Received 29 August 2002/Accepted 3 December 2002

Transcription

1 JOURNAL OF VIROLOGY, Mar. 2003, p Vol. 77, No X/03/$ DOI: /JVI Copyright 2003, American Society for Microbiology. All Rights Reserved. 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 Kristina Abel, 1,2 Lara Compton, 1,2 Tracy Rourke, 1,2 David Montefiori, 3 Ding Lu, 1,2 Kristina Rothaeusler, 2 Linda Fritts, 1,2 Kristen Bost, 1 and Christopher J. Miller 1,2,4 * Center for Comparative Medicine, 1 California National Primate Research Center, 2 and Department of Pathology, Microbiology, and Immunology, 4 School of Veterinary Medicine, University of California Davis, Davis, California, and Department of Surgery, Duke University Medical Center, Durham, North Carolina 3 Received 29 August 2002/Accepted 3 December 2002 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- )-secreting cells during the acute phase postchallenge. Vaccinated-protected animals also had a more pronounced increase in peripheral blood mononuclear cell IFN- 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 * Corresponding author. Mailing address: University of California Davis, California National Primate Research Center, County Rd. 98/ Hutchison Dr., Davis, CA Phone: (530) Fax: (530) cjmiller@ucdavis.edu. 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 , 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 3099

2 3100 ABEL ET AL. J. VIROL. 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- / ], proinflammatory cytokines, chemokines, and CD8 T-cell-mediated noncytolytic antiviral activity) as well as adaptive cellular (antigenspecific cytotoxicity, IFN- 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- -secreting cells, and a greater ability to increase IFN- 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 1 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 , a highly cytopathic, M-tropic variant (13). The SHIV HXBc2 stock contained 4,800 50% tissue culture infective doses (TCID 50 )/ml, and the two SHIV89.6 stocks contained 1,800 and 10 4 TCID 50 /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 TCID 50 (51). 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 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 (SIVmac239 nef, SIVmac239 3) 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 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 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 1). In addition, 18 vaccine-naïve, SIV-infected animals were included in the study as challenge controls (see Tables 1 and 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). 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 10 5 TCID 50 /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.

