Temporal Analyses of Virus Replication, Immune Responses, and Efficacy in Rhesus Macaques Immunized with a Live, Attenuated Simian Immunodeficiency Virus Vaccine

Despite evidence that live, attenuated simian immunodeficiency virus (SIV) vaccines can elicit potent protection against pathogenic SIV infection, detailed information on the replication kinetics of attenuated SIV in vivo is lacking. In this study, we measured SIV RNA in the plasma of 16 adult rhesus macaques immunized with a live, attenuated strain of SIV (SIVmac239Δnef). To evaluate the relationship between replication of the vaccine virus and the onset of protection, four animals per group were challenged with pathogenic SIVmac251 at either 5, 10, 15, or 25 weeks after immunization. SIVmac239Δnef replicated efficiently in the immunized macaques in the first few weeks after inoculation. SIV RNA was detected in the plasma of all animals by day 7 after inoculation, and peak levels of viremia (10⁵ to 10⁷ RNA copies/ml) occurred by 7 to 12 days. Following challenge, SIVmac251 was detected in all of the four animals challenged at 5 weeks, in two of four challenged at 10 weeks, in none of four challenged at 15 weeks, and one of four challenged at 25 weeks. One animal immunized with SIVmac239Δnef and challenged at 10 weeks had evidence of disease progression in the absence of detectable SIVmac251. Although complete protection was not achieved at 5 weeks, a transient reduction in viremia (approximately 100-fold) occurred in the immunized macaques early after challenge compared to the nonimmunized controls. Two weeks after challenge, SIV RNA was also reduced in the lymph nodes of all immunized macaques compared with control animals. Taken together, these results indicate that host responses capable of reducing the viral load in plasma and lymph nodes were induced as early as 5 weeks after immunization with SIVmac239Δnef, while more potent protection developed between 10 and 15 weeks. In further experiments, we found that resistance to SIVmac251 infection did not correlate with the presence of antibodies to SIV gp130 and p27 antigens and was achieved in the absence of significant neutralizing activity against the primary SIVmac251 challenge stock.     

Highly attenuated vaccine strains of simian immunodeficiency virus protect against vaginal challenge: inverse relationship of degree of protection with level of attenuation

Three different deletion mutants of simian immunodeficiency virus (SIV) that vary in their levels of attenuation were tested for the ability to protect against mucosal challenge with pathogenic SIV. Four female rhesus monkeys were vaccinated by intravenous inoculation with SIVmac239Delta3, four with SIVmac239Delta3X, and four with SIVmac239Delta4. These three vaccine strains exhibit increasing levels of attenuation: Delta3 < Delta3X 相似文献   

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.     

vpr deletion mutant of simian immunodeficiency virus induces AIDS in rhesus monkeys.

In previous experiments, animals infected with SIVmac239 containing a point mutation in the vpr and nef genes developed AIDS-like symptoms after early reversion of the vpr and nef genes. Here we show that two animals in which the nef gene but not the vpr gene had reverted in the first few months did not develop disease during a 3-year observation period even after reversion to a functional vpr gene 70 weeks postinfection. To study the influence of a stable vpr mutation on virus load and pathogenesis, a 43-bp deletion was introduced into the vpr gene of SIVmac239on, a nef-open mutant of SIVmac239. Four rhesus monkeys were inoculated with the vpr deletion mutant (SIV delta vpr), and two control animals were infected with SIVmac239on. Both control animals had persistent antigenemia, high cell-associated virus loads, and elevated neopterin levels. They had to be euthanized 20 and 30 weeks postinfection because of AIDS-related symptoms. However, all four rhesus monkeys inoculated with SIV delta vpr showed only transiently detectable antigenemia. The cell-associated virus loads were high in three of the four animals. Two animals with AIDS-like symptoms had to be euthanized 71 and 73 weeks postinfection. The two remaining monkeys infected with SIV delta vpr were still alive 105 weeks postinfection. In contrast to the SIVmac239on-infected animals, SIV delta vpr-infected animals had strong humoral immune responses and intermittent cellular immune responses to SIV antigens. Our data show that a functional vpr gene is not necessary for pathogenesis. However, vpr-deficient SIVmac239 variants might be slightly attenuated, allowing some animals to resist progression to disease for an extended period of time.     

