The TRIM5 gene modulates penile mucosal acquisition of simian immunodeficiency virus in rhesus monkeys

There is considerable variability in host susceptibility to human immunodeficiency virus type 1 (HIV-1) infection, but the host genetic determinants of that variability are not well understood. In addition to serving as a block for cross-species retroviral infection, TRIM5 was recently shown to play a central role in limiting primate immunodeficiency virus replication. We hypothesized that TRIM5 may also contribute to susceptibility to mucosal acquisition of simian immunodeficiency virus (SIV) in rhesus monkeys. We explored this hypothesis by establishing 3 cohorts of Indian-origin rhesus monkeys with different TRIM5 genotypes: homozygous restrictive, heterozygous permissive, and homozygous permissive. We then evaluated the effect of TRIM5 genotype on the penile transmission of SIVsmE660. We observed a significant effect of TRIM5 genotype on mucosal SIVsmE660 acquisition in that no SIV transmission occurred in monkeys with only restrictive TRIM5 alleles. In contrast, systemic SIV infections were initiated after preputial pocket exposures in monkeys that had at least one permissive TRIM5 allele. These data demonstrate that host genetic factors can play a critical role in restricting mucosal transmission of a primate immunodeficiency virus. In addition, we used our understanding of TRIM5 to establish a novel nonhuman primate penile transmission model for AIDS mucosal pathogenesis and vaccine research.     

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.     

Antibody-dependent cell-mediated cytotoxicity in simian immunodeficiency virus-infected rhesus monkeys

With the recent demonstration in the RV144 Thai trial that a vaccine regimen that does not elicit neutralizing antibodies or cytotoxic T lymphocytes may confer protection against human immunodeficiency virus type 1 (HIV-1) infection, attention has turned to nonneutralizing antibodies as a possible mechanism of vaccine protection. In the current study, we evaluated the kinetics of the antibody-dependent cell-mediated cytotoxicity (ADCC) response during acute and chronic SIVmac251 infection of rhesus monkeys. We first adapted a flow cytometry-based ADCC assay, evaluating the use of different target cells as well as different strategies for quantitation of activated natural killer (NK) cells. We found that the use of SIVmac251 Env gp130-coated target cells facilitates analyses of ADCC activity with a higher degree of sensitivity than the use of simian immunodeficiency virus (SIV)-infected target cells; however, the kinetics of the measured responses were the same using these different target cells. By comparing NK cell expression of CD107a with NK cell expression of other cytokines or chemokine molecules, we found that measuring CD107a expression is sufficient for evaluating the anti-SIV function of NK cells. We also showed that ADCC responses can be detected as early as 3 weeks after SIVmac251 infection and that the magnitude of this antibody response is inversely associated with plasma viral RNA levels in animals with moderate to high levels of viral replication. However, we also demonstrated an association between NK cell-mediated ADCC responses and the amount of SIVmac251 gp140 binding antibody that developed after viral infection. This final observation raises the possibility that the antibodies that mediate ADCC are a subset of the antibodies detected in a binding assay and arise within weeks of infection.     

Interactions between Mycobacterium leprae and simian immunodeficiency virus (SIV) in rhesus monkeys

