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Deglycosylated, live-attenuated SIV vaccines elicited protective immune responses against heterologous SIVsmE543-3, which differs from the vaccine strain SIVmac239 to levels similar to those across HIV-1 clades. Two thirds of the vaccinees contained the chronic SIVsmE543-3 infection (controllers), whereas one third did not (noncontrollers). In this study, we investigated immune correlates of heterologous challenge control in rhesus macaques of Burmese origin. Because depletion of CD8+ cells in the controllers by administration of anti-CD8α Ab abrogated the control of viral replication, CD8+ cells were required for the protective immune response. However, classical SIV-specific CD8+ T cells did not account for the protective immune response in all controllers. Instead, IL-15–responding CD8α+ cells, including CD8+ T and NK cells, were significantly higher in the controllers than those in the noncontrollers, before and after vaccination with deglycosylated SIV. It is well established that IL-15 signal transduction occurs through “trans-presentation” in which IL-15 complexed with IL-15Rα on monocytes, macrophages, and dendritic cells binds to IL-15 Rβ/γ expressed on CD8+ T and NK cells. Accordingly, levels of IL-15 stimulation were strongly affected by the depletion of monocytes from PBMCs, implying key roles of innate immune cells. These results suggest that intrinsic IL-15 responsiveness may dictate the outcome of protective responses and may lead to optimized formulations of future broadly protective HIV vaccines.

Wall turbulence has a sublayer where one-point statistics, e.g., the mean velocity and the variances of some velocity fluctuations, vary logarithmically with the distance from the wall. This logarithmic scaling is found here for two-point statistics or specifically two-point cumulants of those fluctuations by means of experiments in a wind tunnel. As for corresponding statistics of the rate of the energy dissipation, the scaling is found to be not logarithmic. We reproduce these scaling laws with some mathematics and also with a model of energy-containing eddies that are attached to the wall., 11 pages, to appear in Physical Review E

Human immunodeficiency virus (HIV) encephalitis and degeneration of cerebral cortex are established histopathologies of HIV-associated neurocognitive disorders (HAND). We previously reported decreased excitatory amino acid transporter-2 (EAAT-2) and astrocytic apoptosis in cortical degeneration using SIVmac239 and simian-human immunodeficiency virus (SHIV)-infected macaques and human AIDS autopsy cases. In the present study, we added highly pathogenic SIVsm543-3-infected macaques. These animals showed similar degenerative changes in the frontal cortex. Using 11 SIV-infected macaques, three SIVsm543-3, five SIVmac239 and three SHIV, we compared brain pathology caused by three different viruses and further analyzed the pathogenic process of HAND. We noticed vacuolar changes in perivascular processes of astrocytes by electron microscopy, and examined expression of astrocyte-specific protein aquaporin-4 (AQP4) by immunohistochemistry. APQ4 was diffusely positive in the neuropil and perivascular area in control brains. There was patchy or diffuse decrease of AQP4 staining in the neuropil of SIV-infected macaques, which was associated with EAAT-2 staining by double immunostaining. A quantitative analysis demonstrated significant positive correlation between areas of AQP4 and EAAT-2. Some astrocytes express EAAT-2 but not AQP4, and decrease of EAAT-2 expression tended to be less than the decrease of AQP4. Active-caspase-3 immunostaining demonstrated apoptosis of neurons and astrocytes in the area of AQP4/EAAT-2 reduction. These results suggest that AQP4 is damaged first and decrease of EAAT-2 may follow in pathogenesis of cortical degeneration. This is the first demonstration of decrease of AQP4 and its association with EAAT-2 decrease in AIDS brain, suggesting a role in the pathogenesis of HAND.

Infant humans and rhesus macaques infected with the human or simian immunodeficiency virus (HIV or SIV), respectively, express higher viral loads and progress more rapidly to AIDS than infected adults. Activated memory CD4