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.     

Persistence of pathogenic challenge virus in macaques protected by simian immunodeficiency virus SIVmacDeltanef

Live attenuated simian immunodeficiency virus (SIV) is the most efficient vaccine yet developed in monkey models of human immunodeficiency virus infection. In all successful vaccine trials, attenuation was achieved by inactivating at least the nef gene. We investigated some virological and immunological characteristics of five rhesus macaques immunized with a nef-inactivated SIVmac251 molecular clone (SIVmac251Deltanef) and challenged 15 months later with the pathogenic SIVmac251 isolate. Three animals were killed 2 weeks postchallenge (p.c.) to search for the challenge virus and to assess immunological changes in various organs. The other two animals have been monitored up for 7 years p.c., with clinical and nef gene changes being noted. The animals killed showed no increase in viral load and no sign of a secondary immune response, although the challenged virus was occasionally detected by PCR. In one of the monkeys being monitored, the vaccine virus persisted and an additional deletion occurred in nef. In the other monkey that was monitored, the challenge and the vaccine (Deltanef) viruses were both detected by PCR until a virus with a hybrid nef allele was isolated 48 months p.c. This nef hybrid encodes a 245-amino-acid protein. Thus, our results show (i) that monkeys were not totally protected against homologous virus challenge but controlled the challenge very efficiently in the absence of a secondary immune response, and (ii) that the challenge and vaccine viruses may persist in a replication-competent form for long periods after the challenge, possibly resulting in recombination between the two viruses.     

Neutralizing Antibodies in Sera from Macaques Immunized with Attenuated Simian Immunodeficiency Virus

Infection with attenuated simian immunodeficiency virus (SIV) in rhesus macaques has been shown to raise antibodies capable of neutralizing an animal challenge stock of primary SIVmac251 in CEMx174 cells that correlate with resistance to infection after experimental challenge with this virulent virus (M. S. Wyand, K. H. Manson, M. Garcia-Moll, D. C. Montefiori, and R. C. Desrosiers, J. Virol. 70:3724–3733, 1996). Here we show that these neutralizing antibodies are not detected in human and rhesus peripheral blood mononuclear cells (PBMC). In addition, neutralization of primary SIVmac251 in human and rhesus PBMC was rarely detected with plasma samples from a similar group of animals that had been infected either with SIVmac239Δnef for 1.5 years or with SIVmac239Δ3 for 3.2 years, although low-level neutralization was detected in CEMx174 cells. Potent neutralization was detected in CEMx174 cells when the latter plasma samples were assessed with laboratory-adapted SIVmac251. In contrast to primary SIVmac251, laboratory-adapted SIVmac251 did not replicate in human and rhesus PBMC despite its ability to utilize CCR5, Bonzo/STRL33, and BOB/gpr15 as coreceptors for virus entry. These results illustrate the importance of virus passage history and the choice of indicator cells for making assessments of neutralizing antibodies to lentiviruses such as SIV. They also demonstrate that primary SIVmac251 is less sensitive to neutralization in human and rhesus PBMC than it is in established cell lines. Results obtained in PBMC did not support a role for neutralizing antibodies as a mechanism of protection in animals immunized with attenuated SIV and challenged with primary SIVmac251.     

Depo-Provera abrogates attenuated lentivirus-induced protection in male rhesus macaques challenged intravenously with pathogenic SIVmac239

BACKGROUND: Progesterone administration prior to intravaginal challenge with pathogenic SIVmac239 decreases the protective efficacy of live attenuated vaccines in rhesus macaques. METHODS: To determine if progesterone alters the efficacy of live attenuated vaccines through local or systemic effects, seven male rhesus macaques were immunized with SHIV89.6 and then challenged intravenously with SIVmac239. Three of these animals were treated with Depo-Provera 30 days prior to the SIV challenge. RESULTS: The SHIV animals had significantly lower plasma viral RNA levels than the unimmunized control monkeys, but the Depo-Provera treated, SHIV-immunized animals did not. Despite the lack of protection, the Depo-Provera SHIV animals had strong SIV specific T-cell responses. However, altered patterns of NK frequency and CD38 T-cell expression prior to SIV challenge were observed in Depo-Provera SHIV animals. CONCLUSIONS: Depo-Provera eliminates live-attenuated lentivirus vaccine efficacy in male rhesus monkeys through systemic effects on antiviral immunity and/or viral replication.     

