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Live-attenuated viruses derived from SIV and SHIV have provided the most consistent protection against challenge with pathogenic viruses, but concerns regarding their long-term safety and efficacy have hampered their clinical usefulness. We report a longitudinal study in which we evaluated the long-term safety and efficacy of DeltavpuSHIV(PPC), a live virus vaccine derived from SHIV(PPC). Macaques were administered two inoculations of DeltavpuSHIV(PPC), three years apart, and followed for eight years. None of the five vaccinated macaques developed an AIDS-like disease from the vaccine. At eight years, macaques were challenged with pathogenic SIV and SHIV. None of the four macaques with detectable cellular-mediated immunity prior to challenge had detectable viral RNA in the plasma. This study demonstrates that multiple inoculations of a live vaccine virus can be used safely and can significantly extend the efficacy of the vaccine, as compared to a single inoculation, which is efficacious for approximately three years.

Factors explaining why human immunodeficiency virus (HIV) enhances the risk of reactivated tuberculosis (TB) are poorly understood. Unfortunately, experimental models of HIV-induced reactivated TB are lacking. We examined whether cynomolgus macaques, which accurately model latent TB in humans, could be used to model pathogenesis of HIV infection in the lungs and associated lymph nodes. These experiments precede studies modeling the effects of HIV infection on latent TB. We infected two groups of macaques with chimeric simian-human immunodeficiency viruses (SHIV-89.6P and SHIV-KU2) and followed viral titers and immunologic parameters including lymphocytes numbers and phenotype in the blood, bronchoalveolar lavage cells, and lymph nodes over the course of infection. Tissues from the lungs, liver, kidney, spleen, and lymph nodes were similarly examined at necropsy. Both strains produced dramatic CD4(+) T cell depletion. Plasma titers were not different between viruses, but we found more SHIV-89.6P in the lungs. Both viruses induced similar patterns of cell activation markers. SHIV-89.6P induced more IFN-gamma expression than SHIV-KU2. These results indicate SHIV-89.6P and SHIV-KU2 infect cynomolgus macaques and may be used to accurately model effects of HIV infection on latent TB.

Morphine is known to prevent the development of cell-mediated immune (CMI) responses and enhance expression of the CCR5 receptor in monocyte macrophages. We undertook a study to determine the effect of morphine on the neuropathogenesis and immunopathogenesis of simian immunodeficiency virus (SIV) infection in Indian Rhesus Macaques. Hypothetically, the effect of morphine would be to prevent the development of CMI responses to SIV and to enhance the infection in macrophages. Sixteen Rhesus Macaques were divided into three experimental groups: M (morphine only, n = 5), VM (morphine + SIV, n = 6), and V (SIV only, n = 5). Animals in groups M and VM were given 2.5 mg/kg of morphine sulfate, four times daily, for up to 59 weeks. Groups VM and V were inoculated with SIVmacR71/17E 26 weeks after the beginning of morphine administration. Morphine prevented the development of enzyme-linked immunosorbent spot-forming cell CMI responses in contrast to virus control animals, all of which developed CMI. Whereas morphine treatment had no effect on viremia, cerebrospinal fluid viral titers or survival over the time course of the study, the drug was associated with a tendency for greater build-up of virus in the brains of infected animals. Histopathological changes in the brains of animals that developed disease were of a demyelinating type in the VM animals compared to an encephalitic type in the V animals. This difference may have been associated with the immunosuppressive effect of the drug in inhibiting CMI responses.

To explore the effects of antisense (AS) interleukin (IL)-4 on virus replication and CD8+ T-cell responses in lymph nodes and blood of macaques infected with simian human immunodeficiency virus, SHIV(89.6)P.Six macaques were inoculated with simian human immunodeficiency virus (SHIV(89.6)P). Seven days later, four of the animals were given 1 mg AS IL-4 plasmid complexed with Megafectin liposome, intravenously, and two of these received a second injection of the same material on day 9. All six macaques were killed at 2 weeks post infection (pi) and monitored for viral RNA and CD8+ T cells in blood and lymph nodes by real-time reverse transcriptase-polymerase chain reaction, flow cytometry and immunohistochemistry.In contrast to the lymph nodes from virus control animals, the lymph nodes of AS IL-4-treated animals had a significant reduction in viral loads and reduced depletion of cells from the nodes. There was an increase in CD8+ T cells in the nodes, and many of the cells expressed granzyme B, suggesting functional activation. This trend of virus reduction and increased CD8+ T cell numbers was also reflected in blood.The therapeutic effect of the AS IL-4 suggests indirectly that the acute immunosuppressive disease caused by SHIVs is mediated, in part, by IL-4 that causes enhanced virus replication by suppressing anti-viral CD8+ T-cell responses, and that this effect was reduced by treatment of the animals with AS IL-4.

Chemokines play a key role in the regulation of central nervous system disease. CXCL10 over-expression has been observed in several neurodegenerative diseases, including multiple sclerosis, Alzheimer's disease and HIV-associated dementia. More recent studies by others and us have shown that CXCL10 elicits apoptosis in fetal neurons. The mechanism of CXCL10-mediated neurotoxicity, however, remains unclear. In this study, we provide evidence for the direct role of Ca(2+) dysregulation in CXCL10-mediated apoptosis. We demonstrate that treatment of fetal neuronal cultures with exogenous CXCL10 produced elevations in intracellular Ca(2+) and that this effect was modulated via the binding of CXCL10 to its cognate receptor, CXCR3. We further explored the association of intracellular Ca(2+) elevations with the caspases that are involved in CXC10-induced neuronal apoptosis. Our data showed that increased Ca(2+), which is available for uptake by the mitochondria, is associated with membrane permeabilization and cytochrome c release from this compartment. The released cytochrome c then activates the initiator active caspase-9. This initiator caspase sequentially activates the effector caspase-3, ultimately leading to apoptosis. This study identifies the temporal signaling cascade involved in CXCL10-mediated neuronal apoptosis and provides putative targets for pharmaceutical intervention of neurological disorders associated with CXCL10 up-regulation.

Simian immunodeficiency virus strain smmPGm can induce neuropathology in macaques and is a model for the development of human HIV-related brain injury. For quantitative studies of proviral presence and expression in the central nervous system (CNS), we inoculated 8 macaques intravenously with the virus. Three animals were necropsied 2 to 4 weeks after development of infection, and we obtained lymphoid tissue biopsies from 5 animals before 5 weeks after infection. Peak plasma viral loads averaged 10 6.76 viral RNA Eq/mL at week 2, whereas cerebrospinal fluid viral loads peaked at 10 4.4 viral RNA Eq/mL. The proviral DNA loads and viral gag mRNA expression in tissues were quantified by real-time polymerase chain reaction. Two animals developed neurologic disease characterized by meningoencephalitis and meningitis. Proviral DNA levels in CNS tissues of these animals at necropsy revealed 10 5.1 and 10 3.4 copies/μg of DNA, respectively, whereas viral RNA expression in the CNS reached 100 to 1000 times higher levels than those seen in early necropsies. In sharp contrast, in 2 animals necropsied at later times without CNS disease, virus mRNA expression was not detected in any CNS tissue. Our results are consistent with the hypothesis that active virus expression in the CNS is strongly correlated with neurologic disease and that the event occurs at variable periods after infection.