Infant humans and rhesus macaques infected with the human or simian immunodeficiency virus (HIV or SIV), respectively, express higher viral loads and progress more rapidly to AIDS than infected adults. Activated memory CD4 + T cells in intestinal tissues are major primary target cells for SIV/HIV infection, and massive depletion of these cells is considered a major cause of immunodeficiency. Monocytes and macrophages are important cells of innate immunity and also are targets of HIV/SIV infection. We reported previously that a high peripheral blood monocyte turnover rate was predictive for the onset of disease progression to AIDS in SIV-infected adult macaques. The purpose of this study was to determine if earlier or higher infection of monocytes/macrophages contributes to the more rapid progression to AIDS in infants. We observed that uninfected infant rhesus macaques exhibited higher physiologic baseline monocyte turnover than adults. Early after SIV infection, the monocyte turnover further increased, and it remained high during progression to AIDS. A high percentage of terminal deoxynucleotidyltransferase dUTP nick end label (TUNEL)-positive macrophages in the lymph nodes (LNs) and intestine corresponded with an increasing number of macrophages derived from circulating monocytes (bromodeoxyuridine positive [BrdU + ] CD163 + ), suggesting that the increased blood monocyte turnover was required to rapidly replenish destroyed tissue macrophages. Immunofluorescence analysis further demonstrated that macrophages were a significant portion of the virus-producing cells found in LNs, intestinal tissues, and lungs. The higher baseline monocyte turnover in infant macaques and subsequent macrophage damage by SIV infection may help explain the basis of more rapid disease progression to AIDS in infants. IMPORTANCE HIV infection progresses much more rapidly in pediatric cases than in adults; however, the mechanism for this difference is unclear. Using the rhesus macaque model, this work was performed to address why infants infected with SIV progress more quickly to AIDS than do adults. Earlier we reported that in adult rhesus macaques, increasing monocyte turnover reflected tissue macrophage damage by SIV and was predictive of terminal disease progression to AIDS. Here we report that uninfected infant rhesus macaques exhibited a higher physiological baseline monocyte turnover rate than adults. Furthermore, once infected with SIV, infants displayed further increased monocyte turnover that may have facilitated the accelerated progression to AIDS. These results support a role for monocytes and macrophages in the pathogenesis of SIV/HIV and begin to explain why infants are more prone to rapid disease progression.

Glycosylation of Env defines pathogenic properties of simian immunodeficiency virus (SIV). We previously demonstrated that pathogenic SIVmac239 and a live-attenuated, quintuple deglycosylated Env mutant (Δ5G) virus target CD4 + T cells residing in different tissues during acute infection. SIVmac239 and Δ5G preferentially infected distinct CD4 + T cells in secondary lymphoid organs (SLOs) and within the lamina propria of the small intestine, respectively (C. Sugimoto et al., J Virol 86:9323–9336, 2012, https://doi.org/10.1128/JVI.00948-12 ). Here, we studied the host responses relevant to SIV targeting of CXCR3 + CCR5 + CD4 + T cells in SLOs. Genome-wide transcriptome analyses revealed that Th1-polarized inflammatory responses, defined by expression of CXCR3 chemokines, were distinctly induced in the SIVmac239-infected animals. Consistent with robust expression of CXCL10, CXCR3 + T cells were depleted from blood in the SIVmac239-infected animals. We also discovered that elevation of CXCL10 expression in blood and SLOs was secondary to the induction of CD14 + CD16 + monocytes and MAC387 + macrophages, respectively. Since the significantly higher levels of SIV infection in SLOs occurred with a massive accumulation of infiltrated MAC387 + macrophages, T cells, dendritic cells (DCs), and residential macrophages near high endothelial venules, the results highlight critical roles of innate/inflammatory responses in SIVmac239 infection. Restricted infection in SLOs by Δ5G also suggests that glycosylation of Env modulates innate/inflammatory responses elicited by cells of monocyte/macrophage/DC lineages. IMPORTANCE We previously demonstrated that a pathogenic SIVmac239 virus and a live-attenuated, deglycosylated mutant Δ5G virus infected distinct CD4 + T cell subsets in SLOs and the small intestine, respectively (C. Sugimoto et al., J Virol 86:9323–9336, 2012, https://doi.org/10.1128/JVI.00948-12 ). Accordingly, infections with SIVmac239, but not with Δ5G, deplete CXCR3 + CCR5 + CD4 + T (Th1) cells during the primary infection, thereby compromising the cellular immune response. Thus, we hypothesized that distinct host responses are elicited by the infections with two different viruses. We found that SIVmac239 induced distinctly higher levels of inflammatory Th1 responses than Δ5G. In particular, SIVmac239 infection elicited robust expression of CXCL10, a chemokine for CXCR3 + cells, in CD14 + CD16 + monocytes and MAC387 + macrophages recently infiltrated in SLOs. In contrast, Δ5G infection elicited only modest inflammatory responses. These results suggest that the glycosylation of Env modulates the inflammatory/Th1 responses through the monocyte/macrophage subsets and elicits marked differences in SIV infection and clinical outcomes.