Groups of rhesus monkeys were inoculated with: 1) simian immunodeficiency virus (SIV)B670 alone; 2) Mycobacterium leprae alone; 3) SIV plus M. leprae on the same day; and 4) M. leprae 2 weeks after SIV. Animals were monitored at intervals for virus loads, antibody responses to M. leprae glycolipid antigens and to SIV Gp120, T-cell CD4+ and CD4+ CD29+ subset percentages, leprosy and acquired immunodeficiency syndrome (AIDS) clinical symptoms. Five out of six animals developed leprosy in each co-inoculated group, compared to one out of six in the M. leprae-only-inoculated group, indicating that M. leprae/SIV co-infection increases the susceptibility to leprosy, regardless of the timing of the two infections. Animals in the co-infected group that received M. leprae 2 weeks after SIV had a significantly slower rate of AIDS progression and long-term survival was significantly greater (three out of six) compared to the group inoculated with SIV alone (zero out of seven). All M. leprae-only-inoculated animals (six out of six) survived. Post-SIV-inoculation, a rapid decrease in the percentages of CD4 + and CD4 + CD29 + T-cells was observed in the SIV-only-inoculated group that was significantly blocked by co-inoculation with M. leprae 2 weeks after SIV, but not by SIV on the same day. The virus load set point was increased by approximately two logs in the group inoculated with M. leprae and SIV on the same day compared to SIV 2 weeks prior to M. leprae or the SIV-only-inoculated group. The results indicate that M. leprae, inoculated 2 weeks after SIV, decreased the pathogenicity of SIV compared to inoculation of M. leprae and SIV on the same day or SIV alone. The decreased pathogenicity correlated with a diminished loss of CD4 + and CD4 + CD29 + T-cell subsets in the group inoculated with M. leprae 2 weeks after SIV compared to the group inoculated with SIV alone. IgG antibody responses to M. leprae-specific cell wall phenolic glycolipid-I antigen were inhibited by 2-week-prior or same-day SIV co-inoculation compared to M. leprae-only inoculated animals. The IgG anti-lipoarabinomannan antibody response was enhanced in the group inoculated with M. leprae and SIV on the same day compared to the groups inoculated with M. leprae alone or SIV 2 weeks prior to M. leprae. Antibody responses to SIV Gp120 antigen were unimpaired in both co-inoculated groups compared to SIV-only-inoculated groups. The antibody results show that the immune responses to SIV and M. leprae are interrelated in SIV/M. leprae co-infected animals.     

The gag-specific cytotoxic T lymphocytes in rhesus monkeys infected with the simian immunodeficiency virus of macaques

The simian immunodeficiency virus of macaques (SIVmac) is a lentivirus which induces an AIDS-like disease in rhesus monkeys. We have explored the virus-specific cellular immune response in SIVmac-infected rhesus monkeys. Con A-activated, IL-2 expanded PBL of some SIVmac-infected rhesus monkeys lyse autologous B lymphoblastoid cell lines infected with a recombinant vaccinia virus that carries the SIVmac gag gene. This lysis is mediated by CD8+ lymphocytes and is MHC class I restricted. Moreover, these effector lymphocytes do not express the NK cell-associated molecules NKH1 or CD16. These cells are, therefore, CTL. In a limited prospective study of SIVmac-infected rhesus monkeys, the presence of the SIVmac gag-specific CTL activity in PBL correlated with both a reduced efficiency in isolating SIVmac from PBL of these monkeys and their extended survival. This method for assessing SIVmac gag-specific cellular immunity in rhesus monkeys will be important not only in investigating the immunopathogenesis of SIVmac-induced disease, but also in evaluating the capacity of candidate AIDS vaccines to elicit a cell-mediated immune response in this animal model.     

Selection of genetic variants of simian immunodeficiency virus in persistently infected rhesus monkeys.

Genetic and antigenic variation may be one means by which lentiviruses that cause AIDS avoid elimination by host immune responses. Genetic variation in the envelope gene (env) was studied by comparing the nucleotide sequences of 27 clones obtained from two rhesus monkeys infected with molecularly cloned simian immunodeficiency virus. All 27 clones differed from each other and differed from the input clone in the gp120 (SU) portion of the envelope gene. Nucleotide substitutions were shown to accumulate with time at an average rate of 8.5 per 1,000 per year in SU. Surprisingly, the majority of nucleotide substitutions (81%) resulted in amino acid changes. Variation in SU was not random but occurred predominantly in five discrete regions. Within these variable regions, a remarkable 98% of the nucleotide substitutions changed the amino acid. These results demonstrate that extensive sequence variability accumulates in vivo after infection with molecularly cloned virus and that selection occurs in vivo for changes in distinct variable regions in env.     