Chemokines and cytokines play a critical role in HIV infection, serving both to modulate virus replication and to recruit target cells to the site of infection. Platelet-derived growth factor (PDGF), a mitogen and chemoattractant for a wide variety of cells, is secreted by macrophages. Since macrophages are the target cells for lentiviral infection in the brain and PDGF is a known inducer of macrophage chemoattractant protein-1 (MCP)-1, a potent chemokine closely associated with HIV encephalitis, we investigated the association of PDGF-B chain (PDGF-B) with encephalitis in macaques caused by simian human immunodeficiency virus (SHIV), a chimera of HIV and SIV. Northern blot analysis confirmed elevated expression of PDGF-B chain mRNA in the brains from encephalitic macaques. Validation of these in vivo studies was confirmed in rhesus macrophage cultures infected with SHIV(KU2) in which we demonstrated heightened expression of PDGF-B chain mRNA. Nuclear run-off analysis established transcriptional up-regulation of PDGF-B chain in virus-inoculated macrophage cultures. Reciprocally, addition of exogenous PDGF enhanced virus replication and MCP-1 expression in these cells. Inhibition of virus replication by tyrosine kinase inhibitor, STI-571, and by PDGF-B antisense oligonucleotides confirmed the specificity of the PDGF effect. Relevance of these findings was confirmed by analysis of archival brain tissue from SHIV encephalitic and non-encephalitic macaques for PDGF-B chain expression. PDGF-B chain protein expression was observed in the virus-infected cells in microglial nodules in the brains of SHIV-encephalitic macaques.

To detect the major sites of viral replication in immunodeficiency virus-infected individuals, we quantified proviral DNA and infectious viruses using quantitative PCR and a plaque assay, respectively, in various tissues of SHIV(KU-2)-infected monkeys in the early and AIDS stages of infection. Compared the quantity of infectious virus among PBMC and the lymphoid tissues, the mesenteric lymph node had the largest number of infectious viruses at the AIDS stage more than at the early stage of infection. These results suggested that the gastrointestinal tract was a major site of viral replication. In the brain, proviral DNA was detected at the early and AIDS stage of infection, but infectious viruses were detected at only the AIDS stage. Moreover, we analyzed the nucleotide sequences of the env V3 region in infectious virus clones isolated from each plaque. The viruses in the lymphoid tissues of the monkey that developed AIDS diverged from the inoculated virus and had the same three amino acid substitutions. However, the viruses in the brain were almost identical to the inoculated virus, suggesting that the virus entered the brain early after infection and persisted without replication and genetic diversion until the AIDS stage.

Comparative studies were performed to determine the neuropathogenesis of infection in macaques with simian human immunodeficiency virus (SHIV)89.6P and SHIV(KU). Both viruses utilize the CD4 receptor and CXCR4 co-receptor. However, in addition, SHIV89.6P uses the CCR5 co-receptor. Both agents are dual tropic for CD4+ T cells and blood-derived macrophages of rhesus macaques. Following inoculation into macaques, both caused rapid elimination of CD4+ T cells but they varied greatly in mechanisms of neuropathogenesis. Two animals infected with SHIV89.6P developed typical lentiviral encephalitis in which multinucleated giant cell formation, nodular accumulations of microglial cells, activated macrophages and astrocytes, and perivascular accumulations of mononuclear cells were present in the brain. Many of the macrophages in these lesions contained viral RNA. Three macaques infected with SHIV(KU) and killed on days 6, 11 and 18, respectively, developed a slowly progressive infection in the CNS but macrophages were not productively infected and there were no pathological changes in the brain. Two other animals infected with this virus and killed several months later showed minimal infection in the brain even though one of the two developed encephalitis of unknown etiology. The basic difference in the mechanisms of neuropathogenesis by the two viruses may be related to co-receptor usage. SHIV89.6P, in utilizing the CCR5 co-receptor, caused neuropathogenic effects that are similar to other neurovirulent primate lentiviruses.

Human immunodeficiency virus (HIV)-encephalitis results from a cascade of viral-host interactions that lead to cytokine and chemokine imbalance, which then leads to neuropathologic manifestations of the disease. These include macrophage/microglia activation, astrocytosis and neuronal dysfunction or death. As the molecular mechanisms of this process are poorly understood, we used Atlas human cytokine or cytokine receptor microarray analysis to highlight gene expression profiles that accompanied encephalitis in Simian human immunodeficiency virus (SHIV) 89.6P-infected macaques. Of the 277 genes screened, marked upregulation of monocyte chemoattractant protein-1, interferon-inducible peptide IP-10 and interleukin-4 were observed specifically in the encephalitic brains. These genes are collectively known to promote macrophage infiltration and activation and virus replication. In contrast, genes regulating neurotrophic functions, such as brain-derived neurotrophic factor were downregulated. We also found that some of the apoptosis genes were up- or down-regulated. These data provide a comprehensive spectrum of gene expression that underscores the two major clinical manifestations of this unique syndrome: enhanced virus replication in brain macrophages and dystrophic changes in neurons.

Neurological disease associated with lentiviral infection occurs mainly as a consequence of primary replication of the virus or a combination of the virus infection and replication of opportunistic pathogens in the central nervous system. Recent studies have shown that whereas the disease can be caused by CCR5 tropic viruses alone, its induction by CXCR4 (X4) tropic viruses occurred usually in association with infections caused by opportunistic pathogens and in the presence of a Th2 cytokine, interleukin (IL)-4. 1,2 Further, X4-mediated neurological disease developed preferentially in rhesus compared to pig-tailed macaques. Because macrophages are the target cells for lentiviral infection in the brain and because macrophage chemoattractant protein (MCP)-1 is one of the major chemokines that is closely associated with acquired immune deficiency syndrome (AIDS) dementia, we tested for correlations between MCP-1 production and virus tropism in macrophages from the two species of macaques. The studies showed that the higher susceptibility of rhesus macaques to X4 virus-mediated encephalitis correlated with heightened production of virus and MCP-1 in cultured macrophages from this species and that these effects were further enhanced with treatment with IL-4. However, the latter effect was restricted to macrophages infected with X4 viruses. IL-4 may therefore be a basic requirement for X4 viruses to cause central nervous system disease.

To evaluate the vaccine potential of SHIVs attenuated by deletion of viral accessory genes, seven rhesus macaques were sequentially immunized with Delta vpu Delta nefSHIV-4 (vaccine-I) followed by Delta vpuSHIV(PPC) (vaccine-II). Despite the absence of virological evidence of productive infection with the vaccine strains, based on analysis of infectivity among peripheral blood mononuclear cells (PBMC) of the vaccinated animals, all seven animals developed binding as well as neutralizing antibodies against both vaccine-I and -II. The animals also developed vaccine virus-specific CTLs that recognized homologous as well as heterologous pathogenic SHIVs and SIV, and also soluble inhibitory factors that blocked the in vitro replication of the vaccine strains and different challenge viruses. Virus-specific cellular and humoral responses were sustained throughout a 58-week prechallenge period. To model aspects of natural transmission, the animals received a mucosal (rectal) challenge, with a mixture of three challenge viruses, SHIV(KU), SHIV(89.6)P, and SIV(mac)R71/17E. Two mock-vaccinated control animals inoculated with the same mixture of challenge viruses developed large numbers of infectious PBMC, high plasma viremia, and precipitous loss of CD4(+) T cells. The control animals did not develop any immune responses and succumbed to AIDS between 6 and 7 weeks postchallenge. All seven vaccinated animals became infected with challenge viruses as indicated by the presence of infectious cells in the PBMC and/or viral RNA in plasma. However, peak plasma viremia in vaccinates was two to nearly five logs lower than in the control animals and later plasma viral RNA became undetectable in all vaccinates. Vaccinated animals maintained normal CD4(+) T cell levels throughout the study. Challenge with pathogenic viruses caused massive anamnestic responses as determined by quantitation of virus-specific CD4(+) and CD8(+) T cells by intracellular IFN-gamma staining, and these cells persisted for at least 74 weeks. The study is still in progress and at this time DNA of SIV has become undetectable in lymph nodes of six of the seven vaccinates, SHIV(89.6)P in five of the seven, and SHIV(KU) in three of the seven animals.

