Long-term follow-up study on SIV intestinal proviral load in rhesus macaques

After experimental infection with simian immunodeficiency virus (SIV), intestinal endoscopy proved to be an easily tolerated, minimal invasive procedure to obtain biopsies from the gastrointestinal tract of rhesus macaques during all stages of disease. As the GI tract is affected by many opportunistic infections and immunological impairment after SIV/human immunodeficiency virus (HIV) infection, knowledge on the proviral load is an important parameter for a better understanding of disease pathogenesis. In this paper, we describe the set-up and evaluation of a quantitative competitive polymerase chain reaction (PCR) and the quantification of SIV intestinal proviral load in a long-term follow-up study of eight rhesus monkeys (Macaca mulatta) after two different routes of virus inoculation. A SIV-specific signal could be detected as early as day 3 after infection. Of 143 biopsies from the follow-up study, 85.3% showed a positive PCR. DNA copy numbers ranged from 300 to 15,000 molecules per 100,000 cells. No significant influence of the inoculation route could be shown on either proviral load or survival time, but higher SIV proviral load was associated with a more rapid progression to disease. Therefore, the amount of proviral load in intestinal biopsies may be an important prognostic value for the further course of the disease.     

Viral factors determine progression to AIDS in simian immunodeficiency virus-infected newborn rhesus macaques.

To evaluate how viral variants may affect disease progression in human pediatric AIDS, we studied the potential of three simian immunodeficiency virus (SIV) isolates to induce simian AIDS in newborn rhesus macaques. The three virus isolates were previously shown to range from pathogenic (SIVmac251 and SIVmac239) to nonpathogenic (SIVmac1A11) when inoculated intravenously into juvenile and adult rhesus macaques. Six newborn macaques inoculated with pathogenic, uncloned SIVmac251 developed persistent, high levels of cell-associated and cell-free viremia, had no detectable antiviral antibodies, and had poor weight gain; these animals all exhibited severe clinical disease and pathologic lesions diagnostic for simian AIDS and were euthanatized 10 to 26 weeks after inoculation. Two newborns inoculated with pathogenic, molecularly cloned SIVmac239 developed persistent high virus load in peripheral blood, but both animals had normal weight gain and developed antiviral antibodies. One of the SIVmac239-infected neonates exhibited pathologic lesions diagnostic for SAIDS and was euthanatized at 34 weeks after inoculation; the other SIVmac239-infected neonate remained alive and exhibited no significant clinical disease for more than 1 year after inoculation. In contrast, three newborn rhesus macaques inoculated with the nonpathogenic molecular clone, SIVmac1A11, had transient, low-level viremia, seroconverted by 10 weeks after inoculation, had normal weight gain, and remained healthy for over 1 year. These results indicate that (i) newborn rhesus macaques infected with an uncloned, virulent SIVmac isolate have a more rapid, fulminant disease course than do adults inoculated with the same virus, (ii) the most rapid disease progression is associated with lack of a detectable humoral immune response in SIV-infected infant macaques, (iii) a molecularly cloned, attenuated SIV isolate is nonpathogenic in neonatal macaques, and (iv) SIV-infected neonatal macaques exhibit patterns of infection, virus load, and disease progression similar to those observed in human immunodeficiency virus-infected children. This SIV/neonatal rhesus model of pediatric AIDS provides a rapid, sensitive model with which to compare the virulence of SIV isolates and to study the mechanisms underlying the differences in disease progression in human immunodeficiency virus-infected infants.     

Experimental infection of rhesus and pig-tailed macaques with macaque rhadinoviruses