// Jun Wang 1, * , Yu Zhang 1, * , Qiping Xu 1, * , Jinhua Qiu 1 , Honghua Zheng 1 , Xiang Ye 1, * , Yuhua Xue 2 , Yongmei Yin 3 , Zhou Zhang 4 , Ying Liu 4 , Yanling Hao 4 , Qiang Wei 5 , Wei Wang 5 , Kazuyasu Mori 6 , Shuji Izumo 7 , Ryuji Kubota 7 , Yiming Shao 4 , Hui Qin Xing 1 1 Fujian Provincial Key Laboratory of Neurodegenerative Disease and Aging Research, Institute of Neuroscience, Department of Pathology, Basic Medicine, Medical College, Xiamen University, Xiamen, Fujian 361102, China 2 School of Pharmaceutical Sciences at Xiamen University, Xiamen, Fujian 361102, China 3 The Fifth People’s Hospital of Wuxi, Affiliated to Jiangnan University, Wuxi, Jiangsu 214005, China 4 State Key Laboratory for Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, National Center for AIDS/STD Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China 5 Institute of Laboratory Animal Sciences of Chinese Academy of Medical Science, Beijing 100021, China 6 AIDS Research Center, National Institute of Infectious Disease, Tokyo 862-1640, Japan 7 Division of Molecular Pathology, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan * These authors have contributed equally to this work Correspondence to: Hui Qin Xing, email: xhqw63@hotmail.com Yiming Shao, email: yshao08@gmail.com Keywords: menin, Tat, neuron, apoptosis, human immunodeficiency virus-associated neurocognitive disorder Received: July 26, 2016 Accepted: January 03, 2017 Published: February 02, 2017 ABSTRACT The molecular mechanisms involved in human immunodeficiency virus (HIV)-associated neurocognitive disorder (HAND) remain poorly understood. It has been recently reported that HIV-1 Tat transactivation requires menin, suggesting that menin may be involved in HAND pathogenesis. But the role of menin is not clear. Here, we found that protein level of menin was increased in simian-human immunodeficiency chimeric virus (SHIV) -SF162.P4 and simian immunodeficiency virus (SIV) sm543-3 -infected rhesus macaques compared with the controls by immunohistochemistry (IHC) and western blot. Menin mainly expressed in the frontal cortex neurons of the brain, more importantly, the number of menin-staining cells was positively correlated with cleaved-caspase-3-positive cells while it was negatively correlated with a neuron-specific nuclear protein NeuN-positive cells, suggesting that expression of menin may induce neuronal apoptosis. Further studies showed that menin level was significantly increased during Tat-induced apoptosis, while downregulation of menin by pll3.7-MEN1-shRNA attenuated the Tat-induced cleavage of caspase-3 and caspase-8 in SY5Y cells and primary neuron cultures. Together, our findings reveal a pro-apoptotic role of menin in the brains of the SIV-infected macaques and the cultured neurons, indicating that targeting menin may be potential to block the HIV-1 Tat induced neuronal damage in HAND.

Human immunodeficiency virus (HIV) encephalitis and degeneration of cerebral cortex are established histopathologies of HIV-associated neurocognitive disorders (HAND). We previously reported decreased excitatory amino acid transporter-2 (EAAT-2) and astrocytic apoptosis in cortical degeneration using SIVmac239 and simian-human immunodeficiency virus (SHIV)-infected macaques and human AIDS autopsy cases. In the present study, we added highly pathogenic SIVsm543-3-infected macaques. These animals showed similar degenerative changes in the frontal cortex. Using 11 SIV-infected macaques, three SIVsm543-3, five SIVmac239 and three SHIV, we compared brain pathology caused by three different viruses and further analyzed the pathogenic process of HAND. We noticed vacuolar changes in perivascular processes of astrocytes by electron microscopy, and examined expression of astrocyte-specific protein aquaporin-4 (AQP4) by immunohistochemistry. APQ4 was diffusely positive in the neuropil and perivascular area in control brains. There was patchy or diffuse decrease of AQP4 staining in the neuropil of SIV-infected macaques, which was associated with EAAT-2 staining by double immunostaining. A quantitative analysis demonstrated significant positive correlation between areas of AQP4 and EAAT-2. Some astrocytes express EAAT-2 but not AQP4, and decrease of EAAT-2 expression tended to be less than the decrease of AQP4. Active-caspase-3 immunostaining demonstrated apoptosis of neurons and astrocytes in the area of AQP4/EAAT-2 reduction. These results suggest that AQP4 is damaged first and decrease of EAAT-2 may follow in pathogenesis of cortical degeneration. This is the first demonstration of decrease of AQP4 and its association with EAAT-2 decrease in AIDS brain, suggesting a role in the pathogenesis of HAND.