3 VOL. 77, 2003 ROLE OF CTL AND IFN- IN PROTECTION FROM SIV CHALLENGE 3101 TABLE 1. Virological assessment of SHIV89.6-vaccinated animals at the time of challenge Animal no. Origin Age (yrs) a Route of immunization b Time of challenge c Plasma vrna d Virus isolation SIV Gag PCR HIV Env e Indian 15 i.v. 16 mo e Indian 9 i.v. 16 mo e Indian 15 i.v. 16 mo e Indian 5 i.v. 16 mo e Indian 5 i.v. 16 mo Indian 15 i.v. 14 mo Indian 11 i.v. 14 mo Indian 11 i.v. 14 mo Indian 10 i.v. 14 mo Indian 8 i.v. 14 mo Indian 8 i.v. 14 mo f Indian 7 i.v. 6 mo f Indian 6 i.v. 6 mo f Chinese 5 i.v. 6 mo f Chinese 5 i.v. 6 mo g Indian 8 i.n. 14 mo g Indian 5 i.n. 14 mo g 1/2 Chinese 5 i.n. 14 mo h Indian 6 i.n. 14 mo h 1/2 Chinese 6 i.n. 14 mo h Indian 5 i.n. 14 mo Indian 7 i.n. 14 mo Indian 7 i.n. 14 mo /2 Chinese 8 i.n. 14 mo Indian 10 i.n. 14 mo Indian 6 i.n. 14 mo Indian 14 i.vag. 14 mo Indian 12 i.vag. 14 mo Indian 11 i.vag. 14 mo Indian 6 i.vag. 17 mo Indian 5 i.vag. 17 mo Indian 5 i.vag. 17 mo Indian 9 i.vag. 15 mo Indian 10 i.vag. 9 mo Indian 7 i.vag. 6 mo Chinese 5 i.vag. 6 mo. 2.7 ND i Chinese 5 i.vag. 6 mo. 2.7 ND Chinese 5 i.vag. 6 mo. 2.7 ND Chinese 4 i.vag. 9 mo f Indian 7 i.vag. 6 mo f 1/4 Chinese 7 i.vag. 6 mo f Chinese 6 i.vag. 6 mo Chinese 5 None Day 0 N/A j N/A N/A N/A Indian 7 None Day 0 N/A N/A N/A N/A Indian 13 None Day 0 N/A N/A N/A N/A Indian 10 None Day 0 N/A N/A N/A N/A /2 Chinese 10 None Day 0 N/A N/A N/A N/A Indian 6 None Day 0 N/A N/A N/A N/A Chinese 6 None Day 0 N/A N/A N/A N/A /4 Chinese 7 None Day 0 N/A N/A N/A N/A Indian 11 None Day 0 N/A N/A N/A N/A Indian 7 None Day 0 N/A N/A N/A N/A Indian 7 None Day 0 N/A N/A N/A N/A Indian 11 None Day 0 N/A N/A N/A N/A Indian 13 None Day 0 N/A N/A N/A N/A Indian 9 None Day 0 N/A N/A N/A N/A Indian 5 None Day 0 N/A N/A N/A N/A Indian 6 None Day 0 N/A N/A N/A N/A Indian 6 None Day 0 N/A N/A N/A N/A Indian 9 None Day 0 N/A N/A N/A N/A a Age of the animal at the time of immunization. b Animals were immunized with 10 4 TCID 50 of SHIV89.6 if not indicated otherwise. c Intravaginal challenge with 10 5 TCID 50 of SIVmac239; time refers to the time postimmunization when the animal was challenged. d Plasma viral RNA levels in log 10 vrna copies/ml of plasma. e First immunization, 2,400 TCID 50 of SHIV HXBc2; second immunization, 900 TCID 50 of SHIV89.6 at 14 weeks post SHIV HXBc2 immunization. f Animals were challenged two times per day at every other day on 4 days. g Three immunizations with 1,800 TCID 50 of SHIV89.6. h Three immunizations with 1,800 TCID 50 of SHIV89.6 plus 10 ng of CT. i ND, not done. j N/A, not applicable.

4 3102 ABEL ET AL. J. VIROL. 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/10 5 PBMC, 100 to 1,000 SIV env copies/10 5 PBMC, and 10,000 HIV env copies/10 5 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 p28 CA concentration as determined by ELISA (Coulter Corporation, Miami, Fla.). In most batches of the AT-2 SIV, 10 ng of p28 CA antigen/ml corresponds to about 1 g of total protein/ml. The following p28 CA antigen concentrations were used: 0.1, 1.0, and 10.0 ng of p28 CA /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- ELISPOT assay. The number of IFN- -secreting cells in PBMC was determined by using an IFN- 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 p28 CA peptide pool at a concentration of 1 g 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 p28 CA 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- -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- -secreting cells/well exceeded 50 cells per PBMC and if the number of positive IFN- 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- SFC per PBMC. For reporting purposes, the background IFN- spot numbers observed in medium-only wells were subtracted from the IFN- 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 p28 CA -specific IFN- secretion (data not shown). In every assay, PBMC from SIV-negative monkeys and SIVpositive 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 g 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 g/ml) for 30 min at 37 C and then infected for 3 h with SIVmac239 (100 TCID 50 /10 6 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 l 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- l reaction volume containing 5 l of cdna and 20 l 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 (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