The recognition of naturally occurring rhadinoviruses in macaque monkeys has spurred interest in their use as models for human infection with Kaposi sarcoma-associated herpesvirus (human herpesvirus 8). Rhesus macaques (Macaca mulatta) and pig-tailed macaques (Macaca nemestrina) were inoculated intravenously with rhadinovirus isolates derived from these species (rhesus rhadinovirus [RRV] and pig-tailed rhadinovirus [PRV]). Nine rhadinovirus antibody-negative and two rhadinovirus antibody-positive monkeys were used for these experimental inoculations. Antibody-negative animals clearly became infected following virus inoculation since they developed persisting antibody responses to virus and virus was isolated from peripheral blood on repeated occasions following inoculation. Viral sequences were also detected by PCR in lymph node, oral mucosa, skin, and peripheral blood mononuclear cells following inoculation. Experimentally infected animals developed peripheral lymphadenopathy which resolved by 12 weeks following inoculation, and these animals have subsequently remained free of disease. No increased pathogenicity was apparent from cross-species infection, i.e., inoculation of rhesus macaques with PRV or of pig-tailed macaques with RRV, whether the animals were antibody positive or negative at the time of virus inoculation. Coinoculation of additional rhesus monkeys with simian immunodeficiency virus (SIV) isolate SIVmac251 and macaque-derived rhadinovirus resulted in an attenuated antibody response to both agents and shorter mean survival compared to SIVmac251-inoculated controls (155.5 days versus 560.1 days; P < 0.019). Coinfected and immunodeficient macaques died of a variety of opportunistic infections characteristic of simian AIDS. PCR analysis of sorted peripheral blood mononuclear cells indicated a preferential tropism of RRV for CD20(+) B lymphocytes. Our results demonstrate persistent infection of macaque monkeys with RRV and PRV following experimental inoculation, but no specific disease was readily apparent from these infections even in the context of concurrent SIV infection.     

Comparison of virology and immunology in SHIV 89.6 proviral DNA and virus-inoculated rhesus macaques

Inoculation of cats, goats and monkeys with plasmids encoding full-length proviral genomes results in persistent lentiviral infections. This system could be used as a method for administration of an attenuated human immunodeficiency virus (HIV) vaccine. Here, we compare the virology and immunology in rhesus macaques inoculated with either simian/human immunodeficiency virus 89.6 (SHIV 89.6) virus or a plasmid containing the SHIV 89.6 proviral genome. There was a delay in appearance of systemic infection in DNA-inoculated animals compared with virus-inoculated animals, but otherwise the pattern of infection was similar. The serum immunoglobulin G anti-simian immunodeficiency virus (SIV) binding antibody response in DNA-inoculated animals was also delayed compared with virus-inoculated animals, but ultimately there was no difference between live virus and DNA-inoculation in the ability to induce the anti-SIV immune responses that were measured. Thus, the data support the concept that plasmid DNA encoding an attenuated virus could be used instead live virus for vaccination.     

In Vivo Replication Capacity Rather Than In Vitro Macrophage Tropism Predicts Efficiency of Vaginal Transmission of Simian Immunodeficiency Virus or Simian/Human Immunodeficiency Virus in Rhesus Macaques

We used the rhesus macaque model of heterosexual human immunodeficiency virus (HIV) transmission to test the hypothesis that in vitro measures of macrophage tropism predict the ability of a primate lentivirus to initiate a systemic infection after intravaginal inoculation. A single atraumatic intravaginal inoculation with a T-cell-tropic molecular clone of simian immunodeficiency virus (SIV), SIVmac239, or a dualtropic recombinant molecular clone of SIV, SIVmac239/1A11/239, or uncloned dualtropic SIVmac251 or uncloned dualtropic simian/human immunodeficiency virus (SHIV) 89.6-PD produced systemic infection in all rhesus macaques tested. However, vaginal inoculation with a dualtropic molecular clone of SIV, SIVmac1A11, resulted in transient viremia in one of two rhesus macaques. It has previously been shown that 12 intravaginal inoculations with SIVmac1A11 resulted in infection of one of five rhesus macaques (M. L. Marthas, C. J. Miller, S. Sutjipto, J. Higgins, J. Torten, B. L. Lohman, R. E. Unger, H. Kiyono, J. R. McGhee, P. A. Marx, and N. C. Pedersen, J. Med. Primatol. 21:99–107, 1992). In addition, SHIV HXBc2, which replicates in monkey macrophages, does not infect rhesus macaques following multiple vaginal inoculations, while T-cell-tropic SHIV 89.6 does (Y. Lu, P. B. Brosio, M. Lafaile, J. Li, R. G. Collman, J. Sodroski, and C. J. Miller, J. Virol. 70:3045–3050, 1996). These results demonstrate that in vitro measures of macrophage tropism do not predict if a SIV or SHIV will produce systemic infection after intravaginal inoculation of rhesus macaques. However, we did find that the level to which these viruses replicate in vivo after intravenous inoculation predicts the outcome of intravaginal inoculation with each virus.     