Use of the macaque model of human immunodeficiency virus (HIV) pathogenesis has shown that the accessory genes nef and vpu are important in the pathogenicity of simian immunodeficiency virus (SIV) and simian–human immunodeficiency virus (SHIV). We examined the ability of two nonpathogenic SHIVs, SHIV PPC and Δ vpuΔnef SHIV PPC , to gain pathogenicity by rapid serial passage in macaques. In this study, each virus was passaged by blood intravenously four times at 4-week intervals in macaques. Animals were monitored for 40 weeks for levels of CD4 T cells and quantitative measures of virus infection. Δ vpuΔnef SHIV PPC maintained a limited phase of productive replication in the four animals, with no loss of CD4 + T cells, whereas SHIV PPC became more pathogenic in later passages, judging by plasma viral load and viral mRNA in lymph nodes, infectious peripheral blood mononuclear cells and CD4 + T cell loss. The nef, LTR, and env of the SHIV PPC viruses underwent numerous mutations, compared to Δ vpuΔnef SHIV PPC . This study confirms the seminal role that nef, LTR, and vpu could play in regulation of pathogenesis of HIV infection.

Previous reports from our lab had shown that sera obtained from SIV mac -infected animals neutralized SIV mac infectivity in CD4 + T cells but failed to protect monkey primary macrophages from infection with the virus. However, the antibodies could inhibit completion of the viral life cycle in the macrophages at the postentry stage(s). In this report we examined the mechanisms of the late effect of the antibodies. Using monoclonal antibodies (MAbs), we demonstrated that only antibodies to the SIV envelope protein (KK17 and KK42) but not antibody to the viral core protein (FA2) had the same inhibitory effect as that of the anti-SIV sera. To identify the stage of the viral replication cycle that was inhibited by anti-SIV antibodies in macrophages, we used various PCR techniques to study viral entry/reverse transcription (by amplifying the viral gag gene), viral genome nuclear transport (by amplifying 2-LTR circular forms), viral integration (by Alu-PCR assay), and viral protein expression (by RIPA). We found that in macrophage cultures inoculated with SIV mac 251 that were preincubated with antienvelope MAbs, viral DNA was detected at 8 h postinoculation but the 2-LTR circular forms and integrated viral DNAs were undetectable, and viral proteins were not expressed in these infected macrophages. These results strongly suggested that anti-SIV antibodies inhibited SIV mac replication in macrophages by blocking nuclear transport of viral genomes since viral DNA could not be detected in the nuclei of treated cultures. Furthermore, we showed that although viral replication in macrophages was interrupted by the antibodies, when cocultured with permissive T cells, the viral genomes presented in the cytoplasm of the macrophages could readily transfer to T cells during cell–cell contact. Importantly, this transfer could not be prevented by the antibodies. These results might explain the failure of passive antibody immunization against SIV mac 251—a critical obstacle in AIDS vaccine development.

Four rhesus macaques were sequentially immunized with live vaccines Δ vpu Δ nef SHIV-4 (vaccine-I) and Δ vpu SHIV PPC (vaccine-II). The vaccine viruses did not replicate productively in the peripheral blood mononuclear cells (PBMCs) of the vaccinated animals. All four animals developed binding antibodies against both the vaccine-I and -II envelope glycoproteins but neutralizing antibodies only against vaccine-I. They developed vaccine virus-specific CTLs that also recognized homologous as well as heterologous pathogenic SHIVs. Thirty weeks after the last immunization, the vaccinated animals and three unvaccinated control animals were challenged iv with a highly virulent heterologous SHIV 89.6 P. As expected, the three unvaccinated control animals developed large numbers of infectious PBMCs, high plasma viremia, and precipitous loss of CD4 + T cells. Two controls did not develop any immune response and succumbed to AIDS in about 6 months. The third control animal developed neutralizing antibodies and had a more chronic disease course, but eventually succumbed to AIDS-related complications 81 weeks after inoculation. The four vaccinated animals became infected with challenge virus as indicated by the presence of challenge virus-specific DNA in the PBMCs and RNA in plasma. However, virus in these animals replicated approximately 200- to 60,000-fold less efficiently than in control animals and eventually, plasma viral RNA became undetectable in three of the four vaccinates. The animals maintained normal CD4 + T-cell levels throughout the observation period of 85 weeks after a transient drop at Week 3 postchallenge. They also maintained CTL responses throughout the observation period. These studies thus showed that the graded immunization schedule resulted in a safe and highly effective long-lasting immune response that was associated with protection against AIDS by highly pathogenic heterologous SHIV 89.6 P.

In an in vitro coculture model of monocyte-derived, cultured human dendritic cells (DC) with autologous CD4(+) resting T cells, CCR5 (R5)-tropic strains of HIV-1, but not CXCR4 (X4)-tropic strains, were transmitted to resting CD4+ T cells, leading to prolific viral output, although DC were susceptible to infection with either strain. Macrophages, which were also infectable with either R5- or X4-tropic strains, did not transmit infection to CD4+ cells. Highly productive HIV infection in this model appeared to be a consequence of heterokaryotic syncytium formation between infected DC and T cells since syncytia formation developed only in R5-infected DC/CD4+ cocultures. These results suggested that the unique microenvironment derived from the fusion between the infected DC and CD4+ cell was highly permissive and selective for replication of R5-tropic viruses. The apparent selectivity for R5-tropic strains in such syncytia was attributable neither to differential DC-mediated activation nor to selective modulation of induction of alpha- or beta-chemokines in the infected DC. This model of HIV replication may provide useful insights into in vitro correlates of HIV pathogenicity.

Vpr is an HIV-1 auxiliary regulatory protein packaged in the virion. It has been shown to enhance the nuclear transport of the HIV-1 pre-integration complex, activate transcription of cellular and viral promoters, and arrest the cell cycle at the G2/M check-point. We previously identified a cellular protein of 180 kDa (RIP) that interacted with HIV-1 Vpr specifically. We now rename this cellular protein as Vpr-binding protein, or VprBP. In this report, we describe the cloning of the VprBP cDNA that encodes 1507 aa residues and is identical to the previously cloned cDNA KIAA0800. We demonstrate that Vpr specifically interacts with recombinantly expressed VprBP in vitro as well as in vivo. Furthermore, Vpr interacts with the cellular endogenous VprBP in the context of the HIV-1 life cycle. Mutational analysis of VprBP suggests that the Vpr binding domain is located within the C-terminal half of VprBP, which has a Pro-rich domain and several Phe-x-x-Phe repeats. Subcellular fractionation studies show that both the endogenous VprBP and the adenovirus-expressed VprBP are distributed predominantly in the cytoplasmic fraction. Consistent with previous reports, the adenovirus-expressed Vpr is distributed in both the cytoplasmic and the nuclear fractions. However, when VprBP and Vpr are expressed together, Vpr is found almost exclusively in the cytoplasm. Expression of VprBP does not affect the nuclear transport of the adenoviral nuclear protein, pTP. VprBP expressed in insect cells also blocks the nuclear transport of a Vpr-GFP fusion protein, and VprBP mutants incapable of interacting with Vpr fail to block Vpr-GFP nuclear transport. We hypothesize that Vpr interaction with VprBP may cause changes in the host cell cytoplasm that affect HIV-1 pathogenesis as well as HIV-1 replication.

