Prior infection with a nonpathogenic chimeric simian-human immunodeficiency virus does not efficiently protect macaques against challenge with simian immunodeficiency virus.

Prior infection with a nef-deleted simian immunodeficiency virus (SIV) protects macaques not only against a homologous pathogenic SIV challenge but also against challenge with a chimeric SIV expressing a human immunodeficiency virus type 1 env gene (SHIV). Since this SHIV is itself nonpathogenic, we sought to explore the use of a nonpathogenic SHIV as a live, attenuated AIDS virus vaccine. Four cynomolgus monkeys infected for greater than 600 days with a chimeric virus composed of SIVmac 239 expressing the human immunodeficiency virus type 1 HXBc2 env, tat, and rev genes were challenged intravenously with 100 animal infectious doses of the J5 clone of SIVmac 32H, an isolate derived by in vivo passage of SIVmac 251. Three of the four monkeys became infected with SIVmac. This observation underlines the difficulty, even with a live virus vaccine, in protecting against an AIDS virus infection.     

Expression of gamma interferon by simian immunodeficiency virus increases attenuation and reduces postchallenge virus load in vaccinated rhesus macaques.

Simian immunodeficiency virus (SIV) infection of macaques is a model for human immunodeficiency virus (HIV) infection. We have previously reported the construction and characterization of an SIV vector with a deletion in the nef gene (SIV(delta nef)) and expressing gamma interferon (SIV(HyIFN)) (L. Giavedoni and T. Yilma, J. Virol. 70:2247-2251, 1996). We now show that rhesus macaques vaccinated with SIV(HyIFN) have a lower viral load than a group similarly immunized with SIV(delta nef). Viral loads remained low in the SIV(HyIFN)-vaccinated group even though SIV expressing gamma interferon could not be isolated after 6 weeks postimmunization in these animals. All immunized and two naive control macaques became infected when challenged with virulent SIV(mac251), at 25 weeks postvaccination. In contrast to the two naive controls that died by 12 and 18 weeks postchallenge, all vaccinated animals remained healthy for more than 32 weeks. In addition, postchallenge cell-associated virus load was significantly lower in SIV(HyIFN)-immunized animals than in the group vaccinated with SIV(delta nef). These findings indicate that cytokine-expressing viruses can provide a novel approach for development of safe and efficacious live attenuated vaccines for AIDS.     

Complement-mediated, infection-enhancing antibodies in plasma from vaccinated macaques before and after inoculation with live simian immunodeficiency virus.

Rhesus monkeys vaccinated against infection with simian immunodeficiency virus (SIV) were examined, in retrospect, for the presence of infection-enhancing antibodies and possible consequences associated with the presence of these antibodies. At the time of experimental inoculation with live virus, complement-mediated, infection-enhancing antibodies were detected in plasma specimens from six of six animals vaccinated with detergent-inactivated whole virus, from nine of nine animals vaccinated with Formalin-inactivated whole virus, and from seven of eight animals immunized with two SIV subunit preparations. The presence of infection-enhancing antibodies at the time of viral challenge gave no indication of predicting vaccine success or failure. After live-virus challenge, titers of infection-enhancing antibodies, like enzyme-linked immunosorbent assay titers, increased in unprotected animals and decreased or became undetectable in animals protected by vaccination. Thus, vaccine protection against SIV infection can still be achieved in the presence of detectable complement-mediated, infection-enhancing antibodies.     

Protection against Simian Immunodeficiency Virus Vaginal Challenge by Using Sabin Poliovirus Vectors

Here we provide the first report of protection against a vaginal challenge with a highly virulent simian immunodeficiency virus (SIV) by using a vaccine vector. New poliovirus vectors based on Sabin 1 and 2 vaccine strain viruses were constructed, and these vectors were used to generate a series of new viruses containing SIV gag, pol, env, nef, and tat in overlapping fragments. Two cocktails of 20 transgenic polioviruses (SabRV1-SIV and SabRV2-SIV) were inoculated into seven cynomolgus macaques. All monkeys produced substantial anti-SIV serum and mucosal antibody responses. SIV-specific cytotoxic T-lymphocyte responses were detected in three of seven monkeys after vaccination. All 7 vaccinated macaques, as well as 12 control macaques, were challenged vaginally with pathogenic SIVmac251. Strikingly, four of the seven vaccinated animals exhibited substantial protection against the vaginal SIV challenge. All 12 control monkeys became SIV positive. In two of the seven SabRV-SIV-vaccinated monkeys we found no virological evidence of infection following challenge, indicating that these two monkeys were completely protected. Two additional SabRV-SIV-vaccinated monkeys exhibited a pronounced reduction in postacute viremia to <10(3) copies/ml, suggesting that the vaccine elicited an effective cellular immune response. Three of six control animals developed clinical AIDS by 48 weeks postchallenge. In contrast, all seven vaccinated monkeys remained healthy as judged by all clinical parameters. These results demonstrate the efficacy of SabRV as a potential human vaccine vector, and they show that the use of a vaccine vector cocktail expressing an array of defined antigenic sequences can be an effective vaccination strategy in an outbred population.     