Inoculation of baboons and macaques with simian immunodeficiency virus/Mne, a primate lentivirus closely related to human immunodeficiency virus type 2

A primate lymphotropic lentivirus was isolated on the human T-cell line HuT 78 after cocultivation of a lymph node from a pig-tailed macaque (Macaca nemestrina) that had died with malignant lymphoma. This isolate, originally designated M. nemestrina immunodeficiency virus (MnIV) and now classified as simian immunodeficiency virus (SIV/Mne), was inoculated intravenously into three juvenile rhesus monkeys (Macaca mulatta), three juvenile pig-tailed macaques (M. nemestrina), and two juvenile baboons (Papio cynocephalus). All six macaques became viremic by 3 weeks after inoculation, whereas neither of the baboons developed viremia. One pig-tailed macaque died at 15 weeks with suppurative peritonitis secondary to ulcerative, necrotizing colitis. Immunologic abnormalities included a marked decrease in CD4+ peripheral blood lymphocytes. Although five macaques mounted an antibody response to SIV/Mne, the animal that died at 15 weeks remained antibody negative. Three other macaques (two rhesus and one pig-tailed) died 66 to 87 weeks after inoculation after exhibiting progressive weight loss, anemia, and diarrhea. Histopathologic findings at necropsy included various manifestations of immune deficiency, nephropathy, subacute encephalitis, pancreatitis, adenocarcinoma, and lymphoid atrophy. SIV/Mne could be readily isolated from the spleens and lymph nodes of all necropsied macaques, and from the cerebrospinal fluid, brains, bone marrow, livers, and pancreas of some of the animals. SIV antigens were localized by avidin-biotin immunohistochemistry to pancreatic islet cells and to bone marrow endothelial cells. The data suggest that African baboons may be resistant to infection by SIV/Mne, whereas Asian macaques are susceptible to infection with this pathogenic primate lentivirus.     

Genital mucosal transmission of simian immunodeficiency virus: animal model for heterosexual transmission of human immunodeficiency virus.

An animal model for the heterosexual transmission of human immunodeficiency virus (HIV) was developed by the application of simian immunodeficiency virus (SIV) onto the genital mucosas of both mature and immature, male and female rhesus macaques. Virus preparations were infused into the vaginal vaults or the urethras (males) of the animals through a soft plastic pediatric nasogastric feeding tube. The macaques that were infected by this route (six males and nine females) developed SIV-specific antibodies, and SIV was isolated from peripheral mononuclear cells of all seropositive animals. One male and one female infected by this route developed severe acquired immunodeficiency syndrome-like disease with retroviral giant-cell pneumonia. As few as two inoculations of cell-free SIV containing 50 50% tissue culture infective doses induced persistent viremia. Cell-free virus preparations were capable of producing infection by the genital route. Much higher doses of virus were required to transmit SIV by this route than are required for transmission by intravenous inoculation. Thus, it appears that the mucous membranes of the genital tract act as a barrier to SIV infection. Spermatozoa and seminal plasma were not required for the genital transmission of SIV. Rarely, SIV was recovered from mononuclear cells in semen and vaginal secretions. The SIV-rhesus macaque model is suitable for assessing the role of cofactors in heterosexual transmission of HIV and will be useful for testing the effectiveness of spermicides, pharmacologic agents, and vaccines in preventing the heterosexual transmission of HIV.     

Rhesus macaques previously infected with simian/human immunodeficiency virus are protected from vaginal challenge with pathogenic SIVmac239.