The simian human immunodeficiency virus (SHIV) macaque model of AIDS has provided a very useful system for evaluation of envelope-based candidate vaccines against HIV-1. Eight rhesus macaques were immunized with monomeric recombinant gp120 of HIV-1(LAI) (rgp120) and used to evaluate whether this vaccine conferred protection against challenge with pathogenic SHIVs (SHIV(KU-2) and SHIV(89.6)P). The vaccinated macaques developed high titers of antibodies against rgp120 that reacted efficiently with the envelope proteins of homologous SHIV (SHIV(KU-2)) and poorly with the SHIV(89.6)P envelope, a heterologous strain of SHIV. This vaccine also induced neutralizing antibodies but only against SHIV(KU-2). Vaccine-induced antibodies were of high avidity and predominantly against linear epitopes on the protein. Vaccinated macaques developed gp120-specific T-helper cells but no consistent cytotoxic T lymphocytes. However, cellular immune responses were short-lived in all eight vaccinates. At week 22 postimmunization, four vaccinates were challenged with SHIV(KU-2) and the other four with SHIV(89.6)P. Four unvaccinated control macaques were also infected: two with SHIV(KU-2) and two with SHIV(89.6)P. Vaccinated macaques generally showed anamnestic antibody and T-helper cell responses. However, T-helper responses were again short-lived. Upon challenge, the level of productive virus replication was indistinguishable between vaccine and control groups, suggesting that rgp120 did not confer protection against virus replication when animals were challenged with homologous or heterologous SHIV viruses.

The pattern of neurological disease caused by human immunodeficiency virus (HIV) infection of the central nervous system (CNS) was investigated using a macaque model of acquired immune defiency syndrome (AIDS). Seven of nine macaques inoculated with neurovirulent simian imunodeficiency virus (SIV mac ) developed AIDS within 3 months. Four of these had clinically obvious neurological disease and extensive conduction defects in the form of latency increases in evoked potential (EP) responses. Neuropathologically, all four animals had disseminated white matter disease in the form of multifocal, perivascular and nodular parenchymal mononuclear cell infiltrates, along with extensive involvement of the cortical grey matter, leptomeninges and intracranial portions of cranial nerves. A brisk multinucleated giant cell (MGC) response was a frequent accompaniment in the affected areas. Three of the animals in this group also showed spongiform vacuolation in the occipital grey matter, a lesion described only rarely in HIV encephalitis. In the remaining three animals, there was only minimal evidence of overt neurological impairment or conduction defects. These animals had only mild to moderate neuropathological changes and lesions were virtually confined to the white matter regions of the brain. MGC responses were rare or absent in the CNS of these animals. Neuropathological findings in this SIV mac model have therefore shown good correlation with the severity of clinical and neurophysiological changes, and are reminiscent of HIV-1 encephalitis. More importantly, white matter involvement was a consistent finding in the affected macaques, regardless of the duration and severity of disease, or type of virus inoculated, suggesting an unusual susceptibility for lentiviral infection in these regions of the macaque CNS.

Previous work using bone marrow passaged SIVmac239 (simian immunodeficiency virus) has shown that macrophage tropic strains of this virus enter the rhesus macaque brain early following inoculation (Sharma et al, 1992; Desrosiers et al, 1991; Zhu et al, 1995; and Narayan et al, 1997). As part of an effort to more fully characterize the extent of neurologic impairment associated with SIV infection of the brain, we used transcranial electrical stimulation of motor cortex and the spinal cord to evoke EMG potentials in two forelimb (EDC and APB) and two hindlimb (LG and AH) muscles. The latencies, magnitudes and thresholds of motor evoked potentials (MEPs) recorded from nine monkeys infected with neurovirulent SIVmac R71/17E were compared to pre-inoculation records from the same monkeys. Seven of nine monkeys developed simian AIDS within 4 months of inoculation and were euthanized. Two monkeys remained free of AIDS-related clinical illness for over 18 months following inoculation. Six of the seven monkeys with rapidly progressing disease showed post-inoculation latency increases (or = 2 s.d. of control) in at least one cortical MEP. Increases in cortical MEP latency ranged from 21-97% in different monkeys. All seven rapidly progressing animals showed post-inoculation increases in at least one spinal cord MEP latency. Maximum spinal cord MEP latency increases ranged from 22-147%. Increases in central conduction time (CCT) ranged up to 204% and exceeded two standard deviations of control in four monkeys. Neither of the two monkeys with slowly progressing disease showed significant increases in either cortical or spinal cord MEP latency or CCT. Only the monkeys with rapidly progressing disease exhibited classic AIDS-related neuropathology, although there was no consistent relationship between the severity of neuropathology and the extent of MEP abnormalities. In conclusion, our results demonstrate clear deficits in the functional integrity of both central and peripheral motor system structures associated with SIV infection and further support the use of SIV-infected rhesus macaques as a model of neuro-AIDS.

We previously showed that inoculation of rhesus macaques with molecularly cloned lymphocytetropic simian immunodeficiency virus (SIVmac239) results in SIV-associated nephropathy (SIVAN) and that the glomerulosclerotic lesions were associated with the selection of macrophagetropic (M-tropic) variants (V. H. Gattone et al., AIDS Res. Hum. Retroviruses 14:1163–1180, 1998). In the present study, seven rhesus macaques were inoculated with M-tropic SIVmacR71/17E, and the renal pathology was examined at necropsy. All SIVmacR71/17E-infected macaques developed AIDS, and most developed other systemic complications, including SIV-induced encephalitis and lentivirus interstitial pneumonia. There was no correlation between the length of infection (42 to 97 days), circulating CD4+T-cell counts, and renal disease. Of the seven macaques inoculated with SIVmacR71/17E, five developed significant mesangial hyperplasia and expansion of matrix and four were clearly azotemic (serum urea nitrogen concentration of 40 to 112 mg/dl). These same five macaques developed focal segmental to global glomerulosclerotic lesions. Increased numbers of glomerular CD68+cells (monocytes/macrophages) were found in glomeruli but not the tubulointerstitium of the macaques inoculated with SIVmacR71/17E. All macaques had glomerular deposits of immunoglobulin G (IgG), IgM, and tubuloreticular inclusions, and six of seven had IgA deposition. However, there was no correlation between the presence of circulating anti-SIVmacantibodies, immunoglobulin deposition, and glomerular disease. Tubulointerstitial infiltrates were mild, with little or no correlation to azotemia, while microcystic tubules were evident in those with glomerulosclerosis or azotemia. The four most severely affected macaques were positive for diffuse glomerular immunostaining for viral core p27 antigen, and there was intense staining in the glomeruli of the two macaques with the most severe glomerulosclerosis. Viral sequences were isolated from glomerular and tubulointerstitial fractions from macaques with severe glomerulosclerosis but only from the tubulointerstitial compartment of those that did not develop glomerulosclerosis. Interviral recombinant viruses generated withenvsequences isolated from glomeruli confirmed the M-tropic nature of the virus found in the glomeruli. The correlation between the increased number of CD68+cells (monocytes/macrophages) in the glomeruli, the localization of p27 antigen in the glomeruli, and the glomerular pathology confirms and extends our previous observations of an association between glomerular infection and infiltration by M-tropic virus and SIVAN.

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 thevpu and nef genes were important in pathogenesis of the infection, and so we deleted portions of these genes to create two vaccines, ΔvpuΔnefSHIV-4 (vaccine 1) and ΔvpuSHIVPPc (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 (KU-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.