5 VOL. 77, 2003 ROLE OF CTL AND IFN- IN PROTECTION FROM SIV CHALLENGE 3103 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 as 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 log 10 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 1). Six of the 16 i.v. SHIV89.6-immunized monkeys had previously been immunized i.v. with nonpathogenic SHIV HXBc2 (Table 1). Following SHIV HXBc2 immunization, these animals developed peak SHIV HXBc2 viremia at week 2 postinoculation (p.i.) (3.9 to 6.2 log 10 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. 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. 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 log 10 vrna copies/ml) post SHIV89.6 inoculation than all other i.v. immunized monkeys (5.7 to 6.9 log 10 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 1). Peak vrna levels (week 2 p.i.) were 1 to 2 log 10 higher in i.v. and i.n. immunized monkeys than in i.vag. immunized monkeys (Fig. 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). Consistent with the delayed and lower plasma vrna levels in i.vag. immunized monkeys, i.v. and i.n. immunized monkeys 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 log 10 vrna copies per ml of plasma. (B) Serum anti-siv IgG antibody titers are reported as endpoint dilutions. The symbols represent i.v. (F), i.n. (Œ), and i.vag. ( ) immunized monkeys. The 6 i.v. immunized monkeys that received a SHIV HXBc2 inoculation prior to SHIV89.6 immunization were not included in the calculation of mean plasma vrna levels of i.v. 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. 1B). One i.vag. immunized monkey (no ) 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. 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 and endpoint titers of 1,600 in

6 3104 ABEL ET AL. J. VIROL. Downloaded from 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. with SIVmac239. Plasma vrna levels are reported as log 10 vrna copies per ml of plasma. The arrow indicates the detection limit of the assay (500 copies), and the dashed line marks the virological cutoff point for vaccine-induced protection (vrna level of 10 4 copies/ml). The number in the upper right corner represents the number of animals in each group. Each symbol represents results for an individual animal. 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 ) 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 1). At the time of challenge, vrna was detectable in the plasma of 6 of the 43 SHIV89.6- immunized monkeys ( 3.0 log 10 vrna copies/ml in monkeys 21349, 26249, and 26509; 3.0 to 4.0 log 10 vrna copies/ml in monkeys 23744, 26012, and 31413), and 3 of 43 monkeys (21349, 26154, and 31413) were virus isolation positive (Table 1). All of the monkeys tested positive by PCR for viral DNA (SIV gag) in their PBMC at the time of challenge (Table 1). 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. 2), and no further significant change (P 0.05) in plasma vrna levels occurred during the next 6 months (Fig. 2). All naïve monkeys challenged with SIVmac239 had peak plasma vrna levels above 10 4 copies/ml, and the vast majority (16 of 18, or 88%) of these naïve monkeys continued to have vrna levels above on September 18, 2018 by guest

7 VOL. 77, 2003 ROLE OF CTL AND IFN- IN PROTECTION FROM SIV CHALLENGE 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 vaccinemediated 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 10 4 copies/ml at all times during the 6-month period p.c., whereas vaccinated but unprotected animals had plasma vrna levels above 10 4 copies/ml at least once during this time period. 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 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 10 4 copies/ml and were also considered protected (Table 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 1 and 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 1 and 2). 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 (r ; 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 2). All of the vaccinated-unprotected animals tested PCR positive for SIV env in PBMC (Table 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 2). Eight of the 24 SIV env-positive monkeys (protected monkeys 20 to 27) had vrna levels below 10 4 copies/ml and were defined as protected (Table 2). In fact, two of the 8 vaccinated-protected and SIV env PCR positive monkeys (nos 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 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 log 10 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 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 3). 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. 2). For the first 8 weeks p.c., more than 80% of the vaccinated animals (n 36) had plasma vrna levels that remained below 10 4 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 ( 10 4 copies/ml) after week 8 p.c., and thus, these monkeys were considered unprotected (Table 2, unprotected monkeys 1 to 16). Only 1 of 43 vaccinated monkeys (no ) had a plasma vrna pattern that was similar to that of the naïve monkeys. In this animal, peak plasma viremia (5.7 log 10 vrna copies/ml) was detected between weeks 1 and 2 p.c., and vrna levels were consistently high ( 5.5 log 10 vrna copies/ml) up

