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ABSTRACTThe thymus is responsible for de novo production of CD4+and CD8+T cells and therefore is essential for T-cell renewal. The goal of this study was to assess the impact of simian immunodeficiency virus (SIV) infection on the production of T cells by the thymus. Levels of recent thymic emigrants within the peripheral blood were assessed through quantification of macaque T-cell receptor excision circles (TREC). Comparison of SIV-infected macaques (n= 15) to uninfected macaques (n= 23) revealed stable or increased TREC levels at 20 to 34 weeks postinfection. Further assessment of SIV-infected macaques (n= 4) determined that TREC levels decreased between 24 and 48 weeks postinfection. Through the assessment of longitudinal time points in three additional SIVmac239-infected macaques, the SIV infection was divided into two distinct phases. During phase 1 (16 to 30 weeks), TREC levels remained stable or increased within both the CD4 and CD8 T-cell populations. During phase 2 (after 16 to 30 weeks), TREC levels declined in both T-cell populations. As has been described for human immunodeficiency virus (HIV)-infected patients, this decline in TREC levels did at times correlate with an increased level of T-cell proliferation (Ki67+cells). However, not all TREC decreases could be attributed to increased T-cell proliferation. Further evidence for thymic dysfunction was observed directly in a SIVmac239-infected macaque that succumbed to simian AIDS at 65 weeks postinfection. The thymus of this macaque contained an increased number of memory/effector CD8+T cells and an increased level of apoptotic cells. In summary, reduced levels of TREC can be observed beginning at 16 to 30 weeks post-SIV infection and correlate with changes indicative of dysfunction within the thymic tissue. SIV infection of macaques will be a useful model system to elucidate the mechanisms responsible for the thymic dysfunction observed in HIV-infected patients.

ABSTRACTThe frequency of cytomegalovirus (CMV)-specific CD4+T lymphocytes was determined in CMV-seropositive rhesus macaques with or without simian immunodeficiency virus (SIV) infection by using the sensitive assays of intracellular cytokine staining and gamma interferon ELISPOT. Both techniques yielded 3- to 1,000-fold-higher frequencies of CMV-specific CD4+T lymphocytes than traditional proliferative limiting dilution assays. The median frequency of CMV-specific CD4+T lymphocytes in 23 CMV-seropositive SIV-negative macaques was 0.63% (range, 0.16 to 5.8%). The majority of CMV-specific CD4+T lymphocytes were CD95posand CD27lobut expressed variable levels of CD45RA. A significant reduction (P< 0.05) in the frequency of CMV-specific CD4+T lymphocytes was observed in pathogenic SIV-infected macaques but not in macaques infected with live attenuated strains of SIV. CMV-specific CD4+T lymphocytes were not detected in six of nine pathogenic SIV-infected rhesus macaques. CMV DNA was detected in the plasma of four of six of these macaques but in no animal with detectable CMV-specific CD4+T lymphocytes. In pathogenic SIV-infected macaques, loss of CMV-specific CD4+T lymphocytes was not predicted by the severity of CD4+T lymphocytopenia. Neither was it predicted by the pre-SIV infection frequencies of CD45RAnegor CCR5posCMV-specific CD4+T lymphocytes. However, the magnitude of activation, as evidenced by the intensity of CD40L expression on CMV-specific CD4+T lymphocytes pre-SIV infection, was three- to sevenfold greater in the two macaques that subsequently lost these cells after SIV infection than in the two macaques that retained CMV-specific CD4+T lymphocytes post-SIV infection. Future longitudinal studies with these techniques will facilitate the study of CMV pathogenesis in AIDS.

ABSTRACTIn this report, three Mamu-A*01+rhesus macaques were examined to compare the emergence of simian immunodeficiency virus (SIV)-specific CD8+T cells in the intestines and blood in early SIV infection using a major histocompatibility complex class I tetramer complexed with the Gag181–189peptide. Fourteen days after intravenous inoculation with SIVmac251, large numbers of SIV Gag181–189-specific CD8+T cells were detected in the intestinal mucosa (3.1 to 11.5% of CD3+CD8+lymphocytes) as well as in the blood (3.1 to 13.4%) of all three macaques. By 21 days postinoculation, levels of tetramer-binding cells had dropped in both the intestines and blood. At day 63, however, levels of SIV Gag181–189-specific CD8+T cells in the intestines had rebounded in all three macaques to levels that were higher (8.6 to 18.7%) than those at day 21. In contrast, percentages of tetramer-binding cells in the peripheral blood remained comparatively stable (2.5 to 4.5%) at this time point. In summary, SIV Gag181–189-specific CD8+T cells appeared in both the intestinal mucosa and peripheral blood at a comparable rate and magnitude in primary SIV infection. Given that the intestine is a major site of early viral replication as well as the site where most of the total body lymphocyte pool resides, these data indicate that it is also an early and important site of development of antiviral immune responses.