Recently, we developed a highly pathogenic variant of simian–human immunodeficiency virus, SHIV-4 (containing the tat, rev, vpu, and env of the HXB2 strain of HIV-1 in a genetic background of SIV mac 239), through a series of four bone marrow-bone marrow passages—first in rhesus monkeys and then in pig-tailed macaques [Joag et al. (1996) J. Virol. 70, 3189–3197]. Inoculation of pig-tailed macaques with this pathogenic virus (SHIV KU-1 ) causes subtotal elimination of CD4 + T cells and fatal opportunistic infections, usually within 6 months. Genetic characterization of SHIV KU-1 showed that it has a functional vpu gene (the first codon is ATG vs ACG for the vpu of SHIV-4) and several amino acid substitutions in Env and nef [Stephens et al. (1997) Virology 231, 313–321]. Two pig-tailed macaques, PPc and PQc, were the first to develop a severe loss of CD4 + T cells and the acquired immune deficiency syndrome and were euthanized at 26 and 105 weeks, respectively. In this report, we analyzed the changes that occurred in the vpu, nef, and env (gp120) genes of the virus used to inoculate macaques PPc and PQc and established the chronology of changes that occurred in these viral genes as these two animals lost their CD4 + T cells and progressed to develop acquired immune deficiency syndrome. Compared with SHIV-4, the virus used to inoculate macaques PPc and PQc had 0, 3, and 0 consensus amino acid changes in the Vpu, gp120, and Nef, respectively. An analysis of the viral sequences amplified from peripheral blood mononuclear cells samples taken at various times after inoculation of PPc revealed that the vpu had not reverted to an open reading frame (closed vpu, ACG) at 4 weeks after inoculation, but by 16 weeks vpu had reverted to an open reading frame (open vpu, ATG). Macaque PQc, which had a longer course of disease, had a closed vpu at 4 and 16 weeks, but by 28 weeks, both closed and open vpu were detected. From 39 to 105 weeks, only an open vpu was detected. In both macaques, the reversion to an open vpu correlated well with the second phase (major) of CD4 + T cell loss. An analysis of the nef and env sequences isolated from the same times after inoculation revealed an association between the reversion of vpu to an open reading frame and the accumulation of increased numbers of consensus changes in these two viral proteins. These data suggest that the concomitant reversion of vpu to an open reading frame along with increased substitutions in Nef and gp120 were important genetic changes in the viral genome that were responsible for the increased and highly efficient rate of replication of the virus in CD4 + T cells and macrophages, which in turn led to elimination of the CD4 + T cells and profound loss of immunocompetence in the infected animals.

We examined the renal pathology and viral genetic changes following inoculation of six rhesus macaques with lymphocyte-tropic SIVmac239. Portions of the renal cortex were sieved into glomerular and tubulointerstitial (TI) fractions and examined for SIVmac sequences by PCR and for p27 core antigen. SIVmac sequences were detected in renal tissue from five of six macaques (three of five glomerular and five of five TI fractions were positive for SIV by PCR). Glomerulosclerosis (segmental and global) was evident in two macaques that were positive for env sequences in the glomerular fractions. Diffuse mesangial hyperplasia and matrix expansion were present in all three animals with glomerular SIV, as was an increase in glomerular collagen I and collagen IV. Tubulointerstitial inflammation was evident in all virus-inoculated macaques. The TI infiltration of CD68+ cells was most pronounced in the animals with SIVmac present in the glomerulus. All SIVmac-infected macaques exhibited increased glomerular deposition of IgM and to a lesser extent IgG, but no C3 or IgA was evident. Sequence analyses of the viral env gene (gp120) isolated from the glomerular and TI fractions of a macaque that developed glomerulopathy revealed the presence of specific viral variants in glomerular and TI fractions. In addition, chimeric viruses constructed with glomerular but not tubulointerstitial gp120 sequences were converted to a macrophage-tropic phenotype. These results indicate that infection by lymphocyte-tropic SIVmac239 is primarily associated with immunoglobulin deposition in the glomerulus and suggests that when glomerulosclerosis develops there is selection of viral variants that are macrophage tropic in nature.

By subcutaneous inoculation of SHIV(KU-2) in the hands of macaques, we developed a model of human immunodeficiency virus type-1 (HIV-1) occupational infection due to needle-stick injury and used the model to determine whether neutralizing serum to SHIV administered before or after virus inoculation could either prevent or abort infection, respectively. Six rhesus macaques were given 15 ml/kg pooled anti-SHIV plasma and challenged 24 hr later with approximately 300 animal infectious doses of SHIV(KU-2), subcutaneously. Three of the six macaques completely resisted infection with SHIV(KU-2). A fourth animal failed to yield infectious virus, but DNA extracted from its peripheral blood mononuclear cells (PBMC) and lymph nodes had viral sequences. Partial resistance was noted in the other two animals because virus recovery was delayed compared with the control animals. In contrast, six of six macaques given the same dose of anti-SHIV plasma 18 hr after exposure to virus became infected, as did two of two macaques given anti-SHIV plasma only 2 hr after exposure to virus. Our results suggest that neutralizing antibodies may have a prophylactic but not a therapeutic role in HIV-1 infections.

Brains of macaques inoculated with macrophage-tropic, neurovirulent virus 7F, with lymphocyte-tropic SIV mac239, or with dual-tropic SIVmac239/1yE, were examined for microglial activation, astrocyte activation, apoptosis and neuron loss. The brain one animal inoculated with neurovirulent virus 7f showed massive microglial activation as assessed by expression of the major histo-compatibility complex class II (MHC-II). In this animal very numerous, large microglial nodules expressing MHC-II were concentrated in the basal pons and internal capsule. These microglial nodules contained cells undergoing apoptosis detected by in situ end labeling of fragmented DNA. In this animal, neuron loss was apparent near the microglial nodules. In the animals inoculated with SIVmac239 or SIVmac239/17E, pathologic changes such as perivascular cuffing and formation of microglial nodules were absent. However, increased expression of MHC-11 by microglial cells was also concentrated in white matter of the basal pons, midbrain and internal capsule. These results indicate the microglial activation in SIV-infected macaques follows a ventral to dorsal gradient regardless of viral tropism. These results also show that the type and severity of neuropathological changes in SIV-infected macaques is highly dependent on the tropism of the inoculated virus.

SIV smm PBj14 is a highly pathogenic lentivirus which causes acute diarrhea, rash, massive lymphocyte proliferation predominantly in the gastrointestinal tract, and death within 7 to 14 days. In cell culture, the virus has mitogenic effects on resting macaque T lymphocytes. In contrast, SIV mac 239 causes AIDS in rhesus macaques, generally within 2 years after inoculation. In a previous study, replacement of amino acid residues 17 and 18 of the Nef protein of SIV mac 239 with the corresponding amino acid residues of the Nef protein of SIV smm PBj14 yielded a PBj-like virus that caused extensive activation of resting T lymphocytes in cultures and acute PBj-like disease when inoculated into pig-tailed macaques. This study suggested that nef played a major role in both processes. In this study, we replaced the nef /long terminal repeat (LTR) region of a nonpathogenic simian-human immunodeficiency virus (SHIV), SHIV PPc , with the corresponding region from SIV smm PBj14 and examined the biological properties of the resultant virus. Like SIV smm PBj14, SHIV PPc PBjnef caused massive stimulation of resting peripheral blood mononuclear cells (PBMC), which then produced virus in the absence of extraneous interleukin 2. However, when inoculated into macaques, the virus failed to replicate productively or cause disease. Thus, while these results confirmed that the nef /LTR region of SIV smm PBj14 played a major role in the activation of resting PBMC, duplication of the cellular activation process in macaques may require a further interaction between nef and the envelope glycoprotein of simian immunodeficiency virus because SHIV, containing the envelope of human immunodeficiency virus type 1, failed to cause activation in vivo.

By animal to animal passage in rhesus and pig-tailed macaques, we developed a rhesus model of HIV-1 disease in humans. Rhesus macaques infected with a cell-free stock of SHIVKU-2 developed CD4+ T cell loss, primary lentiviral encephalitis and pneumonia, and AIDS. Six of nine rhesus macaques died within eight months post-inoculation, while the remaining three are at five, five, and eight months post-inoculation, respectively. Animals infected by either mucosal or parenteral routes of infection had a similar course of infection.