Natural-killer group 2 member D (NKG2D) is an activating receptor that plays an important role in the immune response mediated by NK cells, γδ(+) T cells, and CD8(+) T cells. In humans, MHC class I chain-related genes and UL-16 binding protein (ULBP)/retinoic acid early transcript 1 (REAT1) gene family encode ligands for NKG2D. The rhesus and crab-eating macaques, which belong to the Old World monkeys, are widely used as non-human primate models in medical researches on the immunological process. In the present study, we investigated the polymorphisms of ULBP4/RAET1E, a member of the ULBP/RAET1 family, and found 25 and 14 alleles from the rhesus and crab-eating macaques, respectively, of which diversities were far more extended than in humans. A phylogenetic study suggested that the allelic diversification of ULBP4/RAET1E predated the divergence of rhesus and crab-eating macaques.

The pathogenesis of acquired immunodeficiency syndrome dementia complex (ADC) is still poorly understood. Many studies suggest that proinflammatory cytokines such as IL-1beta and TNF-alpha released by microglia/macrophages or astrocytes play a role in CNS injury. A microscopic finding of a microglial nodule with multinucleated giant cells (MNGCs) is a histopathologic hallmark of ADC and named HIV encephalitis. However, in vivo expression of these cytokines in this microenvironment of HIV encephalitis is not yet clarified. One of the main reasons is complexities of brain pathology in patients who have died from terminal AIDS. In this study, we infected two macaques with macrophage-tropic Simian immunodeficiency virus SIV239env/MERT and examined expression of TNF-alpha and IL-1beta in inflammatory lesions with MNGCs and its relation to virus-infected cells using immunohistochemistry. One macaque showed typical inflammatory lesions with MNGCs in the frontal white matter. Small microglial nodules were also detected in the basal ganglia and the spinal cord. SIVenv positive cells were detected mainly in inflammatory lesions, and seemed to be microglia/macrophages and MNGCs based on their morphology. Expression of IL-1beta and TNF-alpha were detected in the inflammatory lesions with MNGCs, and these positive cells were found to be negative for SIVenv by double-labeling immunohistochemistry or immunohistochemistry of serial sections. There were a few TNF-alpha positive cells and almost no IL-1beta positive cells in the area other than inflammatory lesions. Another macaque showed scattered CD3+ cells and CD68+ cells in the perivascular regions of the white matter. SIVenv and TNF-alpha was demonstrated in a few perivascular macrophages. These findings indicate that virus-infected microglia/macrophages do not always express IL-1beta and TNF-alpha, which suggests an indirect role of HIV-1-infected cells in cytokine-mediated pathogenesis of ADC. Our macaque model for human ADC may be useful for better understanding of its pathogenesis.

Recent findings established that primary targets of HIV/SIV are lymphoid cells within the gastrointestinal (GI) tract. Focus has therefore shifted to T-cells expressing α4β7 integrin which facilitates trafficking to the GI tract via binding to MAdCAM-1. Approaches to better understand the role of α4β7+ T-cells in HIV/SIV pathogenesis include their depletion or blockade of their synthesis, binding and/or homing capabilities in vivo. Such studies can ideally be conducted in rhesus macaques (RM), the non-human primate model of AIDS. Characterization of α4β7 expression on cell lineages in RM blood and GI tissues reveal low densities of expression by NK cells, B-cells, naive and TEM (effector memory) T-cells. High densities were observed on TCM (central memory) T-cells. Intravenous administration of a single 50 mg/kg dose of recombinant rhesus α4β7 antibody resulted in significant initial decline of α4β7+ lymphocytes and sustained coating of the α4β7 receptor in both the periphery and GI tissues.