Recombinant canarypox vaccine-elicited CTL specific for dominant and subdominant simian immunodeficiency virus epitopes in rhesus monkeys

Since virus-specific CTL play a central role in containing HIV replication, a candidate AIDS vaccine should generate virus-specific CTL responses. In this study, the ability of a recombinant canarypox virus expressing SIV Gag-Pol-Env (ALVAC/SIV gag-pol-env) was assessed for its ability to elicit both dominant and subdominant epitope-specific CTL responses in rhesus monkeys. Following a series of five immunizations, memory CTL responses specific for a dominant Gag epitope could be demonstrated in the peripheral blood of vaccinated monkeys. Memory CTL responses to a subdominant Pol epitope were undetectable in these animals. Following challenge with SIVmac251, the experimentally vaccinated animals developed high frequency CTL responses specific for the dominant Gag epitope that emerged in temporal association with the early containment of viral replication. Interestingly, the experimentally vaccinated, but not the control vaccinated animals, developed CTL responses to the subdominant Pol epitope that were detectable only after containment of early viremia. Thus, recombinant canarypox vaccination elicited low frequency, but durable memory CTL populations. The temporal association of the emergence of the dominant epitope-specific response with early viral containment following challenge suggests that this immune response played a role in the accelerated clearing of early viremia in these animals. The later emerging CTL response specific for the subdominant epitope may contribute to the control of viral replication in the setting of chronic infection.     

Selective amplification of simian immunodeficiency virus genotypes after intrarectal inoculation of rhesus monkeys.

Animal models for sexual transmission of human immunodeficiency virus can define the influences of virus type, dose, and route of inoculation on infection and clinical outcome. We used an uncloned simian immunodeficiency virus stock (SIVmac) to inoculate cells in vitro and to inoculate rhesus monkeys by intravenous and intrarectal routes. The distribution of virus genotypes present in each of these infection examples was characterized by DNA sequence analysis of viral long terminal repeats (LTRs). Our analysis of LTR sequences from in vitro and in vivo infections revealed three main genotypes: one genotype was observed only for in vitro infection, and two other genotypes were recovered only from infected animals. By comparing animals inoculated with high intrarectal doses of SIVmac and those inoculated with low doses, we demonstrated that unique subsets of the stock were selected after intrarectal infection. Our findings indicate that minor genotypes present in the stock cross the rectal mucosa and are amplified selectively to become prominent in peripheral blood mononuclear cells from acutely infected animals. Studies with a molecular recombinant of SIV and human immunodeficiency virus type 1 sequences, SHIV, showed that viral LTR sequences do not undergo especially rapid sequence variation or rearrangement after intrarectal inoculation. The mucosal barrier exerts a significant influence on infection and disease progression by reducing the efficiency of SIVmac infection and by permitting distinct, pathogenic genotypes to become established in the host.     

Emergence of CTL coincides with clearance of virus during primary simian immunodeficiency virus infection in rhesus monkeys

The CTL response was characterized during primary SIV/macaque (SIVmac) infection of rhesus monkeys to assess its role in containing early viral replication using both an epitope-specific functional and an MHC class I/peptide tetramer-binding assay. The rapid expansion of a single dominant viral epitope-specific CTL population to 1.3-8.3% of circulating CD8+ peripheral blood and 0. 3-1.3% of lymph node CD8+ T cells was observed, peaking at day 13 following infection. A subsequent decrease in number of these cells was then demonstrated. Interestingly, the percent of tetramer-binding CD8+ T cells detected in the lymph nodes of all evaluated animals was smaller than the percent detected in PBL. These epitope-specific CD8+ T cells expressed cell surface molecules associated with memory and activation. Early clearance of SIVmac occurred coincident with the emergence of the CTL response, suggesting that CTL may be important in containing virus replication. A higher percent of annexin V-binding cells was detected in the tetramer+ CD8+ T cells (range, from 33% to 75%) than in the remaining CD8+ T cells (range, from 3.3% to 15%) at the time of maximum CTL expansion in all evaluated animals. This finding indicates that the decrease of CTL occurred as a result of the death of these cells rather than their anatomic redistribution. These studies provide strong evidence for the importance of CTL in containing AIDS virus replication.     