Effect of vaccination with recombinant modified vaccinia virus Ankara expressing structural and regulatory genes of SIV(macJ5) on the kinetics of SIV replication in cynomolgus monkeys

The efficacy of a multicomponent vaccination with modified vaccinia Ankara constructs (rMVA) expressing structural and regulatory genes of simian immunodeficiency virus (SIV(mac251/32H/J5)) was investigated in cynomolgus monkeys, following challenge with a pathogenic SIV. Vaccination with rMVA-J5 performed at week 0, 12, and 24 induced a moderate proliferative response to whole SIV, a detectable humoral response to all but Nef SIV antigens, and failed to induce neutralizing antibodies. Two months after the last boost, the monkeys were challenged intravenously with 50 MID50 of SIV(mac251). All control monkeys, previously inoculated with non-recombinant MVA, were infected by week two and seroconverted by weeks four to eight. In contrast a sharp increase of both humoral and proliferative responses at two weeks post-challenge was observed in vaccinated monkeys compared to control monkeys. Although all vaccinated monkeys were infected, vaccination with rMVA-J5 appeared to partially control viral replication during the acute and late phase of infection as judged by cell- and plasma-associated viral load.     

Env-Independent Protection Induced by Live, Attenuated Simian Immunodeficiency Virus Vaccines

Live attenuated simian immunodeficiency viruses (SIV), such as nef deletion mutants, are the most effective vaccines tested in the SIV-macaque model so far. To modulate the antiviral immune response induced by live attenuated SIV vaccines, we had previously infected rhesus monkeys with a nef deletion mutant of SIV expressing interleukin 2 (SIV-IL2) (B. R. Gundlach, H. Linhart, U. Dittmer, S. Sopper, S. Reiprich, D. Fuchs, B. Fleckenstein, G. Hunsmann, S. Stahl-Hennig, and K. Überla, J. Virol. 71:2225–2232, 1997). In the present study, SIV-IL2-infected macaques and macaques infected with the nef deletion mutant SIVΔNU were challenged with pathogenic SIV 9 to 11 months postvaccination. In contrast to the results with naive control monkeys, no challenge virus could be isolated from the SIV-IL2- and SIVΔNU-infected macaques. However, challenge virus sequences could be detected by nested PCR in some of the vaccinated macaques. To determine the role of immune responses directed against Env of SIV, four vaccinated macaques were rechallenged with an SIV-murine leukemia virus (MLV) hybrid in which the env gene of SIV had been functionally replaced by the env gene of amphotropic MLV. All vaccinated macaques were protected from productive infection with the SIV-MLV hybrid in the absence of measurable neutralizing antibodies, while two naive control monkeys were readily infected. Since the SIV-MLV hybrid uses the MLV Env receptor Pit2 and not CD4 and a coreceptor for virus entry, chemokine inhibition and receptor interference phenomena were not involved in protection. These results indicate that the protective responses induced by live attenuated SIV vaccines can be independent of host immune reactions directed against Env.     

Evidence for recombination of live, attenuated immunodeficiency virus vaccine with challenge virus to a more virulent strain

Live, attenuated immunodeficiency virus vaccines, such as nef deletion mutants, are the most effective vaccines tested in the simian immunodeficiency virus (SIV) macaque model. In two independent studies designed to determine the breadth of protection induced by live, attenuated SIV vaccines, we noticed that three of the vaccinated macaques developed higher set point viral load levels than unvaccinated control monkeys. Two of these vaccinated monkeys developed AIDS, while the control monkeys infected in parallel remained asymptomatic. Concomitant with an increase in viral load, a recombinant of the vaccine virus and the challenge virus could be detected. Therefore, the emergence of more-virulent recombinants of live, attenuated immunodeficiency viruses and less-aggressive wild-type viruses seems to be an additional risk of live, attenuated immunodeficiency virus vaccines.     