We recently reported that a chimeric simian/human immunodeficiency virus (SHIV(KU‐1)) developed in our laboratory caused progressive depletion of CD4(+)T lymphocytes and AIDS within 6 months of inoculation into pig‐tailed macaques (M.nemestrina). None of the pig‐tailed macaques showed productive SHIV infection in the central nervous system (CNS). In this report, we show that by further passage of the pathogenic virus in rhesus macaques [M. mulatta], we have derived a new strain of SHIV (SHIV(KU‐2)) that has caused AIDS and productive CNS infection in 3 of 5 rhesus macaques infected with the virus. Productive replication of SHIV in the CNS was clearly shown by high infectivity titers and p27 protein levels in brain homogenates, and in 2 of the 3 rhesus macaques this was associated with disseminated, nodular, demyelinating lesions, including focal multinucleated giant cell reaction, largely confined to the white matter. These findings were reminiscent of HIV‐1 associated neurological disease, and our immunohistochemical and in situ hybridization data indicated that the neuropathological lesions were associated with the presence of SHIV‐specific viral antigens and nucleic acid respectively. However, the concomitant reactivation of opportunistic infections in these macaques suggested that such pathogens may have influenced the replication of SHIV in the CNS, or modified the neuropathological sequelae of SHIV infection in the rhesus species, but not in pig‐tailed macaques. Our findings in the two species of macaques highlight the complexities of lentiviral neuropathogenesis, the precise mechanisms of which are still elusive.

Image texture analysis is used in a wide variety of applications in medical research. Neurovirulent simian immunodeficiency virus (SIV) infection in monkeys is considered a good model for HIV-1 infection in humans and causes neuropathological changes in white matter which can include diffuse myelin pallor, subtle white matter astrocytosis, perivascular macrophage infiltrates, and microglial nodules with multinucleated giant cells. The ability of image texture analysis to quantify these changes was evaluated. Sections of thionin-stained brain tissue from eight male rhesus macaques ranging in age from 42-59 months were used. Four animals served as controls and four animals were infected with neurovirulent SIVmac239/17E-R71 by bone marrow inoculation. Images of cerebral white matter were captured and analyzed by calculating 13 textural features based on statistical analysis of spatial co-occurrence matrices. Statistical analysis of the results included multiple comparisons using the Newman-Keuls multiple range test. The effect of variation in background illumination used at image acquisition was also evaluated. Ten of the 13 textural features used in this study successfully discriminated between tissue from control and SIV-infected animals and were consistent with independent neuropathological assessment. Three textural features were highly sensitive to variation in background illumination and found not useful in this application.

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

SIVmac251 and its closely related derivatives SIVmac239 and SIVmac239/17E vary greatly in their susceptibility to neutralization with homologous and heterologous antisera. Whereas SIVmac251 induces homologous neutralizing antibodies, the antibodies induced by SIVmac239 rarely neutralize infectivity of this virus in lymphocyte cultures. In contrast, SIVmac239/17E is remarkably susceptible to neutralization with homologous and heterologous antisera induced by other strains of SIVmac. In this study, we studied the molecular basis for the neutralization of SIVmac239/17E. Using chimeric viruses in which different regions of theenvgene of both SIVmac239 and SIVmac239/17E were inserted into a background of either of the parental genomes, we showed that the newly acquired neutralization properties of SIVmac239/17E were attributable to amino acid substitutions between the V2 and V4 regions of gp120. Site-directed mutagenesis of theenvgene in this region showed that the arginine substitutions at positions 334 and/or 340 within the “V3” domain were fundamental to virus neutralization but other substitutions in the V2–V4 region added to the ease of its neutralization since it became neutralizable with much higher dilutions of serum. The molecular determinants for neutralization of this virus are distinct from those reported as responsible for neutralization of SIVmac251 and both are distinct from SIVmac239.

By animal-to-animal passage of simian/human immunodeficiency virus (SHIV) in pig-tailed macaques, we have developed a macaque model of human immunodeficiency virus type 1 (HIV-1) disease in humans. Passaging was begun with a chimeric virus containing the env gene of HIV-1 HXBc2 and the gag and pol genes of simian immunodeficiency virus SIVmac239. SHIV was passaged serially in cohorts of two macaques each, using bone marrow-to-bone marrow transfers at 5, 5, and 16 weeks for passages 2, 3, and 4, respectively. The fifth passage was done by using cell-free virus isolated from cerebrospinal fluid of a passage 4 macaque. The virus became more virulent with each passage. Virus replication was restricted in all three animals in passages 1 and 2 but not in five of the six animals in passages 3, 4, and 5. In these animals, intense virus replication in the lymphoid tissues resulted in almost total elimination of CD4+ T cells within weeks of inoculation, and three of these animals developed AIDS in less than 1 year. The more uniform virus-host interaction initiated by the cell-free virus in the passage 5 animals contrasted with a more variable pattern of disease initiated by infectious bone marrow cells during earlier passages. The virulent cell-free SHIV can now be used to screen the efficacy of vaccines directed against the envelope of HIV-1.

Human immunodeficiency virus type 1 (HIV-1) replicates productively in vitro in CD4(+)-T cells and/or macrophages. In the host, however, HIV-1 replication may be restricted by the quiescence of susceptible cells. Vpr is a 15-kDa late viral gene product, which is assembled in the virion and suspected to enhance HIV-1 replication in the infected host. We demonstrated previously that Vpr interacted specifically with the cellular transcription factor Sp1, and activated transcription from the HIV-1 long-terminal-repeat. Both Vpr-Sp1 interaction and trans-activation by Vpr required a central Leu/Ile-rich domain (LR domain, aa 60-81) in Vpr. This domain of Vpr was also found critical for Vpr interaction with another cellular protein of 180 kDa. We now provide biochemical evidence that the Vpr LR-domain has a leucine-zipper-like structure. The leucine-zipper structure has been found in a variety of cellular transcription factors, which use the leucine-zipper domain to form a specific dimer before they can bind to DNA through an upstream basic domain. The LR domain of HIV-1 Vpr, when fused to the basic domain of the cellular transcription factor CREB, was capable of supporting specific DNA binding by the CREB basic domain. Point mutational analysis of the Leu/Ile residues in the LR domain suggested that multiple Leu/Ile residues may be involved in maintaining the leucine-zipper-like structure. Mutagenesis in the context of the full-length Vpr also helped identify Leu/Ile residues may be involved in maintaining the leucine-zipper-like structure. Mutagenesis in the context of the full-length Vpr also helped identify Leu/Ile residues critical for Vpr interaction with the cellular 180-kDa protein. These results suggested that the leucine-zipper-like domain may be an important functional determinant for HIV-1 Vpr.

Since the pathogenesis of SIV mac disease complex is thought to be explained by the tropism of the infecting virus for either CD4 + T-lymphocytes or macrophages or both types of cells, we compared the infection in primary macaque macrophages with molecularly cloned, lymphocyte-tropic SIV mac 239 and a cloned, macrophage-tropic chimeric virus (SIV mac 239/17E) whose env gene was derived from brain of a macaque (17E) dying from SIV-induced encephalopathy. SIV mac 239/17E caused a productive, syncytial cytopathic infection accompanied by accumulation of virus particles within cytoplasmic esicles of the macrophages. Pulse-chase and immune precipitation studies showed that both the viral glycoprotein precursor (gp160) and the gag precursor (p57) were cleaved into gp120 and p27, respectively, and both were released into the culture medium of infected cells, although most of the p27 remained cell associated. SIV mac 239 also infected macrophages, but in comparison to SIV mac 239/17E, minimal virus replication occurred. Immunocytostaining revealed that while occasional syncytia were observed in cultures, the majority of the infected cells were not associated with syncytium formation. Ultrastructural studies did not reveal the accumulation of virions within infected macrophages. Pulse-chase studies showed that both gp160 and p57 were produced but were cleaved inefficiently and only minimal amounts of gp120 and p27 were released into the culture medium, even after prolonged incubation times. The processing of proteins of the two viruses was indistinguishable in lymphocytes. Since these two viruses are identical except for changes within the env gene, these results indicate that efficient assembly and release of SIV from blood-derived macrophages is mediated by changes in the envelope glycoprotein.