Phenotypic and functional studies of the programmed death-1 (PD-1) molecule on CD4(+) and CD8(+) T cells were performed on peripheral blood mononuclear cells from uninfected and simian immunodeficiency virus (SIV)-infected rhesus macaques. These data demonstrated a rapid upregulation of PD-1 expression on tetramer-positive CD8(+) T cells from MamuA.01(+) SIV-infected macaques upon infection. Upregulation of PD-1 on total CD8(+) T cells was not detectable. In contrast, CD4(+) T-cell PD-1 expression was markedly higher in total CD4(+) T cells during chronic, but not acute, infection and there was a correlation between the level of PD-1 expression on naive and central memory CD4(+) T cells and the levels of viral loads. Such association was emphasized further by a marked decrease of PD-1 expression on tetramer-positive CD8 T cells as well as on CD4(+) T cells on longitudinal samples collected before and after the initiation of antiretroviral therapy and downregulation of viral replication in vivo. Cloning of PD-1 and its two ligands from several non-human primate species demonstrated > 95% conservation for PD-1 and PD-L2 and only about 91% homology for PD-L1. Functional studies using soluble recombinant PD-1 protein or PD-1-immunoglobulin G fusion proteins induced marked increases in the SIV-specific proliferative responses of both CD4(+) and CD8(+) T cells from rhesus macaques. The results of these studies serve as a foundation for future in vivo trials of the use of rMamu-PD-1 to potentially enhance and/or restore antiviral immune responses in vivo.

The rhesus macaque exhibits individual differences in susceptibility and resistance to infectious agents such as simian immunodeficiency virus (SIV) under experimental conditions, and these may be genetically determined at least in part by major histocompatibility complex (MHC) class I polymorphism. Although the importance of defining MHC class I polymorphism is well recognized, development of a generic and comprehensive molecular typing method of MHC class I alleles of the rhesus macaque has been hampered because, during the evolution of this species, multiple copies of similar DNA sequences have been generated by duplication events including the coding sequences of Mamu-A and Mamu-B loci. We report here a newly developed reference strand-mediated conformation analysis (RSCA)-based typing method of multiple Mamu-A and Mamu-B cDNAs that allowed us to estimate the number of expressed alleles. This technique detected 1-7 Mamu-A signals and 2-12 Mamu-B signals in a single sample, indicating that the number of functional alleles may vary. By comparing the data from the parents with those from the descendants in the breeding colony, several MHC class I haplotypes consisting of variable numbers of functional Mamu-A and Mamu-B alleles could be assigned.

Monocyte and dendritic cell (DC) development was evaluated using in vivo BrdU pulse-chase analyses in rhesus macaques, and phenotype analyses of these cells in blood also were assessed by immunostaining and flow cytometry for comparisons among rhesus, cynomolgus, and pigtail macaques, as well as African green monkeys and humans. The nonhuman primate species and humans have three subsets of monocytes, CD14+CD16−, CD14+CD16+, and CD14−CD16+ cells, which correspond to classical, intermediate, and nonclassical monocytes, respectively. In addition, there exist presently two subsets of DC, BDCA-1+ myeloid DC and CD123+ plasmacytoid DC, that were first confirmed in rhesus macaque blood. Following BrdU inoculation, labeled cells first appeared in CD14+CD16− monocytes, then in CD14+CD16+ cells, and finally in CD14−CD16+ cells, thus defining different stages of monocyte maturation. A fraction of the classical CD14+CD16− monocytes gradually expressed CD16+ to become CD16+CD14+ cells and subsequently matured into the nonclassical CD14−CD16+ cell subset. The differentiation kinetics of BDCA-1+ myeloid DC and CD123+ plasmacytoid DC were distinct from the monocyte subsets, indicating differences in their myeloid cell origins. Results from studies utilizing nonhuman primates provide valuable information about the turnover, kinetics, and maturation of the different subsets of monocytes and DC using approaches that cannot readily be performed in humans and support further analyses to continue examining the unique myeloid cell origins that may be applied to address disease pathogenesis mechanisms and intervention strategies in humans.