Nontraumatic vaginal inoculation of rhesus macaques with a simian/human immunodeficiency virus (SIV/HIV) chimera containing the envelope gene from HIV-1 89.6 (SHIV 89.6) results in systemic infection (Y. Lu, B. Brosio, M. Lafaile, J. Li, R. G. Collman, J. Sodroski, and C. J. Miller, J. Virol. 70:3045-3050, 1996). A total of five rhesus macaques have each been infected by exposure to at least three intravaginal inoculations of SHIV 89.6. The SHIV 89.6 infection is characterized by a transient viremia that evokes humoral and cellular immune responses to HIV and SIV antigens, but disease does not develop in animals infected with SHIV 89.6. To determine if a previous infection with SHIV 89.6 by vaginal inoculation could protect animals from vaginal challenge with pathogenic SIV, all five animals were intravaginally inoculated twice with pathogenic SIV-mac239. After challenge, all of the SHIV-immunized animals had low or undetectable viral RNA levels in plasma compared to control animals. Three of the five of the SHIV-immunized animals remained virus isolation negative for more than 8 months, while two became virus isolation positive. The presence of SIV Gag-specific cytotoxic T lymphocytes in peripheral blood mononuclear cells and SIV-specific antibodies in cervicovaginal secretions at the time of challenge was associated with resistance to pathogenic SIV infection after vaginal challenge. These results suggest that protection from sexual transmission of HIV may be possible by effectively stimulating both humoral and cellular antiviral immunity in the systemic and genital mucosal immune compartments.     

Route of simian immunodeficiency virus inoculation determines the complexity but not the identity of viral variant populations that infect rhesus macaques

A better understanding of the host and viral factors associated with human immunodeficiency virus (HIV) transmission is essential to developing effective strategies to curb the global HIV epidemic. Here we used the rhesus macaque-simian immunodeficiency virus (SIV) animal model of HIV infection to study the range of viral genotypes that are transmitted by different routes of inoculation and by different types of viral inocula. Analysis of transmitted variants was undertaken in outbred rhesus macaques inoculated intravenously (IV) or intravaginally (IVAG) with a genetically heterogeneous SIVmac251 stock derived from a well-characterized rhesus macaque viral isolate. In addition, we performed serial IV and IVAG passage experiments using plasma from SIV-infected macaques as the inoculum. We analyzed the V1-V2 region of the SIV envelope gene from virion-associated RNA in plasma from infected animals by the heteroduplex mobility assay (HMA) and by DNA sequence analysis. We found that a more diverse population of SIV genetic variants was present in the earliest virus-positive plasma samples from all five IV SIVmac251-inoculated monkeys and from two of five IVAG SIVmac251-inoculated monkeys. In contrast, we found a relatively homogeneous population of SIV envelope variants in three of five monkeys inoculated IVAG with SIVmac251 stock and in two monkeys infected after IVAG inoculation with plasma from an SIV-infected animal. In some IVAG-inoculated animals, the transmitted SIV variant was the most common variant in the inoculum. However, a specific viral variant in the SIVmac251 stock was not consistently transmitted by IVAG inoculation. Thus, it is likely that host factors or stochastic processes determine the specific viral variants that infect an animal after IVAG SIV exposure. In addition, our results clearly demonstrate that the route of inoculation is associated with the extent and breadth of the genetic complexity of the viral variant population in the earliest stages of systemic infection.     

Genital tract sequestration of SIV following acute infection

We characterized the evolution of simian immunodeficiency virus (SIV) in the male genital tract by examining blood- and semen-associated virus from experimentally and sham vaccinated rhesus monkeys during primary infection. At the time of peak virus replication, SIV sequences were intermixed between the blood and semen supporting a scenario of high-level virus "spillover" into the male genital tract. However, at the time of virus set point, compartmentalization was apparent in 4 of 7 evaluated monkeys, likely as a consequence of restricted virus gene flow between anatomic compartments after the resolution of primary viremia. These findings suggest that SIV replication in the male genital tract evolves to compartmentalization after peak viremia resolves.     

Detection of simian immunodeficiency virus DNA in macrophages from infected rhesus macaques.

We have examined the frequency of infection of monocyte-derived and alveolar macrophages isolated from rhesus macaques inoculated with simian immunodeficiency virus (SIVmac) utilizing a semiquantitative PCR methodology. Animals were inoculated with either pathogenic (SIVmac239) or nonpathogenic (SIVmac1A11) molecularly cloned viruses of SIVmac, or with uncloned pathogenic SIVmacBIOL. The frequency of SIV DNA in macrophages was highest early after infection and at terminal stages of disease, whereas during the asymptomatic period, SIV DNA was present at very low levels in macrophages.     

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.     