Adoptive Transfer of Lymphocytes Isolated from Simian Immunodeficiency Virus SIVmac239Δnef-Vaccinated Macaques Does Not Affect Acute-Phase Viral Loads but May Reduce Chronic-Phase Viral Loads in Major Histocompatibility Complex-Matched Recipients

The live attenuated simian immunodeficiency virus (SIV) SIVmac239Δnef is the most effective SIV/human immunodeficiency virus (HIV) vaccine in preclinical testing. An understanding of the mechanisms responsible for protection may provide important insights for the development of HIV vaccines. Leveraging the uniquely restricted genetic diversity of Mauritian cynomolgus macaques, we performed adoptive transfers between major histocompatibility complex (MHC)-matched animals to assess the role of cellular immunity in SIVmac239Δnef protection. We vaccinated and mock vaccinated donor macaques and then harvested between 1.25 × 10⁹ and 3.0 × 10⁹ mononuclear cells from multiple tissues for transfer into 12 naive recipients, followed by challenge with pathogenic SIVmac239. Fluorescently labeled donor cells were detectable for at least 7 days posttransfer and trafficked to multiple tissues, including lung, lymph nodes, and other mucosal tissues. There was no difference between recipient macaques'' peak or postpeak plasma viral loads. A very modest difference in viral loads during the chronic phase between vaccinated animal cell recipients and mock-vaccinated animal cell recipients did not reach significance (P = 0.12). Interestingly, the SIVmac239 challenge virus accumulated escape mutations more rapidly in animals that received cells from vaccinated donors. These results may suggest that adoptive transfers influenced the course of infection despite the lack of significant differences in the viral loads among animals that received cells from vaccinated and mock-vaccinated donor animals.     

Induction of AIDS in Rhesus Monkeys by a Recombinant Simian Immunodeficiency Virus Expressing nef of Human Immunodeficiency Virus Type 1

A nef gene is present in all primate lentiviruses, including human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus of macaque monkeys (SIVmac). However, the nef genes of HIV-1 and SIVmac exhibit minimal sequence identity, and not all properties are shared by the two. Nef sequences of SIVmac239 were replaced by four independent nef alleles of HIV-1 in a context that was optimal for expression. The sources of the HIV-1 nef sequences included NL 4-3, a variant NL 4-3 gene derived from a recombinant-infected rhesus monkey, a patient nef allele, and a nef consensus sequence. Of 16 rhesus monkeys infected with these SHIVnef chimeras, 9 maintained high viral loads for prolonged periods, as observed with the parental SIVmac239, and 6 have died with AIDS 52 to 110 weeks postinfection. Persistent high loads were observed at similar frequencies with the four different SIV recombinants that expressed these independent HIV-1 nef alleles. Infection with other recombinant SHIVnef constructions resulted in sequence changes in infected monkeys that either created an open nef reading frame or optimized the HIV-1 nef translational context. The HIV-1 nef gene was uniformly retained in all SHIVnef-infected monkeys. These results demonstrate that HIV-1 nef can substitute for SIVmac nef in vivo to produce a pathogenic infection. However, the model suffers from an inability to consistently obtain persisting high viral loads in 100% of the infected animals, as is observed with the parental SIVmac239.     

The nef gene products of both simian and human immunodeficiency viruses enhance virus infectivity and are functionally interchangeable.

