The U3 promoter and the nef gene of simian immunodeficiency virus (SIV) smmPBj1.9 do not confer acute pathogenicity upon SIVagm

Two chimeric proviruses comprising the U3 promoter and the nef gene of simian immunodeficiency virus (SIV) smmPBj1.9 in addition to other genomic regions of SIVagm3mc from African green monkeys (Cercopithecus aethiops) were constructed. The derived chimeric viruses (SIVagm3mc/SIVsmmPBj1.9) were both able to replicate in nonstimulated peripheral blood leukocytes from pig-tailed macaques (Macaca nemestrina), a biological property often correlated with acute pathogenicity. However, only one of the chimeric viruses was acutely pathogenic, inducing a rapid depletion of the peripheral CD4+ T cells in two infected pig-tailed macaques within 10 days after infection in a manner similar to infection with SIVsmmPBj1.9 itself. The other chimeric virus actively replicated during the first 8 weeks after experimental infection of two pig-tailed macaques but induced neither acute disease nor CD4+ T-cell depletion for 113 weeks after infection. Thus, the U3 promoter and the nef gene of SIVsmmPBj1.9 alone appear to be insufficient to confer acute pathogenicity to SIVagm3mc.     

Infection of baboons with a simian immunodeficiency virus/HIV-1 chimeric virus constructed with an HIV-1 Thai subtype E envelope

OBJECTIVE: To construct an infectious chimeric simian immunodeficiency virus/HIV-1 (SHIV) with the envelope of a Thai subtype E HIV-1 strain for use in a non-human primate model. METHODS: A novel SHIV genome was derived using the sequences of the ectodomain of the envelope gene from the Thai subtype E strain, HIV-1(9466). This SHIV (SHIV9466.33) was recovered by cocultivation from human peripheral blood mononuclear cells (PBMC) after transfection of human rhabdosarcoma cells. Rhesus macaque and baboon PBMC were screened in vitro for susceptibility to infection with SHIV9466.33. After successful infection of baboon PBMC, four animals were inoculated intravenously with SHIV9466.33 and monitored for plasma viral RNA, virus isolation from the PBMC, seroconversion, T-cell subsets, and signs of disease. RESULTS: SHIV9466.33 was able to infect PBMC from 12 out of 14 baboons. All four of the baboons selected for in vivo inoculation became infected. Peak plasma viral RNA levels of 8000 to 700,000 RNA copies/ml were measured at 2 weeks post-inoculation. Virus was isolated from the PBMC of all four baboons during acute infection, and all seroconverted. Although transient declines in CD4+ T-cells were observed during early infection, CD4+ levels remained within normal ranges thereafter. In contrast, in vitro cultures of PBMC of four rhesus macaques were not susceptible to infection with SHIV9466.33. CONCLUSION: SHIV9466.33 containing an HIV-1 subtype E envelope displayed tropism for baboon PBMC but not for rhesus macaque PBMC. This chimeric virus established infection and induced antiviral antibodies in baboons inoculated by the intravenous route with cell-free virus. Thus, infection of baboons with SHIV9466.33 will serve as an important animal model for future studies of HIV-1 vaccine efficacy.     

Oral immunization of macaques with attenuated vaccine virus induces protection against vaginally transmitted AIDS

The chimeric simian-human immunodeficiency virus SHIVKU-1, bearing the envelope of human immunodeficiency virus type 1 (HIV-1), causes fulminant infection with subtotal loss of CD4(+) T cells followed by development of AIDS in intravaginally inoculated macaques and thus provides a highly relevant model of sexually transmitted disease caused by HIV-1 in human beings. Previous studies using this SHIV model had shown that the vpu and nef genes were important in pathogenesis of the infection, and so we deleted portions of these genes to create two vaccines, DeltavpuDeltanefSHIV-4 (vaccine 1) and DeltavpuSHIVPPc (vaccine 2). Six adult macaques were immunized subcutaneously with vaccine 1, and six were immunized orally with vaccine 2. Both viruses caused infection in all inoculated animals, but whereas vaccine 1 virus caused only a nonproductive type of infection, vaccine 2 virus replicated productively but transiently for a 6- to 10-week period. Both groups were challenged 6 to 7 months later with pathogenic SHIVKU-1 by the intravaginal route. All four unvaccinated controls developed low CD4(+) T-cell counts (<200/microliter) and AIDS. The 12 vaccinated animals all became infected with SHIVKU-1, and two in group 1 developed a persistent productive infection followed by development of AIDS in one. The other 10 have maintained almost complete control over virus replication even though spliced viral RNA was detected in lymph nodes. This suppression of virus replication correlated with robust antiviral cell-mediated immune responses. This is the first demonstration of protection against virulent SHIV administered by the intravaginal route. This study supports the concept that sexually transmitted HIV disease can be prevented by parenteral or oral immunization.     