ABSTRACTWe report a pilot evaluation of a DNA vaccine producing genetically inactivated simian immunodeficiency virus (SIV) particles in primates, with a focus on eliciting mucosal immunity. Our results demonstrate that DNA vaccines can be used to stimulate strong virus-specific mucosal immune responses in primates. The levels of immunoglobulin A (IgA) detected in rectal secretions of macaques that received the DNA vaccine intradermally and at the rectal mucosa were the most striking of all measured immune responses and were higher than usually achieved through natural infection. However, cytotoxic T lymphocyte responses were generally low and sporadically present in different animals. Upon rectal challenge with cloned SIVmac239, resistance to infection was observed, but some animals with high SIV-specific IgA levels in rectal secretions became infected. Our results suggest that high levels of IgA alone are not sufficient to prevent the establishment of chronic infection, although mucosal IgA responses may have a role in reducing the infectivity of the initial viral inoculum.

ABSTRACTInduction of virus-specific T-cell responses in mucosal as well as systemic compartments of the immune system is likely to be a critical feature of an effective AIDS vaccine. We investigated whether virus-specific CD8+lymphocytes induced in rhesus macaques by immunization with attenuated simian immunodeficiency virus (SIV), an approach that is highly effective in eliciting protection against mucosal challenge, express the mucosa-homing receptor a4ß7 and traffic to the intestinal mucosa. SIV-specific CD8+T cells expressing a4ß7 were detected in peripheral blood and intestine of macaques infected with attenuated SIV. In contrast, virus-specific T cells in blood of animals immunized cutaneously by a combined DNA-modified vaccinia virus Ankara regimen did not express a4ß7. These results demonstrate the selective induction of SIV-specific CD8+T lymphocytes expressing a4ß7 by a vaccine approach that replicates in mucosal tissue and suggest that induction of virus-specific lymphocytes that are able to home to mucosal sites may be an important characteristic of a successful AIDS vaccine.

ABSTRACTAlthough lymphocyte turnover in chronic human immunodeficiency virus and simian immunodeficiency virus (SIV) infection has been extensively studied, there is little information on turnover in acute infection. We carried out a prospective kinetic analysis of lymphocyte proliferation in 13 rhesus macaques inoculated with pathogenic SIV. A short-lived dramatic increase in circulating Ki-67+lymphocytes observed at 1 to 4 weeks was temporally related to the onset of SIV replication. A 5- to 10-fold increase in Ki-67+CD8+T lymphocytes and a 2- to 3-fold increase in Ki-67+CD3-CD8+natural killer cells accounted for >85% of proliferating lymphocytes at peak proliferation. In contrast, there was little change in the percentage of Ki-67+CD4+T lymphocytes during acute infection, although transient increases in Ki-67-and Ki-67+CD4+T lymphocytes expressing CD69, Fas, and HLA-DR were observed. A two- to fourfold decline in CD4+T lymphocytes expressing CD25 and CD69 was seen later in SIV infection. The majority of Ki-67+CD8+T lymphocytes were phenotypically CD45RA-CD49dhiFashiCD25-CD69-CD28-HLA-DR-and persisted at levels twofold above baseline 6 months after SIV infection. Increased CD8+T-lymphocyte proliferation was associated with cell expansion, paralleled the onset of SIV-specific cytotoxic T-lymphocyte activity, and had an oligoclonal component. Thus, divergent patterns of proliferation and activation are exhibited by CD4+and CD8+T lymphocytes in early SIV infection and may determine how these cells are differentially affected in AIDS.

ABSTRACTAn effective vaccine for AIDS may require development of novel vectors capable of eliciting long-lasting immune responses. Here we report the development and use of replication-competent and replication-defective strains of recombinant herpes simplex virus (HSV) that express envelope and Nef antigens of simian immunodeficiency virus (SIV). The HSV recombinants induced antienvelope antibody responses that persisted at relatively stable levels for months after the last administration. Two of seven rhesus monkeys vaccinated with recombinant HSV were solidly protected, and another showed a sustained reduction in viral load following rectal challenge with pathogenic SIVmac239 at 22 weeks following the last vaccine administration. HSV vectors thus show great promise for being able to elicit persistent immune responses and to provide durable protection against AIDS.