A formalin inactivated whole SIVmac vaccine in Ribi adjuvant protects against homologous and heterologous SIV challenge.

Eight monkeys were immunized at 0, 4, 9, and 18 weeks with a total of 2 mg of formalin inactivated SIVmac vaccine with Ribi adjuvant. Two weeks after the last booster four immunized monkeys and two controls were challenged with 10 MID50 of live homologous virus SIVmac, and the remaining four vaccinated animals along with two controls were challenged with the heterologous SIVsm strain. All eight vaccinated monkeys resisted the virus challenge, whereas all controls became infected. Three months after the first challenge the monkeys were rechallenged with the same virus strain, without further boosting. Two of four vaccinated monkeys were still resistant to the homologous SIV strain, and three of four monkeys were resistant to the heterologous SIVsm strain. This study demonstrates vaccine induced cross-protection between SIV strains.     

Protection by Live, Attenuated Simian Immunodeficiency Virus against Heterologous Challenge

We examined the ability of a live, attenuated deletion mutant of simian immunodeficiency virus (SIV), SIVmac239Delta3, which is missing nef and vpr genes, to protect against challenge by heterologous strains SHIV89.6p and SIVsmE660. SHIV89.6p is a pathogenic, recombinant SIV in which the envelope gene has been replaced by a human immunodeficiency virus type 1 envelope gene; other structural genes of SHIV89.6p are derived from SIVmac239. SIVsmE660 is an uncloned, pathogenic, independent isolate from the same primate lentivirus subgrouping as SIVmac but with natural sequence variation in all structural genes. The challenge with SHIV89.6p was performed by the intravenous route 37 months after the time of vaccination. By the criteria of CD4(+) cell counts and disease, strong protection against the SHIV89.6p challenge was observed in four of four vaccinated monkeys despite the complete mismatch of env sequences. However, SHIV89.6p infection was established in all four previously vaccinated monkeys and three of the four developed fluctuating viral loads between 300 and 10,000 RNA copy equivalents per ml of plasma 30 to 72 weeks postchallenge. When other vaccinated monkeys were challenged with SIVsmE660 at 28 months after the time of vaccination, SIV loads were lower than those observed in unvaccinated controls but the level of protection was less than what was observed against SHIV89.6p in these experiments and considerably less than the level of protection against SIVmac251 observed in previous experiments. These results demonstrate a variable level of vaccine protection by live, attenuated SIVmac239Delta3 against heterologous virus challenge and suggest that even live, attenuated vaccine approaches for AIDS will face significant hurdles in providing protection against the natural variation present in field strains of virus. The results further suggest that factors other than anti-Env immune responses can be principally responsible for the vaccine protection by live, attenuated SIV.     

Vaccine protection against functional CTL abnormalities in simian human immunodeficiency virus-infected rhesus monkeys.

Accumulating evidence suggests that HIV-specific CD8(+) CTL are dysfunctional in HIV-infected individuals with progressive clinical disease. In the present studies, cytokine production by virus-specific CTL was assessed in the rhesus monkey model for AIDS to determine its contribution to the functional impairment of CTL. CTL from monkeys infected with nonpathogenic isolates of simian and simian-human immunodeficiency virus expressed high levels of IFN-gamma, TNF-alpha, and IL-2 after in vitro exposure to a nonspecific mitogen or the optimal peptide representing a dominant virus-specific CTL epitope. However, similarly performed studies assessing these capabilities in CTL from monkeys infected with pathogenic immunodeficiency virus isolates demonstrated a significant dysfunction in the ability of the CTL to produce IL-2 and TNF-alpha. Importantly, CTL from vaccinated monkeys that effectively controlled the replication of a highly pathogenic simian-human immunodeficiency virus isolate following challenge demonstrated a preserved capacity to produce these cytokines. These experiments suggest that defects in cytokine production may contribute to CTL dysfunction in chronic HIV or SIV infection. Moreover, an AIDS vaccine that confers protection against clinical disease evolution in this experimental model also preserves the functional capacity of these CTL to produce both IL-2 and TNF-alpha.     