Deletion of nef slows but does not prevent CD4-positive T-cell depletion in human immunodeficiency virus type 1-infected human-PBL-SCID mice

The pathogenicity of four human immunodeficiency virus type 1 (HIV-1) isolates with nef deleted for SCID mice repopulated with human peripheral blood leukocytes (hu-PBL-SCID mice) was studied. Deletion of nef led to a substantial reduction in CD4-positive T-cell depletion and delayed kinetics of plasma viremia in infected hu-PBL-SCID mice. Deletion of the nef gene impacts both the efficiency of primary infection and the cytopathicity of virus for infected CD4-positive T cells in this animal model of HIV-1 infection.     

A humanized form of a CD4-specific monoclonal antibody exhibits decreased antigenicity and prolonged plasma half-life in rhesus monkeys while retaining its unique biological and antiviral properties

Certain monoclonal antibodies (MAbs) directed against CD4 can efficiently block HIV-1 replication in vitro. To explore CD4-directed passive immunotherapy for prevention or treatment of AIDS virus infection, we previously examined the biological activity of a nondepleting CD4-specific murine MAb, mu5A8. This MAb, specific for domain 2 of CD4, blocks HIV-1 replication at a post-gp120-CD4 binding step. When administered to normal rhesus monkeys, all CD4+ target cells were coated with antibody, yet no cell clearance or measurable immunosuppression occurred. However, strong anti-mouse Ig responses rapidly developed in all monkeys. In the present study, we report a successfully humanized form of mu5A8 (hu5A8) that retains binding to both human and monkey CD4 and anti-AIDS virus activity. When administered intravenously to normal rhesus monkeys, hu5A8 bound to all target CD4+ cells without depletion and showed a significantly longer plasma half-life than mu5A8. Nevertheless, an anti-hu5A8 response directed predominantly against V region determinants did eventually appear within 2 to 4 weeks in most animals. However, when hu5A8 was administered to rhesus monkeys chronically infected with the simian immunodeficiency virus of macaques, anti-hu5A8 antibodies were not detected. Repeated administration of hu5A8 in these animals resulted in sustained plasma levels and CD4+ cell coating with humanized antibody for 6 weeks. These studies demonstrate the feasibility of chronic administration of CD4-specific MAb as a potential means of treating or preventing HIV-1 infection.     

Animal model of mucosally transmitted human immunodeficiency virus type 1 disease: intravaginal and oral deposition of simian/human immunodeficiency virus in macaques results in systemic infection, elimination of CD4+ T cells, and AIDS

Chimeric simian/human immunodeficiency virus (SHIV) consists of the env, vpu, tat, and rev genes of human immunodeficiency virus type 1 (HIV-1) on a background of simian immunodeficiency virus (SIV). We derived a SHIV that caused CD4+ cell loss and AIDS in pig-tailed macaques (S. V. Joag, Z. Li, L. Foresman, E. B. Stephens, L. J. Zhao, I. Adany, D. M. Pinson, H. M. McClure, and O. Narayan, J. Virol. 70:3189-3197, 1996) and used a cell-free stock of this virus (SHIV(KU-1)) to inoculate macaques by the intravaginal route. Macaques developed high virus burdens and severe loss of CD4+ cells within 1 month, even when inoculated with only a single animal infectious dose of the virus by the intravaginal route. The infection was characterized by a burst of virus replication that peaked during the first week following intravenous inoculation and a week later in the intravaginally inoculated animals. Intravaginally inoculated animals died within 6 months, with CD4+ counts of <30/microl in peripheral blood, anemia, weight loss, and opportunistic infections (malaria, toxoplasmosis, cryptosporidiosis, and Pneumocystis carinii pneumonia). To evaluate the kinetics of virus spread, we inoculated macaques intravaginally and euthanized them after 2, 4, 7, and 15 days postinoculation. In situ hybridization and immunocytochemistry revealed cells expressing viral RNA and protein in the vagina, uterus, and pelvic and mesenteric lymph nodes in the macaque euthanized on day 2. By day 4, virus-infected cells had disseminated to the spleen and thymus, and by day 15, global elimination of CD4+ T cells was in full progress. Kinetics of viral replication and CD4+ loss were similar in an animal inoculated with pathogenic SHIV orally. This provides a sexual-transmission model of human AIDS that can be used to study the pathogenesis of mucosal infection and to evaluate the efficacy of vaccines and drugs directed against HIV-1.     