Simian immunodeficiency virus in kidney cell cultures from highly infected rhesus macaques (Macaca mulatta)

Trace amounts of simian immunodeficiency virus (SIV) proviral DNA were detected in monolayers of primary kidney cells from two rhesus macaques (Macaca mulatta) heavily infected with the highly pathogenic strain SIVmac251. There was no detectable infectious SIV in the supernatant from the kidney cell cultures obtained from either monkey. However, infectious SIV was rescued by co-culture of kidney cells with a permissive lymphoid cell line. Macrophages, present in these cultures, may be the reservoir for the proviral genomes.     

Infection of rhesus monkeys with topical instillation of simian immunodeficiency virus (SIV)B670 into the conjunctival sac

We tested the ability of SIV to cause local and systemic infection in three rhesus monkeys after topical instillation of cell-free virus into the conjunctival cul-de-sac. Conjunctivitis or other signs of infection were monitored after inoculation. Conjunctiva were swabbed for virus culture and biopsied for PCR. Changes in lymphocyte subsets, seroconversion, antigenemia, and virus isolation from PBL were assessed systemically postinoculation. Viral DNA was detected in conjunctival biopsy by PCR in one of three animals that later developed systemic infection. The other two animals remained uninfected. These data demonstrate that the conjunctiva is a route by which SIV (and perhaps HIV) may cause systemic infection.     

Simian immunodeficiency virus rapidly penetrates the cervicovaginal mucosa after intravaginal inoculation and infects intraepithelial dendritic cells

Despite recent insights into mucosal human immunodeficiency virus (HIV) transmission, the route used by primate lentiviruses to traverse the stratified squamous epithelium of mucosal surfaces remains undefined. To determine if dendritic cells (DC) are used by primate lentiviruses to traverse the epithelial barrier of the genital tract, rhesus macaques were intravaginally exposed to cell-free simian immunodeficiency virus SIVmac251. We examined formalin-fixed tissues and HLA-DR(+)-enriched cell suspensions to identify the cells containing SIV RNA in the genital tract and draining lymph nodes within the first 24 h of infection. Using SIV-specific fluorescent in situ hybridization combined with immunofluorescent antibody labeling of lineage-specific cell markers, numerous SIV RNA(+) DC were documented in cell suspensions from the vaginal epithelium 18 h after vaginal inoculation. In addition, we determined the minimum time that the SIV inoculum must remain in contact with the genital mucosa for the virus to move from the vaginal lumen into the mucosa. We now show that SIV enters the vaginal mucosa within 60 min of intravaginal exposure, infecting primarily intraepithelial DC and that SIV-infected cells are located in draining lymph nodes within 18 h of intravaginal SIV exposure. The speed with which primate lentiviruses penetrate mucosal surfaces, infect DC, and disseminate to draining lymph nodes poses a serious challenge to HIV vaccine development.     

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.     

A period of transient viremia and occult infection precedes persistent viremia and antiviral immune responses during multiple low-dose intravaginal simian immunodeficiency virus inoculations

In rhesus macaques, classic systemic infection, characterized by persistent viremia and seroconversion, occurred after multiple low-dose (10(3) 50% tissue culture infective doses) intravaginal (IVAG) inoculations with simian immunodeficiency virus (SIV) strain SIVmac251. Monkeys developed classic SIV infections after a variable number of low-dose IVAG exposures to SIVmac251. Once established, the systemic infection was identical to SIV infection following high-dose IVAG SIV inoculation. However, occult systemic infection characterized by transient cell-associated or cell-free viremia consistently occurred early in the series of multiple vaginal SIV exposures. Further, antiviral cellular immune responses were present prior to the establishment of a classic systemic infection in the low-dose vaginal SIV transmission model.     

Reversion in vivo after inoculation of a molecular proviral DNA clone of simian immunodeficiency virus with a cytotoxic-T-lymphocyte escape mutation

Vaccine-based control of the replication of a simian immunodeficiency virus (SIV), SIVmac239, in macaques has recently been shown. In the process of the control, a mutant virus escaping from epitope-specific cytotoxic-T-lymphocyte (CTL) responses was rapidly selected and contained. In this study, we show that the wild-type virus appeared and became predominant in the absence of the epitope-specific CTL after inoculation of naive macaques with a molecular clone DNA of the CTL escape mutant SIV. This is the first report describing reversion in vivo from an inoculated, molecular proviral DNA clone of immunodeficiency virus with a CTL escape mutation.     

