (Table2).2). Further, the magnitude of the increase in β-chemokine mRNA levels was similar in monkeys of all groups. Thus, increased β-chemokine mRNA levels p.c. were not predictive of challenge outcome but seemed to be part of an innate, proinflammatory response to pathogen exposure. In contrast to our results, some vaccine studies in rhesus and cynomolgous macaques found that immunization led to increased chemokine levels and that these chemokine levels correlated with protection (3, 21, 29, 41). However, technical differences in the analysis used likely contributed to the disparate results. In the latter studies, β-chemokine levels were measured in supernatants of in vitro stimulated PBMC, whereas in the present study β-chemokine mRNA levels were measured directly in cells isolated from vaccinated and naive monkeys without in vitro stimulation. Our data are consistent with studies of HIV-1-infected patients in which a lack of correlation between β-chemokine levels and disease progression has been described (9, 64, 65). Similarly, a recent study in SIV-infected rhesus macaques showed that increased β-chemokine PBMC mRNA levels correlated with disease progression and low CD4 counts (31).

It should be noted that the induction of any immune response in vivo is characterized by the induction of multiple cytokines and chemokines. Therefore, we have to consider not only the expression levels and the function of each individual cytokine, but also the interaction between multiple cytokines (61, 68, 77, 85). Further, these effects may differ depending on the anatomic location. This complexity should be addressed in more detail in future studies.

We found no association between serum anti-SIV binding or neutralizing antibody titers with challenge outcome. This is consistent with earlier studies that have shown that protection from pathogenic virus can be achieved in the absence of strong antibody responses with attenuated SHIVs and SIV challenge (60, 69, 78) or with attenuated SIV and SHIV challenge (15, 88). In fact, the higher p.c. anti-SIV IgG binding antibody titers in naïve and vaccinated-unprotected animals suggest that antibody titers were related to the amount of viral antigen present. Thus, the animals with higher plasma vRNA levels also had higher antibody titers (Table (Table2).2). This is consistent with our observation of delayed postimmunization antibody induction in i.vag. SHIV89.6-immunized monkeys that had lower plasma vRNA levels than i.v. and i.n. immunized monkeys during the acute phase of infection. Similar data have been reported after infection with other attenuated SIV viruses (14, 18, 91). It should be noted that the simple measurement of SIV-specific binding antibody titers might be insufficient in assessing their immunological function (11, 12). However, it seems unlikely that affinity maturation of the anti-HIV Env responses in the SHIV-immunized animals contributed to protection from the SIV Env heterologous virus challenge. Further, despite the presence of neutralizing antibodies to the vaccine virus SHIV89.6, we found no neutralizing antibody responses to the challenge virus SIVmac239 in vaccinated monkeys. This is consistent with earlier reports showing potent neutralizing antibody responses in SHIV89.6-infected monkeys to related laboratory-adapted HIV-1 strains but not to primary HIV-1 isolates (62); it is also consistent with reports showing that neutralizing antibodies develop only after prolonged SIVmac239 infection in macaques and are not detectable during the acute phase of infection (57). More importantly, and consistent with our results, the protection achieved by immunization with attenuated SIV (SIVmacΔ3 or SIVmacΔnef) against pathogenic SIVmac251 (17, 98) cannot be explained by the induction of neutralizing antibodies.

Despite the analysis of multiple immune effector mechanisms (Table (Table2)2) in a large number of vaccinated animals, no single immune response was found to be responsible for protection against i.vag. challenge with pathogenic SIVmac239 in all animals. It is possible that immune mechanisms other than the ones we measured contribute to the observed protection against infection with pathogenic virus. The data presented in the present study are also limited to immune responses measured in PBMC, and this may not accurately reflect the immune responses in the lymphoid tissues, the main sites of HIV and SIV replication (73, 74, 82). Further, the results of the present study support the fact that although live attenuated vaccines provide the most consistent protection from SIV challenge, the protection is neither uniform nor absolute. The coexistence of the vaccine and challenge virus after challenge has been described (37), and recent reports indicate that superinfection with HIV can occur (79).

The present study clearly demonstrates that the route of immunization with an attenuated lentivirus vaccine does not influence mucosal challenge outcome if the monkeys have been systemically infected for a prolonged period of time. Further, the results of the study show that multiple host immune effector mechanisms contribute to live attenuated lentivirus-induced protection against i.vag. challenge with pathogenic SIVmac239. Thus, it seems imprudent to judge vaccine immunogenicity and predict efficacy based on a single immune parameter. We have shown that innate as well as cellular immune responses contribute to the observed protection. The systemic and local effects of these immune responses need further evaluation and are likely to be critical in our understanding of the observed vaccine-mediated protection.

Acknowledgments

This work was supported by Public Health Science grant RR00169, grant RR14555 from the National Center for Research Resources, and grant AI44480 from the National Institute of Allergy and Infectious Diseases.

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