ABSTRACTWe demonstrate that human immunodeficiency virus type 1 (HIV-1)-specific CD8+cytotoxic T lymphocytes (CTL) suppress HIV-1 replication in primary lymphocytes, monocytes, and dendritic cells individually. Viral inhibition is significantly diminished in lymphocyte-dendritic cell clusters, suggesting that these clusters in vivo could be sites where viral replication is more difficult to control by CTL.

ABSTRACTMost humans and Old World nonhuman primates are infected for life with Epstein-Barr virus (EBV) or closely related gammaherpesviruses in the same lymphocryptovirus (LCV) subgroup. Several potential strategies for immune evasion and persistence have been proposed based on studies of EBV infection in humans, but it has been difficult to test their actual contribution experimentally. Interest has focused on the EBV nuclear antigen 1 (EBNA1) because of its essential role in the maintenance and replication of the episomal viral genome in latently infected cells and because EBNA1 endogenously expressed in these cells is protected from presentation to the major histocompatibility complex class-I restricted cytotoxic T-lymphocyte (CTL) response through the action of an internal glycine-alanine repeat (GAR). Given the high degree of biologic conservation among LCVs which infect humans and Old World primates, we hypothesized that strategies essential for viral persistence would be well conserved among viruses of this subgroup. We show that the rhesus LCV EBNA1 shares sequence homology with the EBV and baboon LCV EBNA1 and that the rhesus LCV EBNA1 is a functional homologue for EBV EBNA1-dependent plasmid maintenance and replication. Interestingly, all three LCVs possess a GAR domain, but the baboon and rhesus LCV EBNA1 GARs fail to inhibit antigen processing and presentation as determined by using three different in vitro CTL assays. These studies suggest that inhibition of antigen processing and presentation by the EBNA1 GAR may not be an essential mechanism for persistent infection by all LCV and that other mechanisms may be important for immune evasion during LCV infection.

ABSTRACTThree 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 SIVmac239?3, four with SIVmac239?3X, and four with SIVmac239?4. These three vaccine strains exhibit increasing levels of attenuation: ?3 < ?3X

ABSTRACTImmune escape from cytotoxic T-lymphocyte (CTL) responses has been shown to occur not only by changes within the targeted epitope but also by changes in the flanking sequences which interfere with the processing of the immunogenic peptide. However, the frequency of such an escape mechanism has not been determined. To investigate whether naturally occurring variations in the flanking sequences of an immunodominant human immunodeficiency virus type 1 (HIV-1) Gag CTL epitope prevent antigen processing, cells infected with HIV-1 or vaccinia virus constructs encoding different patient-derived Gag sequences were tested for recognition by HLA-A*0201-restricted, p17-specific CTL. We found that the immunodominant p17 epitope (SL9) and its variants were efficiently processed from minigene expressing vectors and from six HIV-1 Gag variants expressed by recombinant vaccinia virus constructs. Furthermore, SL9-specific CTL clones derived from multiple donors efficiently inhibited virus replication when added to HLA-A*0201-bearing cells infected with primary or laboratory-adapted strains of virus, despite the variability in the SL9 flanking sequences. These data suggest that escape from this immunodominant CTL response is not frequently accomplished by changes in the epitope flanking sequences.

ABSTRACTWe examined the ability of a live, attenuated deletion mutant of simian immunodeficiency virus (SIV), SIVmac239?3, which is missingnefand vprgenes, 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 envsequences. 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 SIVmac239?3 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.