Viral escape from dominant simian immunodeficiency virus epitope-specific cytotoxic T lymphocytes in DNA-vaccinated rhesus monkeys

Virus-specific cytotoxic T lymphocytes (CTL) are critical for control of human immunodeficiency virus type 1 replication. However, viral escape from CTL recognition can undermine this immune control. Here we demonstrate the high frequency and pattern of viral escape from dominant epitope-specific CTL in SIV gag DNA-vaccinated rhesus monkeys following a heterologous simian immunodeficiency virus (SIV) challenge. DNA-vaccinated monkeys exhibited initial effective control of the SIV challenge, but this early control was lost by serial breakthroughs of viral replication over a 3-year follow-up period. Increases in plasma viral RNA correlated temporally with declines of dominant SIV epitope-specific CD8(+) T-lymphocyte responses and the emergence of viral mutations that escaped recognition by dominant epitope-specific CTL. Viral escape from CTL occurred in a total of seven of nine vaccinated and control monkeys, including three animals that initially controlled viral replication to undetectable levels of plasma viral RNA. These data suggest that CTL exert selective pressure on viral replication and that viral escape from CTL may be a limitation of CTL-based AIDS vaccine strategies.     

Quantitative evaluation of Enterocytozoon bieneusi infection in simian immunodeficiency virus-infected rhesus monkeys

The association of the microsporidia Enterocytozoon bieneusi with chronic diarrhea and wasting in individuals with acquired immunodeficiency syndrome (AIDS) has been demonstrated. The disease caused by E. bieneusi has been linked to decreased levels of circulating CD4+ T lymphocytes. In this study, we investigated the relationship between the extent of excretion of E. bieneusi in feces of simian immunodeficiency virus (SIV)-infected juvenile macaques and the CD4+ T lymphocyte counts in the peripheral blood. Twelve juvenile rhesus monkeys (Macaca mulatta) were intravenously inoculated with the pathogenic molecular clone SIVmac239. Numbers of CD4+ T lymphocytes were assessed by three-color flow cytometry. The presence of E. bieneusi DNA in feces was assessed by nested PCR. In addition, selected samples of feces were examined by competitive quantitative PCR to assess the level of E. bieneusi infection. Low (n = 5) to undetectable (n = 7) quantities of E. bieneusi were present in feces of the twelve animals in prior to inoculation with SIV. After SIV inoculation the number of animals shedding E. bieneusi increased (n = 10) as did the quantity of E. bieneusi shedding in the feces. Of the twelve juvenile animals, five animals died within 8 months post-SIV inoculation with symptoms of AIDS. Four of the five deceased animals showed shedding of E. bieneusi DNA in feces (> or =100 spores/g) for at least three consecutive months. Increased number of E. bieneusi in feces was accompanied by decreased counts of circulating CD4+ T lymphocytes and increased SIV plasma viral load.     

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.     

Short- and long-term clinical outcomes in rhesus monkeys inoculated with a highly pathogenic chimeric simian/human immunodeficiency virus

A highly pathogenic simian/human immunodeficiency virus (SHIV), SHIV(DH12R), isolated from a rhesus macaque that had been treated with anti-human CD8 monoclonal antibody at the time of primary infection with the nonpathogenic, molecularly cloned SHIV(DH12), induced marked and rapid CD4(+) T cell loss in all rhesus macaques intravenously inoculated with 1.0 50% tissue culture infective dose (TCID(50)) to 4.1 x 10(5) TCID(50)s of virus. Animals inoculated with 650 TCID(50)s of SHIV(DH12R) or more experienced irreversible CD4(+) T lymphocyte depletion and developed clinical disease requiring euthanasia between weeks 12 and 23 postinfection. In contrast, the CD4(+) T-cell numbers in four of five monkeys receiving 25 TCID(50)s of SHIV(DH12R) or less stabilized at low levels, and these surviving animals produced antibodies capable of neutralizing SHIV(DH12R). In the fifth monkey, no recovery from the CD4(+) T cell decline occurred, and the animal had to be euthanized. Viral RNA levels, subsequent to the initial peak of infection but not at peak viremia, correlated with the virus inoculum size and the eventual clinical course. Both initial infection rate constants, k, and decay constants, d, were determined, but only the latter were statistically correlated to clinical outcome. The attenuating effects of reduced inoculum size were also observed when virus was inoculated by the mucosal route. Because the uncloned SHIV(DH12R) stock possessed the genetic properties of a lentivirus quasispecies, we were able to assess the evolution of the input virus swarm in animals surviving the acute infection by monitoring the emergence of neutralization escape viral variants.     