Adult rhesus macaques infected with nef-defective simian immunodeficiency virus (SIV) exhibit extremely low levels of steady-state virus replication, do not succumb to immunodeficiency disease, and are protected from experimental challenge with pathogenic isolates of SIV. Similarly, rare humans found to be infected with nef-defective human immunodeficiency virus type 1 (HIV-1) variants display exceptionally low viral burdens and do not show evidence of disease progression after many years of infection. HIV-1 Nef induces the rapid endocytosis and lysosomal degradation of cell surface CD4 and enhances virus infectivity in primary human T cells and macrophages. Although expression of SIV Nef also leads to down-modulation of cell surface CD4 levels, no evidence for SIV Nef-induced enhancement of virus infectivity was observed in earlier studies. Thus, it remains unclear whether fundamental differences exist between the activities of HIV-1 and SIV Nef. To establish more clearly whether the SIV and HIV-1 nef gene products are functionally analogous, we compared the replication kinetics and infectivity of variants of SIVmac239 that either do (SIVnef+) or do not (SIV delta nef) encode intact nef gene products. SIVnef+ replicates more rapidly than nef-defective viruses in both human and rhesus peripheral blood mononuclear cells (PBMCs). As previously described for HIV-1 Nef, SIV Nef also enhances virus infectivity within each cycle of virus replication. As a strategy for evaluating the in vivo contribution of HIV-1 nef alleles and long terminal repeat regulatory sequences to the pathogenesis of immunodeficiency disease, we constructed SIV-HIV chimeras in which the nef coding and U3 regulatory regions of SIVmac239 were replaced by the corresponding regions from HIV-1/R73 (SIVR7nef+). SIVR7nef+ displays enhanced infectivity and accelerated replication kinetics in primary human and rhesus PBMC infections compared to its nef-defective counterpart. Converse chimeras, containing SIV Nef in an HIV-1 background (R7SIVnef+) also exhibit greater infectivity than matched nef-defective viruses (R7SIV delta nef). These data indicate that SIV Nef, like that of HIV-1, does enhance virus replication in primary cells in tissue culture and that HIV-1 and SIV Nef are functionally interchangeable in the context of both HIV-1 and SIV.     

Influence of Mismatch of Env Sequences on Vaccine Protection by Live Attenuated Simian Immunodeficiency Virus

Vaccine/challenge experiments that utilize live attenuated strains of simian immunodeficiency virus (SIV) in monkeys may be useful for elucidating what is needed from a vaccine in order to achieve protective immunity. Derivatives of SIVmac239 and SIVmac239Δnef were constructed in which env sequences were replaced with those of the heterologous strain E543; these were then used in vaccine/challenge experiments. When challenge occurred at 22 weeks, 10 of 12 monkeys exhibited apparent sterilizing immunity despite a mismatch of Env sequences, compared to 12 of 12 monkeys with apparent sterilizing immunity when challenge virus was matched in its Env sequence. However, when challenge occurred at 6 weeks, 6 of 6 SIV239Δnef-immunized monkeys became superinfected by challenge virus mismatched in its Env sequence (SIV239/EnvE543). These results contrast markedly not only with the results of the week 22 challenge but also with the sterilizing immunity observed in 5 of 5 SIV239Δnef-immunized rhesus monkeys challenged at 5 weeks with SIV239, i.e., with no mismatch of Env sequences. We conclude from these studies that a mismatch of Env sequences in the challenge virus can have a dramatic effect on the extent of apparent sterilizing immunity when challenge occurs relatively early, 5 to 6 weeks after the nef-deleted SIV administration. However, by 22 weeks, mismatch of Env sequences has little or no influence on the degree of protection against challenge virus. Our findings suggest that anti-Env immune responses are a key component of the protective immunity elicited by live attenuated, nef-deleted SIV.     

Retroviral recombination in vivo: viral replication patterns and genetic structure of simian immunodeficiency virus (SIV) populations in rhesus macaques after simultaneous or sequential intravaginal inoculation with SIVmac239Deltavpx/Deltavpr and SIVmac239Deltanef