International audience; Increasing the safety and the efficacy of existing HIV vaccines is one of the strategies that could help to promote the development of a vaccine for human use. We developed a HIV DNA vaccine (Delta 4-SHIVKU2) that has been shown to induce potent polyfunctional HIV-specific T cell responses following a single dose immunization of mice and macaques. Delta 4-SHIVKU2 also induced protection when immunized macaques were challenged with homologous pathogenic viruses. In the present study, our aim was to examine whether a chimeric HIV DNA vaccine (CAL-Delta 4-SHIVKU2) whose genome is driven by the LTR of the goat lentivirus, caprine arthritis encephalitis (CAEV) expresses efficiently the vaccine antigens and induces potent immune responses in animal models for HIV vaccine. Data of radioimmunoprecipitation assays clearly show that this chimeric genome drives efficient expression of all HIV antigens in the construct. In addition, evaluation of the p24 Gag protein in the supernatant of HEK-293-T cells transfected in parallel with Delta 4-SHIVKU2 and CAL-Delta 4-SHIVKU2 showed no difference suggesting that these two LTRs are inducing equally the expression of the viral genes. Immunization of mice and macaques using our single dose immunization regimen resulted in induction of similar IFN-gamma ELISPOT responses in Delta 4-SHIVKU2- and CAL-Delta 4-SHIVKU2-treated mice. Similar profiles of T cell responses were also detected both in mice and macaques when multiparametric flow cytometry analyses were performed. Since CAEV LTR is not dependent of Tat to drive viral gene expression and is not functional for integration with HIV integrase, this new vector increases the safety and efficacy of our vaccine vectors and vaccination strategy. (C) 2012 Elsevier Ltd. All rights reserved.

The human immunodeficiency virus, type 1 (HIV-1) genome encodes a 15-kDa accessory gene product, Vpr, that is essential for virus replication in primary monocytes/macrophages. Being present in the virion, Vpr is believed to function in the early phases of HIV-1 replication, including nuclear migration of the pre-integration complex and/or transcription of the provirus genome. By gel filtration analysis of highly purified Vpr protein and its mutants, we demonstrate that HIV-1 Vpr exists as an oligomer. The N-terminal domain of Vpr (amino acids (aa) 1-42) is sufficient for oligomerization; however, deletion of aa 36-76 from Vpr disrupts oligomerization, suggesting that aa 36-42 are critical for Vpr oligomerization. As a result of Vpr oligomerization, basic aa residues within Vpr aa 1-73 are highly resistant to trypsin digestion, while those within Vpr aa 74-96 are easily accessible. Mutations within the leucine-/isoleucine-rich domain (aa 60-81), which was previously identified to be involved in Vpr interaction with a host cellular protein, rendered Arg62 more susceptible to trypsin digestion. Thus, the Vpr oligomeric structure must be extended into this domain. These results suggest a novel feature of HIV-1 Vpr that may be important for its functions.

The ovine lentiviruses cause encephalitis, pneumonia, and arthritis in sheep worldwide. Visna virus is a prototype of this family and the pathogenesis and molecular biology of the virus has been well characterized. The envelope proteins of visna virus are responsible for binding of virus to host cells and for causing cell fusion. The surface glycoprotein also elicits cellular and humoral immune responses to the virus, the former being thought to be responsible for eliminating infected cells as well as causing inflammatory lesions. In this study, transgenic sheep were constructed that expressed the envelope genes of visna virus under the control of the visna LTR to investigate the role of the env gene in the pathogenesis of lentiviral disease in its natural host. Three transgenic lambs were identified that contain the env transgene and express the envelope glycoproteins. These transgenic animals have remained healthy and expression of the viral gene has had no obvious deleterious effect. Expression of the visna envelope protein was demonstrated by cell fusion mediated by the envelope gene as well as by immunoprecipitation of the envelope proteins with monoclonal antibodies and immunofluorescence analyses of Env protein in cells. The target cell for visna virus replication in infected animals is the monocyte/macrophage. In natural infection, the level of viral gene expression in these cells increases with cell maturation. In the transgenic sheep, monocytes did not express the envelope glycoproteins until they differentiated into macrophages in vitro. Expression of the env mRNA in macrophages was quantitated by an RNase protection assay. In addition to expression in macrophages, the transgene was expressed by fibroblasts isolated from skin of the transgenic sheep. Expression of both the Env and Rev proteins was detected by immunoprecipitation and immunofluorescence. Two of the three lambs responded immunologically to the expression of the transgene by producing binding antibodies to the envelope glycoproteins. Thus, these transgenic sheep provide a model to study whether a lentivirus glycoprotein will prevent infection or modulate disease in its natural host after virus challenge.

We studied 15 macaques inoculated with SIV and identified three phases of infection. Phase 1 was characterized by activated lymphocytes in blood and infected cells in the CSF. In phase 2, activated cells were not detected but virus was recovered from mitogen-stimulated PBMC, while in phase 3, virus was recovered from mitogen-stimulated PBMC only after depletion of CD8+ lymphocytes, indicating effective control of the virus in peripheral blood. Early development of phase 3 status correlated with a longer period of clinical normalcy.

Nucleotide sequence analyses of the env genes of two neurotropic variants of SIV mac 239 were performed to determine whether molecular changes in these genes could be correlated with neurotropism. Biological characterization of virus from the infectious molecular clone of SIV mac 239 had shown that it is highly lymphocyte-tropic and poorly macrophage-tropic. This virus failed to replicate in the brain after intracerebral inoculation, but passage of this virus in macaques resulted in development of vital variants that had acquired cell tropism for macrophages and were neurovirulent (D. P. Sharma, M. C. Zink, H. Anderson, R. J. Adams, J. E. Clements, S. V. Joag, and O. Narayan, J. Virol. , 66, 3550-3556, 1992). The neurotropic virus SIV mac 239/R71 was obtained from the brain of a monkey after the third in vivo passage of SIV mac 239. Inoculation of this virus into another macaque leads to CNS disease and the isolation of another neurotropic virus SIV mac 239/17E. The viral env sequences obtained by polymerase chain reaction amplification directly from DNA obtained from the brain of R71 and 17E macaques had a limited number of changes dispersed throughout the env gene when compared to the parental virus, SIV mac 239. The most important finding was that there was a common set of nucleotide changes in the env gene of both R71 and 17E. This suggested that viruses containing these changes had a selective growth advantage in the brain and were the predominant species present in the central nervous system of macaques R71 and 17E. Analysis of individual clones containing the R71 env gene revealed that different env genes were present, but all had the changes that were conserved in both R71 and 17E but not present in the original lymphocyte-tropic parental virus, SIV mac 239. Construction of an infectious recombinant virus containing the tat, rev , and env genes from 17E and the remainder of the genome from the parental virus SIV mac 239 resulted in a virus that had the macrophage-tropism of 17E virus isolated from brain. This demonstrates that the env gene of 17E confers the cellular tropism of the virus on the parental virus, SIV mac 239.