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.     

Enteric ganglionitis in rhesus macaques infected with simian immunodeficiency virus

Gastrointestinal (GI) disease is a debilitating feature of human immunodeficiency virus (HIV) infection that can occur in the absence of histopathological abnormalities or identifiable enteropathogens. However, the mechanisms of GI dysfunction are poorly understood. The present study was undertaken to characterize changes in resident and inflammatory cells in the enteric nervous system (ENS) of macaques during the acute stage of simian immunodeficiency virus (SIV) infection to gain insight into potential pathogenic mechanisms of GI disease. Ganglia from duodenum, ileum, and colon were examined in healthy and acutely infected macaques by using a combination of routine histology, double-label immunofluorescence and in situ hybridization. Evaluation of tissues from infected macaques showed progressive infiltration of myenteric ganglia by CD3+ T cells and IBA1+ macrophages beginning as early as 8 days postinfection. Quantitative image analysis revealed that the severity of myenteric ganglionitis increased with time after SIV infection and, in general, was more severe in ganglia from the small intestine than in ganglia from the colon. Despite an abundance of inflammatory cells in myenteric ganglia during acute infection, the ENS was not a target for virus infection. This study provides evidence that the ENS may be playing a role in the pathogenesis of GI disease and enteropathy in HIV-infected people.     

Early control of highly pathogenic simian immunodeficiency virus/human immunodeficiency virus chimeric virus infections in rhesus monkeys usually results in long-lasting asymptomatic clinical outcomes

In contrast to simian immunodeficiency viruses (SIVs), which induce immunodeficiency over a 1- to 2-year period, highly pathogenic simian-human immunodeficiency viruses (SHIVs) cause an irreversible and systemic depletion of CD4(+) T lymphocytes in macaque monkeys within weeks of inoculation. Nonetheless, the seemingly more aggressive SHIVs have proven to be easier to control by the same vaccine regimens which fail to contain SIV. Because early events during in vivo infections may determine both the pathogenic consequences of the challenge virus and its sensitivity to interventions that prevent disease, we have evaluated the effects of inoculum size and a potent antiretroviral drug on the development of disease in monkeys infected with SHIV(DH12R). The results obtained show that in a majority of inoculated animals, suppression of SHIV replication during the first 2 weeks of infection, which prevents complete loss of CD4(+) T cells, leads to very low to undetectable postpeak viremia and an asymptomatic clinical course for periods up to 4 years.     

Potent simian immunodeficiency virus-specific cellular immune responses in the breast milk of simian immunodeficiency virus-infected, lactating rhesus monkeys

Breast milk transmission of HIV is a leading cause of infant HIV/AIDS in the developing world. Remarkably, only a small minority of breastfeeding infants born to HIV-infected mothers contract HIV via breast milk exposure, raising the possibility that immune factors in the breast milk confer protection to the infants who remain uninfected. To model HIV-specific immunity in breast milk, lactation was pharmacologically induced in Mamu-A*01(+) female rhesus monkeys. The composition of lymphocyte subsets in hormone-induced lactation breast milk was found to be similar to that in natural lactation breast milk. Hormone-induced lactating monkeys were inoculated i.v. with SIVmac251 and CD8(+) T lymphocytes specific for two immunodominant SIV epitopes, Gag p11C and Tat TL8, and SIV viral load were monitored in peripheral blood and breast milk during acute infection. The breast milk viral load was 1-2 logs lower than plasma viral load through peak and set point of viremia. Surprisingly, whereas the kinetics of the SIV-specific cellular immunity in breast milk mirrored that of the blood, the peak magnitude of the SIV-specific CD8(+) T lymphocyte response in breast milk was more than twice as high as the cellular immune response in the blood. Furthermore, the appearance of the SIV-specific CD8(+) T lymphocyte response in breast milk was associated with a reduction in breast milk viral load, and this response remained higher than that in the blood after viral set point. This robust viral-specific cellular immune response in breast milk may contribute to control of breast milk virus replication.     