To characterize the occurrence, frequency, and kinetics of retroviral recombination in vivo, we intravaginally inoculated rhesus macaques, either simultaneously or sequentially, with attenuated simian immunodeficiency virus (SIV) strains having complementary deletions in their accessory genes and various degrees of replication impairment. In monkeys inoculated simultaneously with SIVmac239Deltavpx/Deltavpr and SIVmac239Deltanef, recombinant wild-type (wt) virus and wild-type levels of plasma viral RNA (vRNA) were detected in blood by 2 weeks postinoculation. In monkeys inoculated first with SIVmac239Deltavpx/Deltavpr and then with SIVmac239Deltanef, recombination occurred but was associated with lower plasma vRNA levels than plasma vRNA levels seen for monkeys inoculated intravaginally with wt SIVmac239. In one monkey, recombination occurred 6 weeks after the challenge with SIVmac239Deltanef when plasma SIVmac239Deltavpx/Deltavpr RNA levels were undetectable. In monkeys inoculated first with the more highly replicating strain, SIVmac239Deltanef, and then with SIVmac239Deltavpx/Deltavpr, wild-type recombinant virus was not detected in blood or tissues. Instead, a virus that had repaired the deletion in the nef gene by a compensatory mutation was found in one animal. Overall, recombinant SIV was eventually found in four of six animals intravaginally inoculated with the two SIVmac239 deletion mutants. These findings show that recombination can occur readily in vivo after mucosal SIV exposure and thus contributes to the generation of viral genetic diversity and enhancement of viral fitness.     

Importance of the nef gene for maintenance of high virus loads and for development of AIDS

When rhesus monkeys were infected with a form of cloned SIVmac239 having a premature stop signal at the 93rd codon of nef, revertants with a coding codon at this position quickly and universally came to predominate in the infected animals. This suggests that there are strong selective forces for open functional forms of nef in vivo. Although deletion of nef sequences had no detectable effect on virus replication in cultured cells, deletion of nef sequences dramatically altered the properties of virus in infected rhesus monkeys. Our results indicate that nef is required for maintaining high virus loads during the course of persistent infection in vivo and for full pathologic potential. Thus, nef should become a target for antiviral drug development. Furthermore, the properties of virus with a deletion in nef suggest a means for making live-attenuated strains of virus for experimental vaccine testing.     

Determinants of increased replicative capacity of serially passaged simian immunodeficiency virus with nef deleted in rhesus monkeys

Most rhesus macaques infected with simian immunodeficiency virus SIVmac239 with nef deleted (either Delta nef or Delta nef Delta vpr Delta US [Delta 3]) control viral replication and do not progress to AIDS. Some monkeys, however, develop moderate viral load set points and progress to AIDS. When simian immunodeficiency viruses (SIVs) recovered from two such animals (one Delta nef and the other Delta 3) were serially passaged in rhesus monkeys, the SIVs derived from both lineages were found to consistently induce moderate viral loads and disease progression. Analysis of viral sequences in the serially passaged derivatives revealed interesting changes in three regions: (i) an unusually high number of predicted amino acid changes (12 to 14) in the cytoplasmic domain of gp41, most of which were in regions that are usually conserved; these changes were observed in both lineages; (ii) an extreme shortening of nef sequences in the region of overlap with U3; these changes were observed in both lineages; and (iii) duplication of the NF-kappa B binding site in one lineage only. Neither the polymorphic gp41 changes alone nor the U3 deletion alone appeared to be responsible for increased replicative capacity because recombinant SIVmac239 Delta nef, engineered to contain either of these changes, induced moderate viral loads in only one of six monkeys. However, five of six monkeys infected with recombinant SIVmac239 Delta nef containing both TM and U3 changes did develop persisting moderate viral loads. These genetic changes did not increase lymphoid cell-activating properties in the monkey interleukin-2-dependent T-cell line 221, but the gp41 changes did increase the fusogenic activity of the SIV envelope two- to threefold. These results delineate sequence changes in SIV that can compensate for the loss of the nef gene to partially restore replicative and pathogenic potential in rhesus monkeys.     

Low-dose penile SIVmac251 exposure of rhesus macaques infected with adenovirus type 5 (Ad5) and then immunized with a replication-defective Ad5-based SIV gag/pol/nef vaccine recapitulates the results of the phase IIb step trial of a similar HIV-1 vaccine