CD4 down-modulation during infection of human T cells with human immunodeficiency virus type 1 involves independent activities of vpu, env, and nef

The human immunodeficiency virus type 1 (HIV-1) genes vpu, env, and nef have all been implicated in modulating the levels of cell surface CD4 on infected cells. To quantitatively assess the relative contribution of each gene product to the regulation of CD4 during HIV infection of Jurkat T cells and peripheral blood mononuclear cells, we have developed an infectious HIV reporter system which expresses different combinations of these genes. To distinguish infected cells in the early or late stages of infection from uninfected cells, these viruses were designed to express human placental alkaline phosphatase with the kinetics of either early or late viral genes. Flow cytometry to detect placental alkaline phosphatase and CD4 in infected cells showed that vpu, env, and nef are independently capable of down-modulation of CD4. As predicted by their respective expression patterns, nef down-modulated CD4 rapidly during the early phase of virus infection whereas vpu and env functioned late in the infection. In both Jurkat cells and peripheral blood mononuclear cells, a combination of the three genes was more efficient than any one or two genes, demonstrating that all three genes are required to achieve maximal CD4 down-modulation. In primary cells, down-modulation of CD4 was less efficient than in Jurkat cells and there was a stronger dependence on nef function for reducing cell surface CD4. HIV therefore has three genes that are able to independently down-modulate CD4; together, they can eliminate the bulk of cell surface CD4.     

Construction, replication, and immunogenic properties of a simian immunodeficiency virus expressing interleukin-2

To study the effect of interleukin-2 (IL-2) on simian immunodeficiency virus (SIV) replication, pathogenesis, and immunogenicity, we replaced the nef gene of SIVmac239 by the IL-2 coding region. The virus, designated SIV-IL2, stably expressed high levels of IL-2 in cell culture. In comparison to SIVmac239, SIV-IL2 replicated more efficiently in peripheral blood mononuclear cells in the absence of exogenously added IL-2. To determine whether this growth advantage would be of relevance in vivo, four juvenile rhesus monkeys were infected with SIV-IL2 and four monkeys were infected with a nef deletion mutant of SIV (SIVdeltaNU). After a peak in the cell-associated viral load 2 weeks postinfection, the viruses could barely be isolated 3 to 7 months postinfection. Mean capsid antigen levels were higher in the SIV-IL2 group than in the nef deletion group 2 weeks postinfection. Viruses reisolated from the SIV-IL2-infected animals expressed high levels of IL-2 during the acute phase of infection. Deletions in the IL-2 coding region of SIV-IL2 were observed in two of the SIV-IL2-infected macaques 3 months postinfection. Urinary neopterin levels, a marker for unspecific immune stimulation, were higher in the SIV-IL2-infected macaques than in SIVdeltaNU-infected animals during the acute phase of infection. The SIV-specific T-cell-proliferative response and antibody titers were similar in both groups. Cytotoxic T cells directed against viral antigens were detected in all SIV-IL2-infected macaques and in two of the SIVdeltaNU-infected animals. Expression of IL-2 did not seem to alter the attenuated phenotype of nef deletion mutants fundamentally, although there might have been a slight increase in virus replication and immune stimulation during the acute phase of infection. Deletion of the viral IL-2 gene 3 months postinfection could be a consequence of a selective disadvantage due to local coexpression of viral antigen and IL-2 in the presence of an antiviral immune response.     