Experimental coinfection of rhesus macaques with rhesus cytomegalovirus and simian immunodeficiency virus: pathogenesis

Human cytomegalovirus (HCMV) possesses low pathogenic potential in an immunocompetent host. In the immunosuppressed host, however, a wide spectrum of infection outcomes, ranging from asymptomatic to life threatening, can follow either primary or nonprimary infection. The variability in the manifestations of HCMV infection in immunosuppressed individuals implies that there is a threshold of host antiviral immunity that can effectively limit disease potential. We used a nonhuman primate model of CMV infection to assess the relationship between CMV disease and the levels of developing anti-CMV immunity. Naive rhesus macaques were inoculated with rhesus cytomegalovirus (RhCMV) followed 2 or 11 weeks later by inoculation with pathogenic simian immunodeficiency virus SIVmac239. Two of four monkeys inoculated with SIV at 2 weeks after inoculation with RhCMV died within 11 weeks with simian AIDS (SAIDS), including activated RhCMV infection. Neither animal had detectable anti-SIV antibodies. The other two animals died 17 and 27 weeks after SIV inoculation with either SAIDS or early lymphoid depletion, although no histological evidence of activated RhCMV was observed. Both had weak anti-SIV antibody titers. RhCMV antibody responses for this group of monkeys were significantly below those of control animals inoculated with only RhCMV. In addition, all animals of this group had persistent RhCMV DNA in plasma and high copy numbers of RhCMV in tissues. In contrast, animals that were inoculated with SIV at 11 weeks after RhCMV infection rarely exhibited RhCMV DNA in plasma, had low copy numbers of RhCMV DNA in most tissues, and did not develop early onset of SAIDS or activated RhCMV. SIV antibody titers were mostly robust and sustained in these monkeys. SIV inoculation blunted further development of RhCMV humoral responses, unlike the normal pattern of development in control monkeys following RhCMV inoculation. Anti-RhCMV immunoglobulin G levels and avidity were slightly below control values, but levels maintained were higher than those observed following SIV infection at 2 weeks after RhCMV inoculation. These findings demonstrate that SIV produces long-lasting insults to the humoral immune system beginning very early after SIV infection. The results also indicate that anti-RhCMV immune development at 11 weeks after infection was sufficient to protect the host from acute RhCMV sequelae following SIV infection, in contrast to the lack of protection afforded by only 2 weeks of immune response to RhCMV. As previously observed, monkeys that were not able to mount a significant immune response to SIV were the most susceptible to SAIDS, including activated RhCMV infection. Rapid development of SAIDS in animals inoculated with SIV 2 weeks after RhCMV inoculation suggests that RhCMV can augment SIV pathogenesis, particularly during primary infection by both viruses.     

Distinct expression patterns of CD69 in mucosal and systemic lymphoid tissues in primary SIV infection of rhesus macaques

Although the intestinal tract plays a major role in early human immunodeficiency virus (HIV) infection, the role of immune activation and viral replication in intestinal tissues is not completely understood. Further, increasing evidence suggests the early leukocyte activation antigen CD69 may be involved in the development or regulation of important T cell subsets, as well as a major regulatory molecule of immune responses. Using the simian immunodeficiency virus (SIV) rhesus macaque model, we compared expression of CD69 on T cells from the intestine, spleen, lymph nodes, and blood of normal and SIV-infected macaques throughout infection. In uninfected macaques, the majority of intestinal lamina propria CD4+ T cells had a memory (CD95+) phenotype and co-expressed CD69, and essentially all intestinal CCR5+ cells co-expressed CD69. In contrast, systemic lymphoid tissues had far fewer CD69+ T cells, and many had a naïve phenotype. Further, marked, selective depletion of intestinal CD4+CD69+ T cells occurred in early SIV infection, and this depletion persisted throughout infection. Markedly increased levels of CD8+CD69+ T cells were detected after SIV infection in virtually all tissues, including the intestine. Further, confocal microscopy demonstrated selective, productive infection of CD3+CD69+ T cells in the intestine in early infection. Combined, these results indicate CD69+CD4+ T cells are a major early target for viral infection, and their rapid loss by direct infection may have profound effects on intestinal immune regulation in HIV infected patients.