The Step Trial showed that the MRKAd5 HIV-1 subtype B Gag/Pol/Nef vaccine did not protect men from HIV infection or reduce setpoint plasma viral RNA (vRNA) levels but, unexpectedly, it did modestly enhance susceptibility to HIV infection in adenovirus type 5 (Ad5)-seropositive, uncircumcised men. As part of the process to understand the results of the Step Trial, we designed a study to determine whether rhesus macaques chronically infected with a host-range mutant Ad5 (Ad5hr) and then immunized with a replication defective Ad5 SIVmac239 Gag/Pol/Nef vaccine were more resistant or susceptible to SIV infection than unimmunized rhesus macaques challenged with a series of escalating dose penile exposures to SIVmac 251. The Ad5 SIV vaccine induced CD8(+) T cell responses in 70% of the monkeys, which is similar to the proportion of humans that responded to the vaccine in the Step Trial. However, the vaccine did not protect vaccinated animals from penile SIV challenge. At the lowest SIV exposure dose (10(3) 50% tissue culture infective doses), 2 of 9 Ad5-seropositive animals immunized with the Ad5 SIV vaccine became infected compared to 0 of 34 animals infected in the other animal groups (naive animals, Ad5-seropositive animals immunized with the empty Ad5 vector, Ad5-seronegative animals immunized with the Ad5 SIV vaccine, and Ad5-seronegative animals immunized with the empty Ad5 vector). Penile exposure to more concentrated virus inocula produced similar rates of infection in all animal groups. Although setpoint viral loads were unaffected in Step vaccinees, the Ad5 SIV-immunized animals had significantly lower acute-phase plasma vRNA levels compared to unimmunized animals. Thus, the results of the nonhuman primate (NHP) study described here recapitulate the lack of protection against HIV acquisition seen in the Step Trial and suggest a greater risk of infection in the Ad5-seropositive animals immunized with the Ad5 SIV vaccine. Further studies are necessary to confirm the enhancement of virus acquisition and to discern associated mechanisms.     

Importance of vpr for infection of rhesus monkeys with simian immunodeficiency virus.

The importance of the vpr gene for simian immunodeficiency virus (SIV) replication, persistence, and disease progression was examined by using the infectious pathogenic molecular clone called SIVmac239. The ATG start codon of the vpr gene was converted to TTG by site-specific mutagenesis. The constructed Vpr- mutant virus is identical with the parental SIVmac239/nef-stop virus with the exception of this one nucleotide. These viruses replicated with similar kinetics and to similar extents in rhesus monkey lymphocyte cultures and in the human CEMX174 cell line. Five rhesus monkeys were inoculated with the Vpr- variant of SIVmac239/nef-stop, and two monkeys received SIVmac239/nef-stop as controls. Both controls showed reversion of the TAA stop signal in nef by 2 weeks postinfection, as has been observed previously. Reversion of the TAA stop codon in nef also occurred in the five monkeys that received the Vpr- variant, but reversion was delayed on average to about 4 weeks. Thus, the mutation in vpr appeared to delay the rapidity with which reversion occurred in the nef gene. Reversion of the TTG sequence in vpr to ATG was observed in three of the five test animals. Reversion in vpr was first observed in these three animals 4 to 8 weeks postinfection. No vpr revertants were found over the entire 66 weeks of observation in the other two test animals that received the vpr mutant. Antibodies to vpr developed in those three animals in which reversion of vpr was documented, but antibodies to vpr were not observed in the two animals in which reversion of vpr was not detected. Antibody responses to gag and to whole virus antigens were of similar strength in all seven animals. Both control animals and two of the test animals in which vpr reverted maintained high virus loads and developed progressive disease. Low virus burden and no disease have been observed in the two animals in which vpr did not revert and in the one animal in which vpr reversion was first detected only at 8 weeks. The reversion of vpr in three of the five test animals indicates that there is significant selective pressure for functional forms of vpr in vivo. Furthermore, the results suggest that both vpr and nef are important for maximal SIV replication and persistence in vivo and for disease progression.     

Simian immunodeficiency virus DNA vaccine trial in macaques.