Recently, encouraging AIDS vaccine trials in macaques have implicated cytotoxic T lymphocytes (CTLs) in the control of the simian human immunodeficiency virus SHIV89.6P that induces acute CD4+ T cell depletion. However, none of these vaccine regimens have been successful in the containment of replication of the pathogenic simian immunodeficiency viruses (SIVs) that induce chronic disease progression. Indeed, it has remained unclear if vaccine-induced CTL can control SIV replication. Here, we show evidence suggesting that vaccine-induced CTLs control SIVmac239 replication in rhesus macaques. Eight macaques vaccinated with DNA-prime/Gag-expressing Sendai virus vector boost were challenged intravenously with SIVmac239. Five of the vaccinees controlled viral replication and had undetectable plasma viremia after 5 wk of infection. CTLs from all of these five macaques rapidly selected for escape mutations in Gag, indicating that vaccine-induced CTLs successfully contained replication of the challenge virus. Interestingly, analysis of the escape variant selected in three vaccinees that share a major histocompatibility complex class I haplotype revealed that the escape variant virus was at a replicative disadvantage compared with SIVmac239. These findings suggested that the vaccine-induced CTLs had “crippled” the challenge virus. Our results indicate that vaccine induction of highly effective CTLs can result in the containment of replication of a highly pathogenic immunodeficiency virus.

In contrast to the simian immunodeficiency virus SIVmac239, which replicates poorly in rhesus monkey alveolar macrophages, a variant with nine amino acid changes in envelope (SIVmac239/316E) replicates efficiently and to high titer in these same cells. We examined levels of viral DNA, RNA, antigen, and infectious virus to identify the nature of the block to SIVmac239 replication in these cells. Low levels of viral antigen (0.1 to 1.0 ng of p27 per ml) and infectious virus (100 to 1,000 infectious units per ml) were produced in the supernatant 1 to 4 days after SIVmac239 infection, but these levels did not increase subsequently. SIVmac239 DNA was synthesized in these macrophage cultures during the initial 24 h after infection, but the levels did not increase subsequently. Quantitation of the numbers of infectious cells in cultures over time and the results of experiments in which cells were reexposed to SIVmac239 after the initial exposure indicated that only a small proportion of cells were susceptible to SIVmac239 infection in these alveolar macrophage cultures and that the vast majority (>95%) of cells were refractory to SIVmac239 infection. In contrast to the results with SIVmac239, the levels of viral antigen, infectious virus, and viral DNA increased exponentially 2 to 7 days after infection by SIVmac239/316E, reaching levels greater than 100 ng of p27 per ml and 100,000 infectious units per ml. Since SIVmac239/316E has previously been described as a virus capable of infecting cells in a relatively CD4-independent fashion, we examined the levels of CD4 expression on the surface of fresh and cultured alveolar macrophages from rhesus monkeys. The levels of CD4 expression were extremely low, below the limit of detection by flow cytometry, on greater than 99% of the macrophages. CCR5+cells were profoundly depleted only from alveolar macrophage cultures infected with SIVmac239/316E. High concentrations of an antibody to CD4 delayed but did not block replication of SIVmac239/316E. The results suggest that the adaptation of SIVmac316 to efficient replication in alveolar macrophages results from its ability to infect these cells in a CD4-independent fashion or in a CD4-dependent fashion even at extremely low levels of surface CD4 expression. Since resident macrophages in brains and lungs of humans also express little or no CD4, our findings predict the presence of human immunodeficiency virus type 1 that is relatively CD4 independent in the lung and brain compartments of infected people.

We investigated the histological features of lymph nodes, focusing on monocytes/macrophages, in rhesus monkeys (Macaca mulatta) acutely infected with simian immunodeficiency virus (SIV). In monkeys infected with a pathogenic SIV, SIVmac239, MAC387(+) newly blood-derived macrophages markedly increased in number at paracortical areas at 11 to 14 days postinoculation, concomitant with the peak of the primary SIV antigenemia. The MAC387(+) macrophages densely gathered around high endothelial venules and formed cell clusters with CD3(+) T lymphocytes, tingible body macrophages, and plasmacytoid monocytes. In the cell clusters, CD3(+) T lymphocytes which closely adhered to the MAC387(+) macrophages enlarged in size, suggesting a histological manifestation of T-lymphocyte activation by macrophages. By 54 days postinoculation, when SIV antigenemia became undetectable, the MAC387(+) macrophages decreased in number and the cell cluster disappeared from paracortical areas. In contrast, the monkeys infected with a nef-deleted mutant of SIVmac239 showed lower levels of SIV antigenemia and lower numbers of MAC387(+) macrophages in paracortical areas than those infected with SIVmac239. These results indicate that MAC387(+) macrophages accumulate in paracortical areas for the period of the intense primary SIV antigenemia and may play an important role in activating naive T lymphocytes.