8 3106 ABEL ET AL. J. VIROL. TABLE 2. Postchallenge evaluation of virological and immune response parameters Animal no. Peak plasma vrna (log 10 /ml) Parameters evaluated through 6 mo p.c. Proviral DNA (SIV env) Anti-SIV lgg a CD4:CD8 ratio b Parameters evaluated during the acute phase (wk 1 5) p.c. Proliferation c pctl d IFN- SFC e 72-Fold mrna induction IFN- IFN- Mx MIP-1 TNF- IL-6 Protected 1) ND g 2) ND 3) ND 4) ND ND ND 5) ND ND ND 6) ND ND 7) ) ) ) ) ) ) ND ND ND ND ND ND ND ND ND ND 14) ND ND ND ND ND ND ND ND ND ND 15) ND ND ND ND ND ND ND ND ND ND 16) ND ND ND ND ND ND ND ND ND ND 17) ND ND ND ND ND ND ND ND ND ND 18) ND ND ND ND ND ND ND ND ND ND 19) ND ND ND ND ND ND ND ND ND ND 20) ND 21) ND ND 22) ND ND ND 23) ) f ND 25) ) ND 27) Unprotected 1) ND ND ND ND 2) ND 3) ND ND ND ND 4) ND 5) ND 6) ) ) ND 9) ) ) ) ND ND ND ND ND ND ND ND ND ND 13) ND ND ND ND ND ND ND ND ND ND 14) ND ND ND ND ND ND ND ND ND ND 15) ND ND ND ND ND ND ND ND ND ND 16) ND ND ND ND ND ND ND ND ND ND Naïve 1) ND 2) ND 3) ND 4) ND 5) ND 6) ND 7) ND 8) ND ND 9) ND 10) ND ND ND ND ND ND ND ND ND ND ND 11) ND ND ND ND ND ND 12) ND ND ND ND ND ND ND ND ND ND ND 13) ND ND ND ND 14) ND ND ND ND ND ND ND ND ND ND 15) ND ND ND ND ND ND 16) ND ND ND ND ND ND ND ND ND ND ND 17) ND ND ND ND ND ND ND ND ND ND ND 18) ND ND ND ND ND ND ND ND ND ND ND a For endpoint dilution titer:, 10 2 to 10 3 ;, 10 3 to 10 4 ;, b For extent of ratio inversion:, 20%;, 20% but 50%;, 50%. c,si 2.0;, SI 2.0 but 4.0;, SI 4.0. d, 300/10 6 CD8 cells;, 300 but 700/10 6 CD8 cells;, 700/10 6 CD8 cells. e, SPC/10 6 PBMC 50;, SPC/10 6 PBMC 50 but 100;, SPC/10 6 PBMC 100. f At week 24 p.c. g ND, not done.

9 VOL. 77, 2003 ROLE OF CTL AND IFN- IN PROTECTION FROM SIV CHALLENGE 3107 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 deviation in CD4 T-cell numbers in each group relative to the mean CD4 T-cell numbers in that group prechallenge per l of blood. 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 10 4 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 vaccinatedunprotected 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. 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 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 vaccinatedunprotected 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 2). 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 2). Thus, we have made no effort to distinguish immune responses among these two vaccinated-protected groups (Table 2 and data not shown). (i) Anti-SIV antibody responses. All vaccine-naïve control animals, except one rapid progressor monkey (no ), 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 10 4 (range, 2,000 to 800,000), compared to 100% of vaccinatedunprotected monkeys (range, 25,000 to 1,600,000) and 75% of naïve monkeys (range, 4,000 to 32,000) (Table 2). It should be noted that among the vaccinated-protected animals, the animals with plasma vrna levels of 2.7 log 10 copies/ml and undetectable SIV env DNA (animals 1 to 15) had lower anti- SIV binding antibody titers (200 to 200,000) than vaccinatedprotected animals with plasma vrna levels of 2.7 log 10 but 4.0 log 10 copies/ml and/or detectable SIV env DNA (animals 16 to 27; anti-siv binding antibody titers, 16,000 to 800,000) (Table 2). These antibody responses persisted throughout the p.c. period, and at 6 months p.c. antibody titers of 10 4 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 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 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 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. 4). At the time of challenge, the vaccinated-protected and vaccinated-unprotected animals had similar frequencies of SIV Gagspecific precursor CTLs (pctls) (mean, 481 and 497 pctls per 10 6 CD8 T cells, respectively) (Fig. 4). However, vacci-