Common themes of antibody maturation to simian immunodeficiency virus, simian-human immunodeficiency virus, and human immunodeficiency virus type 1 infections

Characterization of virus-specific immune responses to human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) is important to understanding the early virus-host interactions that may determine the course of virus infection and disease. Using a comprehensive panel of serological assays, we have previously demonstrated a complex and lengthy maturation of virus-specific antibody responses elicited by attenuated strains of SIV that was closely associated with the development of protective immunity. In the present study, we expand these analyses to address several questions regarding the nature of the virus-specific antibody responses to pathogenic SIV, SIV/HIV-1 (SHIV), and HIV-1 infections. The results demonstrate for the first time a common theme of antibody maturation to SIV, SHIV, and HIV-1 infections that is characterized by ongoing changes in antibody titer, conformational dependence, and antibody avidity during the first 6 to 10 months following virus infection. We demonstrate that this gradual evolution of virus-specific antibody responses is independent of the levels of virus replication and the pathogenicity of the infection viral strain. While the serological assays used in these studies were useful in discriminating between protective and nonprotective antibody responses during evaluation of vaccine efficacy with attenuated SIV, these same assays do not distinguish the clinical outcome of infection in pathogenic SIV, SHIV, or HIV-1 infections. These results likely reflect differences in the immune mechanisms involved in mediating protection from virus challenge compared to those that control an established viral infection, and they suggest that additional characteristics of both humoral and cellular responses evolve during this early immune maturation.     

Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes

Clinical evidence suggests that cellular immunity is involved in controlling human immunodeficiency virus-1 (HIV-1) replication. An animal model of acquired immune deficiency syndrome (AIDS), the simian immunodeficiency virus (SIV)-infected rhesus monkey, was used to show that virus replication is not controlled in monkeys depleted of CD8+ lymphocytes during primary SIV infection. Eliminating CD8+ lymphocytes from monkeys during chronic SIV infection resulted in a rapid and marked increase in viremia that was again suppressed coincident with the reappearance of SIV-specific CD8+ T cells. These results confirm the importance of cell-mediated immunity in controlling HIV-1 infection and support the exploration of vaccination approaches for preventing infection that will elicit these immune responses.     

Quinolinic acid and lymphocyte subsets in the intrathecal compartment as biomarkers of SIV infection and simian AIDS

Cerebrospinal fluid (CSF) samples were collected from monkeys infected with SIVmac251 (SIV) or HIV-1/SIVmac chimeric viruses (SHIV(HXBc2) and SHIV(89.6P)) to investigate quinolinic acid (QUIN) levels in the intrathecal compartment. CSF levels of QUIN were elevated in the SIV-infected monkeys, especially in animals with end-stage disease, and in those infected with pathogenic SHIV(89.6P), but not after infection with the nonpathogenic construct SHIV(HXBc2). QUIN elevations occurred in association with reduced CD4+ and increased CD8+ lymphocytes, cellular alterations that were more pronounced in CSF than in the blood. These findings support the view that the intrathecal compartment provides a unique window on viral infection, and are in keeping with the a priori prediction that QUIN increases primarily in response to more pathogenic viral strains.     

Rapid and progressive CD4+ decline in a monkey infected with an SIV+HIV-1 chimeric virus

We previously constructed a simian immunodeficiency virus+human immunodeficiency virus type 1 (HIV-1) chimeric virus, NM-3rN to generate a pathogenic HIV-1 in macaque monkeys. During the in vivo passage of this virus in several monkeys, a viral strain, R43-56 was obtained which acquired a better replication ability in vivo. MM121, one of the three monkeys inoculated with the R43-56, showed weight loss, diarrhea and a rapid and continuous decrease in CD4+ lymphocytes at the moribund stage. An autopsy revealed generalized lymphadenopathy, dehydration, and ileocecal intussusception. In situ hybridization showed that the virus infection was in systemic lymphoid organs. We are presently monitoring the survivors to obtain candidates for a more virulent virus. R43-56 may be a better challenge virus and useful tool for human acquired immunodeficiency syndrome research.     