We have previously reported that CD4+CD8+ double-positive (DP) T cells with a resting memory phenotype exist in a substantial proportion of peripheral blood lymphocytes of adult cynomolgus macaques. In this study, we examined the effects of simian immunodeficiency virus of macaque (SIVmac) infection on DP T cells. In vitro, SIVmac239 nef-open (239) and its nef-deletion mutant replicated well in both CD4+CD8- and DP T cells. However, when the macaques were infected with 239, DP, but not CD4+CD8-, T cells were transiently increased in parallel with cell activation and viral replication, followed by depletion within 1 month postinfection. Interestingly, the nef gene was required for depletion but not for the increase and activation of DP T cells. These data suggest that the pathogenic SIV infection may downmodulate production and/or blood circulation of DP T cells by a Nef function-related mechanism(s) different from that for the depletion of CD4+CD8- T cells.

The quantitative and phenotypic changes of peripheral blood monocytes during the acute stage of simian immunodeficiency virus infection were investigated. We inoculated intravenously three cynomolgus monkeys (Macaca fascicularis) with 100 TCID50 of SIVmac239 and collected whole blood twice a week until 35 days postinoculation. We found that the relative number of monocytes in peripheral blood leukocytes significantly increased at 7-17 days postinoculation. This increase was concomitant with the peak of primary SIV antigenemia. To determine if the monocytes observed during the acute stage were phenotypically altered, they were periodically examined for the expression of surface markers (i.e., CD11b, CD14, CD16, CD29, D32, CD56, CD62L, CD64, CD80, and MHC-II-DR) by flow cytometry. The results showed that the expression levels of CD14 and CD56 on most of the monocytes were remarkably reduced at 7-17 days postinoculation, and a new subpopulation, CD14lowCD16+CD80+ monocytes, was clearly detected at 10 days postinoculation. These results indicate that the phenotypic alteration of peripheral blood monocytes occurs during the primary SIV infection.

We examined the expression kinetics of activation antigens CD38 and MHC-IIDR (DR) on circulating CD8+ lymphocytes in rhesus macaques infected with pathogenic simian immunodeficiency virus ...

Glycans of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) play pivotal roles in modulating virus-target cell interactions. We have previously reported that, whereas SIVmac239 is pathogenic, its deglycosylated essentially nonpathogenic mutant (Δ5G) serves as a live-attenuated vaccine, although both replicate similarly during primary infection. These findings prompted us to determine whether such a polarized clinical outcome was due to differences in the immune tissues targeted by these viruses, where functionally and phenotypically different memory CD4 + T cells reside. The results showed that Δ5G replicates in secondary lymphoid tissue (SLT) at 1- to 2-log-lower levels than SIVmac239, whereas SIVmac239-infected but not Δ5G-infected animals deplete CXCR3 + CCR5 + transitional memory (TrM) CD4 + T cells. An early robust Δ5G replication was localized to small intestinal tissue, especially the lamina propria (effector site) rather than isolated lymphoid follicles (inductive site) and was associated with the induction and depletion of CCR6 + CXCR3 − CCR5 + effector memory CD4 + T cells. These results suggest that differential glycosylation of Env dictates the type of tissue-resident CD4 + T cells that are targeted, which leads to pathogenic infection of TrM-Th1 cells in SLT and nonpathogenic infection of Th17 cells in the small intestine, respectively.

A major difference between vaccine and wild-type strains of measles virus (MV) in vitro is the wider cell specificity of vaccine strains, resulting from the receptor usage of the hemagglutinin (H) protein. Wild-type H proteins recognize the signaling lymphocyte activation molecule (SLAM) (CD150), which is expressed on certain cells of the immune system, whereas vaccine H proteins recognize CD46, which is ubiquitously expressed on all nucleated human and monkey cells, in addition to SLAM. To examine the effect of the H protein on the tropism and attenuation of MV, we generated enhanced green fluorescent protein (EGFP)-expressing recombinant wild-type MV strains bearing the Edmonston vaccine H protein (MV-EdH) and compared them to EGFP-expressing wild-type MV strains. In vitro , MV-EdH replicated in SLAM + as well as CD46 + cells, including primary cell cultures from cynomolgus monkey tissues, whereas the wild-type MV replicated only in SLAM + cells. However, in macaques, both wild-type MV and MV-EdH strains infected lymphoid and respiratory organs, and widespread infection of MV-EdH was not observed. Flow cytometric analysis indicated that SLAM + lymphocyte cells were infected preferentially with both strains. Interestingly, EGFP expression of MV-EdH in tissues and lymphocytes was significantly weaker than that of the wild-type MV. Taken together, these results indicate that the CD46-binding activity of the vaccine H protein is important for determining the cell specificity of MV in vitro but not the tropism in vivo . They also suggest that the vaccine H protein attenuates MV growth in vivo .