An experimental vaccine consisting of five DNA plasmids expressing different combinations and forms of simian immunodeficiency virus-macaque (SIVmac) proteins has been evaluated for the ability to protect against a highly pathogenic uncloned SIVmac251 challenge. One vaccine plasmid encoded nonreplicating SIVmac239 virus particles. The other four plasmids encoded secreted forms of the envelope glycoproteins of two T-cell-tropic relatives (SIVmac239 and SIVmac251) and one monocyte/macrophage-tropic relative (SIVmac316) of the uncloned challenge virus. Rhesus macaques were inoculated with DNA at 1 and 3, 11 and 13, and 21 and 23 weeks. Four macaques were inoculated intravenously, intramuscularly, and by gene gun inoculations. Three received only gene gun inoculations. Two control monkeys were inoculated with control plasmids by all three routes of inoculation. Neutralizing antibody titers of 1:216 to 1:768 were present in all of the vaccinated monkeys after the second cluster of inoculations. These titers were transient, were not boosted by the third cluster of inoculations, and had fallen to 1:24 to 1:72 by the time of challenge. Cytotoxic T-cell activity for Env was also raised in all of the vaccinated animals. The temporal appearance of cytotoxic T cells was similar to that of antibody. However, while antibody responses fell with time, cytotoxic T-cell responses persisted. The SIVmac251 challenge was administered intravenously at 2 weeks following the last immunization. The DNA immunizations did not prevent infection or protect against CD4+ cell loss. Long-term chronic levels of infection were similar in the vaccinated and control animals, with 1 in 10,000 to 1 in 100,000 peripheral blood cells carrying infectious virus. However, viral loads were reduced to the chronic level over a shorter period of time in the vaccinated groups (6 weeks) than in the control group (12 weeks). Thus, the DNA vaccine raised both neutralizing antibody and cytotoxic T-lymphocyte responses and provided some attenuation of the acute phase of infection, but it did not prevent the loss of CD4+ cells.     

Effect of CD8+ lymphocyte depletion on virus containment after simian immunodeficiency virus SIVmac251 challenge of live attenuated SIVmac239delta3-vaccinated rhesus macaques

Although live attenuated vaccines can provide potent protection against simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus challenges, the specific immune responses that confer this protection have not been determined. To test whether cellular immune responses mediated by CD8+ lymphocytes contribute to this vaccine-induced protection, we depleted rhesus macaques vaccinated with the live attenuated virus SIVmac239Delta3 of CD8+ lymphocytes and then challenged them with SIVmac251 by the intravenous route. While vaccination did not prevent infection with the pathogenic challenge virus, the postchallenge levels of virus in the plasmas of vaccinated control animals were significantly lower than those for unvaccinated animals. The depletion of CD8+ lymphocytes at the time of challenge resulted in virus levels in the plasma that were intermediate between those of the vaccinated and unvaccinated controls, suggesting that CD8+ cell-mediated immune responses contributed to protection. Interestingly, at the time of challenge, animals expressing the Mamu-A*01 major histocompatibility complex class I allele showed significantly higher frequencies of SIV-specific CD8+ T-cell responses and lower neutralizing antibody titers than those in Mamu-A*01- animals. Consistent with these findings, the depletion of CD8+ lymphocytes abrogated vaccine-induced protection, as judged by the peak postchallenge viremia, to a greater extent in Mamu-A*01+ than in Mamu-A*01- animals. The partial control of postchallenge viremia after CD8+ lymphocyte depletion suggests that both humoral and cellular immune responses induced by live attenuated SIV vaccines can contribute to protection against a pathogenic challenge and that the relative contribution of each of these responses to protection may be genetically determined.     

Immune mechanisms associated with protection from vaginal SIV challenge in rhesus monkeys infected with virulence-attenuated SHIV 89.6

Although live-attenuated human immunodeficiency virus-1 (HIV) vaccines may never be used clinically, these vaccines have provided the most durable protection from intravenous (IV) challenge in the simian immunodeficiency virus (SIV)/rhesus macaque model. Systemic infection with virulence attenuated-simian-human immunodeficiency virus (SHIV) 89.6 provides protection against vaginal SIV challenge. This paper reviews the findings related to the innate and adaptive immune responses and the role of inflammation associated with protection in the SHIV 89.6/SIVmac239 model. By an as yet undefined mechanism, most monkeys vaccinated with live-attenuated SHIV 89.6 mounted effective anti-viral CD8+ T cell responses while avoiding the self-destructive inflammatory cycle found in the lymphoid tissues of unprotected and unvaccinated monkeys.