Function of human immunodeficiency virus type 1 nef gene product in virus replication

We comparatively analyzed the replication kinetics of wild-type (wt) and nef mutant human immunodeficiency virus type 1 (HIV-1) in several CD4-positive cell lines, in order to clarify the molecular function of Nef protein. The delayed growth of nef mutant virus was observed at the initial stage of replication in all cell lines examined. This phenomenon was greatly amplified in the absence of vpu gene. In order to determine the infection stage in viral replication cycle which is specifically affected on virus replication rate in the presence of the Nef protein, we first examined the difference between wt and nef mutant viruses in the virus production rate from transfected cells, and found that the both viruses were produced with equal efficiency. This result showed that Nef protein could be dispensable for virion production. Therefore, early infection stages were focused by single-round infection assay, and the nef mutant virus was found to be much less infectious than wt virus. This indicated that the effect of Nef protein was exhibited in the early phase of a virus replication cycle, during viral adsorption to integration. By entry assay using wt and nef mutant virions, it was revealed that the Nef protein was required for efficient viral entry. These data suggest that the Nef protein might play a role in efficient incorporation of the Env protein into the virions, leading to enhanced viral infectivity.     

Producer cell-dependent requirement of the Nef protein for efficient entry of HIV-1 into cells

A proviral nef gene mutant of human immunodeficiency virus type 1 (HIV-1) was evaluated for its defective early replication step. Virus stocks were prepared from six CD4-positive and -negative cell lines transfected with wild-type (wt) or the nef mutant clone and inoculated into two target CD4-positive cell lines to monitor the efficiency of viral entry process. The nef mutant virions produced in one cell line exhibited a severe defect in the entry process, although those produced in the other five cell lines were only slightly less efficient than the wt virions at entering into cells. These results have demonstrated that the HIV-1 Nef is critical for efficient viral entry in a producer cell-dependent manner.     

Loss of CD4+ T cells in human immunodeficiency virus type 1-infected chimpanzees is associated with increased lymphocyte apoptosis

Supportive evidence that apoptosis contributes to loss of CD4+ lymphocytes in human immunodeficiency virus type 1 (HIV-1)-infected humans comes from an apparent lack of abnormal apoptosis in apathogenic lentivirus infections of nonhuman primates, including HIV-1 infection of chimpanzees. Two female chimpanzees were inoculated, one cervically and the other intravenously, with HIV-1 derived from the LAI/LAV-1b strain, which was isolated from a chimpanzee infected with the virus for 8 years. Within 6 weeks of infection, both recipient chimpanzees developed a progressive loss of CD4+ T cells which correlated with persistently high viral burdens and increased levels of CD4+ T-cell apoptosis both in vitro and in vivo. Lymph nodes from both animals also revealed evidence of immune hyperactivation. Intermediate levels of T-cell apoptosis in both peripheral blood and lymph nodes were seen in a third chimpanzee that had been infected with the LAI/LAV-1b strain for 9 years; this animal has maintained depressed CD4/CD8 T-cell ratios for the last 3 years. Similar analyses of cells from 4 uninfected animals and 10 other HIV-1-infected chimpanzees without loss of CD4+ cells revealed no difference in levels of apoptosis in these two control groups. These results demonstrate a correlation between immune hyperactivation, T-cell apoptosis, and chronic loss of CD4+ T cells in HIV-1-infected chimpanzees, providing additional evidence that apoptosis is an important factor in T-cell loss in AIDS. Furthermore, the results show that some HIV-1 strains are pathogenic for chimpanzees and that this species is not inherently resistant to HIV-1-induced disease.     

Effects of peripheral nerve implants on the regeneration of partially and fully innervated urodele forelimbs

HIV-2 is less pathogenic and less transmissible than HIV-1. Recent research in relation to deletions in the HIV nef gene and to immune cross-reactions between infections by HIV-2, HIV-1 and simian immunodeficiency virus suggests that T cell recognition and the control of viral replication may be more efficient in HIV-2 infection than in HIV-1 infection. These insights may be crucial to the design of effective vaccines.     

A distinct African lentivirus from Sykes' monkeys

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