Eight rhesus macaques inoculated with molecularly cloned SIV mac 239 developed viremia and virus-binding antibod-ies, but only one (macaque 2D) developed neutralizing antibodies to the virus. Viremia persisted in macaque 2D even in the presence of neutralizing antibodies. Neutralizing antibodies in the plasma collected from macaque 2D late in infection neutralized virus isolated early in infection. In contrast, these antibodies failed to neutralize the plasma viruses isolated after the appearance of neutralizing antibodies. Only antigenic variants were isolated from blood, spleen, and lymph nodes. Viruses isolated from other macaques that did not develop neutralizing antibodies were neutralized by 2D serum and were of the parental (SIV mac 239) phenotype. The variant viruses maintained their strict tropism for lymphocytes, similar to the parental virus.

The optimization of immune responses (IR) induced by HIV DNA vaccines in humans is one of the great challenges in the development of an effective vaccine against AIDS. Ideally, this vaccine should be delivered in a single dose to immunize humans. We recently demonstrated that the immunization of mice with a single dose of a DNA vaccine derived from pathogenic SHIVKU2(Δ4SHIVKU2) induced long-lasting, potent, and polyfunctional HIV-specific CD8+T-cell responses (G. Arrode, R. Hegde, A. Mani, Y. Jin, Y. Chebloune, and O. Narayan, J. Immunol.178:2318-2327, 2007). In the present work, we expanded the characterization of the IR induced by this DNA immunization protocol to rhesus macaques. Animals immunized with a single high dose of Δ4SHIVKU2DNA vaccine were monitored longitudinally for vaccine-induced IR using multiparametric flow cytometry-based assays. Interestingly, all five immunized macaques developed broad and polyfunctional HIV-specific T-cell IR that persisted for months, with an unusual reemergence in the blood following an initial decline but in the absence of antibody responses. The majority of vaccine-specific CD4+and CD8+T cells lacked gamma interferon production but showed high antigen-specific proliferation capacities. Proliferative CD8+T cells expressed the lytic molecule granzyme B. No integrated viral vector could be detected in mononuclear cells from immunized animals, and this high dose of DNA did not induce any detectable autoimmune responses against DNA. Taken together, our comprehensive analysis demonstrated for the first time the capacity of a single high dose of HIV DNA vaccine alone to induce long-lasting and polyfunctional T-cell responses in the nonhuman primate model, bringing new insights for the design of future HIV vaccines.

Cultured macaque macrophages are permissive for the replication of SIVmac251, and inoculation with virus is followed by the production of viral p27. Neutralizing macaque polyclonal and murine monoclonal antibodies preincubated with the virus prevented infection but did not prevent cytopathic virus replication when added more than 3 days after inoculation with virus. However, application of the neutralizing antibodies to macrophages 24 h after inoculation with virus resulted in sustained, low-level production of viral antigen. Cell lysates and individual macrophages from treated cultures contained less viral protein by Western blot (immunoblot) and immunocytochemistry than untreated controls. In situ hybridization and polymerase chain reaction procedures for detecting and estimating relative amounts of viral RNA and DNA showed that both viral nucleic acids failed to increase beyond the levels obtained before the addition of neutralizing antibodies. The data suggest that macrophages may need to be infected with a minimum threshold of virus particles in order to reach their full potential for virus replication and that their exposure to neutralizing antibodies prior to reaching this threshold resulted in limited virus replication.

Neurological disease resulting from lentivirus (including human immunodeficiency virus) infections is usually caused by a strain of virus that replicates productively in microglia in vivo and in macrophage cultures in vitro. We undertook this study using the model of simian immunodeficiency virus in macaques (SIVmac) to test the hypothesis that macrophage tropism is a prerequisite for neurotropism of the virus. Using molecularly cloned SIVmac239, a virus which is lymphocyte- but not macrophagetropic, we showed that this virus failed to infect brain after intracerebral (i.c.) inoculation into two macaques. Rather, these inoculations resulted in disseminated infection in lymphoid organs and the bone marrow. Two sequential passages of infected bone marrow cells inoculated i.c. into new macaques resulted in severe neurological disease and classical neuropathological lesions. Virus obtained from affected brain answered the hypothetical question: it was neurotropic and macrophagetropic. New findings in the study were that both lymphocyte- and macrophage-tropic viruses were present in the animals, but the viruses localized in different tissues: the lymphotropic virus in the spleen, lymph nodes, and plasma and the macrophagetropic virus in the brain and lungs. To determine whether the brain virus was preferentially neurotropic and whether it had neuroinvasive properties, infectious brain homogenate was inoculated into one animal i.c. and into two others peripherally. The i.c. inoculated animal developed fatal encephalitis 5 months later, and examination of tissues showed cell-free virus only in brain homogenates. Only microglia were infected despite persistent viremia and infection in bone marrow cells. The two macaques inoculated peripherally remained healthy and were euthanized at 6 months. Virus replication was detected only in the bone marrow cells and peripheral blood mononuclear cells. No infection in any macrophage population in visceral organs was detected, and the virus did not invade the brain. The strictly microglial specificity of this virus suggested that different macrophage populations in the body may select specific phenotypes of lentivirus from the quasispecies of virus in the bone marrow. This could provide the basis for specific disease affecting different organ systems.

Borna disease virus (BDV), which seems to be distinct from all other known viruses, exhibits a unique mechanism of pathogenesis. This review highlights several aspects of the biology of infection with this virus and summarizes the preliminary characterization of the agent. Studies on BDV may help to illuminate several important areas of neurobiology, including the mechanisms regulating the replication of a new type of RNA virus in the nuclei of neural cells, the neuroinvasiveness and neurotropism of such viruses, their T cell-mediated immunopathology, tolerance in newborn animals to persistent viral infection of the central nervous system, and behavioral diseases and eating disorders induced by such agents.

Comparison of neutralization of SIVmac251 in primary macrophage cultures with neutralization in lymphocytes (CEM174 cells) showed that neutralizing antibodies induced by SIV251 in infected rhesus macaques protected both macrophages and T lymphocytes against infection when the virus was preincubated with the antibodies. In macrophages, the neutralizing antibodies also protected against infection when added 1 hour after the virus. Addition of antisera to macrophages between 24 and 48 hours after virus inoculation resulted in infection with continuous release of small amounts of p24 into the supernatant fluids but these antibody-treated cultures failed to exhibit cytopathic virus replication. In contrast, the same neutralizing antisera did not protect lymphocytes against infection and subsequent cytopathic replication of the virus when added only 1 hour after virus inoculation. This distinction in the effect that neutralizing antibodies had on the development of cytopathic infection in lymphocytes and macrophages when added after virus inoculation, suggests that they could alter the dynamics of virus replication and therefore the pathogenesis of disease.

The mechanism for the gradual loss of CD4+ T lymphocytes and the development of the slowly progressive inflammatory/degenerative lesions that accompany human immunodeficiency virus infection are poorly understood. Using the Simian immunodeficiency virus (SIVmac) macaque model of AIDS, we found that persistently infected primary macrophages fuse with primary activated CD4+ lymphocytes and that this interaction results in production of tumour necrosis factor-alpha (TNF alpha) and interleukin 6 (IL-6). An earlier report had shown that SIV-infected macaque macrophages fuse with CEM174 cells (a human CD4+ cell line) and cause their lysis. In the present report, we have shown that TNF-alpha and IL-6 are also produced during the early stages of this interaction. Data from cocultivation of infected macrophages with several CD4+ T cell lines, including CEM174, suggested that the cytokines are produced by the T cells, and that cytokine production is restricted to those cells which not only express CD4, but are also capable of fusing with the infected macrophages. These data suggest that infected macrophages in vivo could fuse with and eliminate activated CD4+ lymphocytes and, during this interaction, release cytokines, which would contribute to the degenerative and inflammatory lesions characteristic of this disease.