ABSTRACTSooty mangabeys naturally infected with simian immunodeficiency virus (SIV) do not develop immunodeficiency despite the presence of viral loads of 105to 107RNA copies/ml. To investigate the basis of apathogenic SIV infection in sooty mangabeys, three sooty mangabeys and three rhesus macaques were inoculated intravenously with SIVmac239 and evaluated longitudinally for 1 year. SIVmac239 infection of sooty mangabeys resulted in 2- to 4-log-lower viral loads than in macaques and did not reproduce the high viral loads observed in natural SIVsmm infection. During acute SIV infection, polyclonal cytotoxic T-lymphocyte (CTL) activity coincident with decline in peak plasma viremia was observed in both macaques and mangabeys; 8 to 20 weeks later, CTL activity declined in the macaques but was sustained and broadly directed in the mangabeys. Neutralizing antibodies to SIVmac239 were detected in the macaques but not the mangabeys. Differences in expression of CD38 on CD8+T lymphocytes or in the percentage of naive phenotype T cells expressing CD45RA and CD62L-selection did not correlate with development of AIDS in rhesus macaques. In macaques, the proportion of CD4+T lymphocytes expressing CD25 declined during SIV infection, while in mangabeys, CD25-expressing CD4+T lymphocytes increased. Longitudinal evaluation of cytokine secretion by flow cytometric analysis of unstimulated lymphocytes revealed elevation of interleukin-2 and gamma interferon in a macaque and only interleukin-10 in a concurrently infected mangabey during acute SIV infection. Differences in host responses following experimental SIVmac239 infection may be associated with the divergent outcome in sooty mangabeys and rhesus macaques.

ABSTRACTCharacterization of immune responses induced by live attenuated simian immunodeficiency virus (SIV) strains may yield clues to the nature of protective immunity induced by this vaccine approach. We investigated the ability of CD8+T lymphocytes from rhesus macaques immunized with the live, attenuated SIV strain SIVmac239?nef or SIVmac239?3 to inhibit SIV replication. CD8+T lymphocytes from immunized animals were able to potently suppress SIV replication in autologous SIV-infected CD4+T cells. Suppression of SIV replication by unstimulated CD8+T cells required direct contact and was major histocompatibility complex (MHC) restricted. However, CD3-stimulated CD8+T cells produced soluble factors that inhibited SIV replication in an MHC-unrestricted fashion as much as 30-fold. Supernatants from stimulated CD8+T cells were also able to inhibit replication of both CCR5- and CXCR4-dependent human immunodeficiency virus type 1 (HIV-1) strains. Stimulation of CD8+cells with cognate cytotoxic T-lymphocyte epitopes also induced secretion of soluble factors able to inhibit SIV replication. Production of RANTES, macrophage inhibitory protein 1a (MIP-1a), or MIP-1ß from stimulated CD8+T cells of vaccinated animals was almost 10-fold higher than that from stimulated CD8+T cells of control animals. However, addition of antibodies that neutralize these ß-chemokines, either alone or in combination, only partly blocked inhibition of SIV and HIV replication by soluble factors produced by stimulated CD8+T cells. Our results indicate that inhibition of SIV replication by CD8+T cells from animals immunized with live attenuated SIV strains involves both MHC-restricted and -unrestricted mechanisms and that MHC-unrestricted inhibition of SIV replication is due principally to soluble factors other than RANTES, MIP-1a, and MIP-1ß.

ABSTRACTDeletion mutants of the pathogenic clone of simian immunodeficiency virus isolate 239 (SIVmac239) were derived that are missingnef, vpr, and upstream sequences (US) in the U3 region of the LTR (SIVmac239?3), nef, vpx, and US (SIVmac239?3x), and nef, vpr,vpx, and US (SIVmac239?4). These multiply deleted derivatives replicated well in the continuously growing CEMx174 cell line and were infectious for rhesus monkeys. However, on the basis of virus load measurements, strength of antibody responses, and lack of disease progression, these mutants were highly attenuated. Measurements of cell-associated viral load agreed well with assays of plasma viral RNA load and with the strengths of the antibody responses; thus, these measurements likely reflected the extent of viral replication in vivo. A derivative of SIVmac239 lacking vifsequences (SIVmac239?vif) could be consistently grown only in avif-complementing cell line. This ?vifvirus appeared to be very weakly infectious for rhesus monkeys on the basis of sensitive antibody tests only. The weak antibody responses elicited by SIVmac239?vifwere apparently in response to low levels of replicating virus since they were not elicited by heat-inactivated virus and the anti-SIV antibody responses persisted for greater than 1 year. These results, and the results of previous studies, allow a rank ordering of the relative virulence of nine mutant strains of SIVmac according to the following order: ?vpr> ?vpx> ?vpr?vpx? ?nef> ?3 > ?3x = ?4 > ?vif> ?5. The results also demonstrate that almost any desired level of attenuation can be achieved, ranging from still pathogenic in a significant proportion of animals (?vprand ?vpx) to not detectably infectious (?5), simply by varying the number and location of deletions in these five loci.