Envelope variable region 4 is the first target of neutralizing antibodies in early simian immunodeficiency virus mac251 infection of rhesus monkeys

A major goal of AIDS vaccine development is to design vaccination strategies that can elicit broad and potent protective antibodies. The initial viral targets of neutralizing antibodies (NAbs) early after human or simian immunodeficiency virus (HIV/SIV) infection are not known. The identification of early NAb epitopes that induce protective immunity or retard the progression of disease is important for AIDS vaccine development. The aim of this study was to determine the Env residues targeted by early SIV NAbs and to assess the influence of prior vaccination on neutralizing antibody kinetics and specificity during early infection. We previously described stereotypic env sequence variations in SIVmac251-infected rhesus monkeys that resulted in viral escape from NAbs. Here, we defined the early viral targets of neutralization and determined whether the ability of serum antibody from infected monkeys to neutralize SIV was altered in the setting of prior vaccination. To localize the viral determinants recognized by early NAbs, a panel of mutant pseudoviruses was assessed in a TZM-bl reporter gene neutralization assay to define the precise changes that eliminate recognition by SIV Env-specific NAbs in 16 rhesus monkeys. Changing R420 to G or R424 to Q in V4 of Env resulted in the loss of recognition by NAbs in vaccinated monkeys. In contrast, mutations in the V1 region of Env did not alter the NAb profile. These findings indicate that early NAbs are directed toward SIVmac251 Env V4 but not the V1 region, and that this env vaccination regimen did not alter the kinetics or the breadth of NAbs during early infection.     

A distinct African lentivirus from Sykes' monkeys.

Asymptomatic infection with simian immunodeficiency virus (SIV) has been demonstrated in African Sykes' monkeys (Cercopithecus mitis albogularis), and virus isolation confirmed infection with a novel SIV from Sykes' monkeys (SIVsyk). Macaques inoculated with SIVsyk became persistently infected but remained clinically healthy. We utilized polymerase chain reaction amplification to generate a full-length, infectious molecular clone of SIVsyk. The genome organization of SIVsyk is similar to that of the other primate lentiviruses, consisting of gag, pol, vif, vpr, tat, rev, env, and nef. A unique feature is the absence of the highly conserved NF-kappa B binding site in the long terminal repeat. SIVsyk is genetically equidistant from other primate lentiviruses. Thus, SIVsyk represents a new group that is distinct from the four previously recognized primate lentivirus groups: human immunodeficiency virus type 1 (HIV-1), SIV from sooty mangabeys (SIVsmm) and HIV-2, SIV from African green monkeys (SIVagm), and SIV from mandrills (SIVmnd). The genetic differences between SIVsyk and SIVagm, isolates derived from monkeys of the same genus, underscore the potential for other distinct SIVs which have yet to be isolated and characterized.     

Gene-based vaccination with a mismatched envelope protects against simian immunodeficiency virus infection in nonhuman primates

The RV144 trial demonstrated that an experimental AIDS vaccine can prevent human immunodeficiency virus type 1 (HIV-1) infection in humans. Because of its limited efficacy, further understanding of the mechanisms of preventive AIDS vaccines remains a priority, and nonhuman primate (NHP) models of lentiviral infection provide an opportunity to define immunogens, vectors, and correlates of immunity. In this study, we show that prime-boost vaccination with a mismatched SIV envelope (Env) gene, derived from simian immunodeficiency virus SIVmac239, prevents infection by SIVsmE660 intrarectally. Analysis of different gene-based prime-boost immunization regimens revealed that recombinant adenovirus type 5 (rAd5) prime followed by replication-defective lymphocytic choriomeningitis virus (rLCMV) boost elicited robust CD4 and CD8 T-cell and humoral immune responses. This vaccine protected against infection after repetitive mucosal challenge with efficacies of 82% per exposure and 62% cumulatively. No effect was seen on viremia in infected vaccinated monkeys compared to controls. Protection correlated with the presence of neutralizing antibodies to the challenge viruses tested in peripheral blood mononuclear cells. These data indicate that a vaccine expressing a mismatched Env gene alone can prevent SIV infection in NHPs and identifies an immune correlate that may guide immunogen selection and immune monitoring for clinical efficacy trials.     