The intracytoplasmic domain of the Env transmembrane protein is a locus for attenuation of simian immunodeficiency virus SIVmac in rhesus macaques

The human and simian immunodeficiency virus (HIV-1 and SIVmac) transmembrane proteins contain unusually long intracytoplasmic domains (ICD-TM). These domains are suggested to play a role in envelope fusogenicity, interaction with the viral matrix protein during assembly, viral infectivity, binding of intracellular calmodulin, disruption of membranes, and induction of apoptosis. Here we describe a novel mutant virus, SIVmac-M4, containing multiple mutations in the coding region for the ICD-TM of pathogenic molecular clone SIVmac239. Parental SIVmac239-Nef+ produces high-level persistent viremia and simian AIDS in both juvenile and newborn rhesus macaques. The ICD-TM region of SIVmac-M4 contains three stop codons, a +1 frameshift, and mutation of three highly conserved, charged residues in the conserved C-terminal alpha-helix referred to as lentivirus lytic peptide 1 (LLP-1). Overlapping reading frames for tat, rev, and nef are not affected by these changes. In this study, four juvenile macaques received SIVmac-M4 by intravenous injection. Plasma viremia, as measured by branched-DNA (bDNA) assay, reached a peak at 2 weeks postinoculation but dropped to below detectable levels by 12 weeks. At over 1.5 years postinoculation, all four juvenile macaques remain healthy and asymptomatic. In a subsequent experiment, four neonatal rhesus macaques were given SIVmac-M4 intravenously. These animals exhibited high levels of viremia in the acute phase (2 weeks postinoculation) but are showing a relatively low viral load in the chronic phase of infection, with no clinical signs of disease for 1 year. These findings demonstrated that the intracytoplasmic domain of the transmembrane Env (Env-TM) is a locus for attenuation in rhesus macaques.     

CD8+ and CD20+ lymphocytes cooperate to control acute simian immunodeficiency virus/human immunodeficiency virus chimeric virus infections in rhesus monkeys: modulation by major histocompatibility complex genotype

We have previously described two isogenic molecularly cloned simian immunodeficiency virus/human immunodeficiency virus chimeric viruses (SHIVs) that differ from one another by 9 amino acids and direct distinct clinical outcomes in inoculated rhesus monkeys. SHIV(DH12R-Clone 7), like other highly pathogenic CXCR4-tropic SHIVs, induces rapid and complete depletions of CD4+ T lymphocytes and immunodeficiency in infected animals. In contrast, macaques inoculated with SHIV(DH12R-Clone 8) experience only partial and transient losses of CD4+ T cells, show prompt control of their viremia, and remain healthy for periods of time extending for up to 4 years. The contributions of CD8+ and CD20+ lymphocytes in suppressing the replication of the attenuated SHIV(DH12R-Clone 8) and maintaining a prolonged asymptomatic clinical course was assessed by treating animals with monoclonal antibodies that deplete each lymphocyte subset at the time of virus inoculation. The absence of either CD8+ or CD20+ cells during the SHIV(DH12R-Clone 8) acute infection resulted in the rapid, complete, and irreversible loss of CD4+ T cells; sustained high levels of postpeak plasma viremia; and symptomatic disease in Mamu-A*01-negative Indian rhesus monkeys. In Mamu-A*01-positive animals, however, the aggressive, highly pathogenic phenotype was observed only in macaques depleted of CD8+ cells; SHIV(DH12R-Clone 8) was effectively controlled in Mamu-A*01-positive monkeys in the absence of B lymphocytes. Taken together, these results indicate that both CD8+ and CD20+ B cells contribute to the control of primate lentiviral infection in Mamu-A*01-negative macaques. Furthermore, the major histocompatibility complex genotype of an infected animal, as exemplified by the Mamu-A*01 allele in this study, has the additional capacity to shift the balance of the composite immune response.