SIV Vpx Is Essential for Macrophage Infection but Not for Development of AIDS

Analysis of rhesus macaques infected with a vpx deletion mutant virus of simian immunodeficiency virus mac239 (SIVΔvpx) demonstrates that Vpx is essential for efficient monocyte/macrophage infection in vivo but is not necessary for development of AIDS. To compare myeloid-lineage cell infection in monkeys infected with SIVΔvpx compared to SIVmac239, we analyzed lymphoid and gastrointestinal tissues from SIVΔvpx-infected rhesus (n = 5), SIVmac239-infected rhesus with SIV encephalitis (7 SIV239E), those without encephalitis (4 SIV239noE), and other SIV mutant viruses with low viral loads (4 SIVΔnef, 2 SIVΔ3). SIV+ macrophages and the percentage of total SIV+ cells that were macrophages in spleen and lymph nodes were significantly lower in rhesus infected with SIVΔvpx (2.2%) compared to those infected with SIV239E (22.7%), SIV239noE (8.2%), and SIV mutant viruses (10.1%). In colon, SIVΔvpx monkeys had fewer SIV+ cells, no SIV+ macrophages, and lower percentage of SIV+ cells that were macrophages than the other 3 groups. Only 2 SIVΔvpx monkeys exhibited detectable virus in the colon. We demonstrate that Vpx is essential for efficient macrophage infection in vivo and that simian AIDS and death can occur in the absence of detectable macrophage infection.     

Dysfunction of simian immunodeficiency virus/simian human immunodeficiency virus-induced IL-2 expression by central memory CD4+ T lymphocytes

Production of IL-2 and IFN-gamma by CD4+ T lymphocytes is important for the maintenance of a functional immune system in infected individuals. In the present study, we assessed the cytokine production profiles of functionally distinct subsets of CD4+ T lymphocytes in rhesus monkeys infected with pathogenic or attenuated SIV/simian human immunodeficiency virus (SHIV) isolates, and these responses were compared with those in vaccinated monkeys that were protected from immunodeficiency following pathogenic SHIV challenge. We observed that preserved central memory CD4+ T lymphocyte production of SIV/SHIV-induced IL-2 was associated with disease protection following primate lentivirus infection. Persisting clinical protection in vaccinated and challenged monkeys is thus correlated with a preserved capacity of the peripheral blood central memory CD4+ T cells to express this important immunomodulatory cytokine.     

Gag-Specific Cellular Immunity Determines In Vitro Viral Inhibition and In Vivo Virologic Control following Simian Immunodeficiency Virus Challenges of Vaccinated Rhesus Monkeys

A comprehensive vaccine for human immunodeficiency virus type 1 (HIV-1) would block HIV-1 acquisition as well as durably control viral replication in breakthrough infections. Recent studies have demonstrated that Env is required for a vaccine to protect against acquisition of simian immunodeficiency virus (SIV) in vaccinated rhesus monkeys, but the antigen requirements for virologic control remain unclear. Here, we investigate whether CD8(+) T lymphocytes from vaccinated rhesus monkeys mediate viral inhibition in vitro and whether these responses predict virologic control following SIV challenge. We observed that CD8(+) lymphocytes from 23 vaccinated rhesus monkeys inhibited replication of SIV in vitro. Moreover, the magnitude of inhibition prior to challenge was inversely correlated with set point SIV plasma viral loads after challenge. In addition, CD8 cell-mediated viral inhibition in vaccinated rhesus monkeys correlated significantly with Gag-specific, but not Pol- or Env-specific, CD4(+) and CD8(+) T lymphocyte responses. These findings demonstrate that in vitro viral inhibition following vaccination largely reflects Gag-specific cellular immune responses and correlates with in vivo virologic control following infection. These data suggest the importance of including Gag in an HIV-1 vaccine in which virologic control is desired.     

Risk of immunodeficiency virus infection may increase with vaccine-induced immune response

To explore the efficacy of novel complementary prime-boost immunization regimens in a nonhuman primate model for HIV infection, rhesus monkeys primed by different DNA vaccines were boosted with virus-like particles (VLP) and then challenged by repeated low-dose rectal exposure to simian immunodeficiency virus (SIV). Characteristic of the cellular immune response after the VLP booster immunization were high numbers of SIV-specific, gamma interferon-secreting cells after stimulation with inactivated SIV particles, but not SIV peptides, and the absence of detectable levels of CD8(+) T cell responses. Antibodies specific to SIV Gag and SIV Env could be induced in all animals, but, consistent with a poor neutralizing activity at the time of challenge, vaccinated monkeys were not protected from acquisition of infection and did not control viremia. Surprisingly, vaccinees with high numbers of SIV-specific, gamma interferon-secreting cells were infected fastest during the repeated low-dose exposures and the numbers of these immune cells in vaccinated macaques correlated with susceptibility to infection. Thus, in the absence of protective antibodies or cytotoxic T cell responses, vaccine-induced immune responses may increase the susceptibility to acquisition of immunodeficiency virus infection. The results are consistent with the hypothesis that virus-specific T helper cells mediate this detrimental effect and contribute to the inefficacy of past HIV vaccination attempts (e.g., STEP study).     

Identification of SIV env-specific CTL in the jejunal mucosa in vaginally exposed, seronegative rhesus macaques (Macaca mulatta)

We previously reported major histocompatibility complex Class I-restricted cytotoxic T lymphocytes (CTL) in jejunal lamina propria (LP) of monkeys following colonic exposure to subinfectious SIV doses. Those monkeys with strong mucosal CTL responses specific for simian immunodeficiency virus (SIV) envelope (env) were protected from later colonic challenge with a heterologous pathogenic virus dose. Here, env-specific CTL were similarly induced in jejunal LP in five of eight non-progesterone treated macaques that were vaginally exposed to SIV, but not infected. Subsequent vaginal challenge following progesterone treatment produced systemic infection. The only two monkeys that had jejunal env-specific CTL detectable post-challenge developed significantly lower plasma virus loads, and had delayed disease progression. Either vaginal or colonic exposure to subinfectious SIV doses can induce CTL detectable in jejunal LP. The association of such CTL with protection or delayed disease upon challenge suggests that successful vaccine protection against SIV/HIV may require CTL responses in the mucosa.     

No evidence for consistent virus-specific immunity in simian immunodeficiency virus-exposed, uninfected rhesus monkeys

Defining the immune correlates of the protection against human immunodeficiency virus type 1 (HIV-1) acquisition in individuals who are exposed to HIV-1 but do not become infected may provide important direction for the creation of an HIV-1 vaccine. We have employed the simian immunodeficiency virus (SIV)/rhesus monkey model to determine whether monkeys can be repeatedly exposed to a primate lentivirus by a mucosal route and escape infection and whether virus-specific immune correlates of protection from infection can be identified in uninfected monkeys. Five of 18 rhesus monkeys exposed 18 times by intrarectal inoculation to SIVmac251 or SIVsmE660 were resistant to infection, indicating that the exposed/uninfected phenotype can be reproduced in a nonhuman primate AIDS model. However, routine peripheral blood lymphocyte gamma interferon enzyme-linked immunospot (ELISPOT), tetramer, and intracellular cytokine staining assays, as well as cytokine-augmented ELISPOT and peptide-stimulated tetramer assays, failed to define a systemic antigen-specific cellular immune correlate to this protection. Further, local cell-mediated immunity could not be demonstrated by tetramer assays of these protected monkeys, and local humoral immunity was not associated with protection against acquisition of virus in another cohort of mucosally exposed monkeys. Therefore, resistance to mucosal infection in these monkeys may not be mediated by adaptive virus-specific immune mechanisms. Rather, innate immune mechanisms or an intact epithelial barrier may be responsible for protection against mucosal infection in this population of monkeys.     

Identification of SIV env-specific CTL in the jejunal mucosa in vaginally exposed, seronegative rhesus macaques (Macaca mulatta)

We previously reported major histocompatibility complex Class I-restricted cytotoxic T lymphocytes (CTL) in jejunal lamina propria (LP) of monkeys following colonic exposure to subinfectious SIV doses. Those monkeys with strong mucosal CTL responses specific for simian immunodeficiency virus (SIV) envelope (env) were protected from later colonic challenge with a heterologous pathogenic virus dose. Here, env-specific CTL were similarly induced in jejunal LP in five of eight non-progesterone treated macaques that were vaginally exposed to SIV, but not infected. Subsequent vaginal challenge following progesterone treatment produced systemic infection. The only two monkeys that had jejunal env-specific CTL detectable post-challenge developed significantly lower plasma virus loads, and had delayed disease progression. Either vaginal or colonic exposure to subinfectious SIV doses can induce CTL detectable in jejunal LP. The association of such CTL with protection or delayed disease upon challenge suggests that successful vaccine protection against SIV/HIV may require CTL responses in the mucosa.