HIV vaccine development has been hampered by issues such as undefined correlates of protection and extensive diversity of HIV. We addressed these issues using a previously established SIV-macaque model in which SIV mutants with deletions of multiple gp120 N-glycans function as potent live attenuated vaccines to induce near-sterile immunity against the parental pathogenic SIVmac239. In this study, we investigated the protective efficacy of these mutants against a highly pathogenic heterologous SIVsmE543-3 delivered intravenously to rhesus macaques with diverse MHC genotypes. All 11 vaccinated macaques contained the acute-phase infection with blood viral loads below the level of detection between 4 and 10 weeks postchallenge (pc), following a transient but marginal peak of viral replication at 2 weeks in only half of the challenged animals. In the chronic phase, seven vaccinees contained viral replication for over 80 weeks pc, while four did not. Neutralizing antibodies against challenge virus were not detected. Although overall levels of SIV specific T cell responses did not correlate with containment of acute and chronic viral replication, a critical role of cellular responses in the containment of viral replication was suggested. Emergence of viruses with altered fitness due to recombination between the vaccine and challenge viruses and increased gp120 glycosylation was linked to the failure to control SIV. These results demonstrate the induction of effective protective immune responses in a significant number of animals against heterologous virus by infection with deglycosylated attenuated SIV mutants in macaques with highly diverse MHC background. These findings suggest that broad HIV cross clade protection is possible, even in hosts with diverse genetic backgrounds. In summary, results of this study indicate that deglycosylated live-attenuated vaccines may provide a platform for the elucidation of correlates of protection needed for a successful HIV vaccine against diverse isolates.

Various types of neuronal damage have been reported in acquired immunodeficiency syndrome (AIDS) dementia. We previously demonstrated that inflammation and cortical damage occur independently according to viral tropism in a simian immunodeficiency virus (SIV)-infected macaque model of AIDS dementia. To elucidate the pathogenesis of cortical degeneration, we examined the frontal cortex of SIV-infected macaques and found apoptosis and decreased expression of the excitatory amino acid transporter 2 in astrocytes and diffuse activation of microglia in association with limited neuronal damage. Some activated microglia also expressed excitatory amino acid transporter 2 but not proinflammatory cytokines. No inflammatory changes were seen in the cortex or the white matter, and SIV-infected cells were not detected in or around cortical lesions either by immunohistochemistry or by the polymerase chain reaction detection of SIV genomes of extracted DNA from microdissected tissue samples. These results indicate that an astrocytic abnormality and a compensatory activation of microglia might provide a protective effect against neuronal degeneration in the frontal cortex of SIV-infected macaques without SIV encephalitis.

Infection of rhesus macaques with a deglycosylation mutant, Δ5G, derived from SIV239, a pathogenic clone of simian immunodeficiency virus (SIV), led to robust acute-phase viral replication followed by a chronic phase with undetectable viral load. This study examined whether humoral responses in Δ5G-infected animals played any role in the control of infection. Neutralizing antibodies (nAbs) were elicited more efficiently in Δ5G-infected animals than in SIV239-infected animals. However, functional nAb measured by 90 % neutralization was prominent in only two of the five Δ5G-infected animals, and only at 8 weeks post-infection (p.i.), when viral loads were already below 104 copies ml−1. These results suggest a minimal role for nAbs in the control of the primary infection. In contrast, whilst Ab responses to epitopes localized to the variable loops V1/V2 were detected in all Δ5G-infected animals at 3 weeks p.i., this response was associated with a concomitant reduction in Ab responses to epitopes in gp41 compared with those in SIV239-infected animals. These results suggest that the altered surface glycosylation and/or conformation of viral spikes induce a humoral response against SIV that is distinct from the response induced by SIV239. More interestingly, whereas V1/V2-specific Abs were induced in all animals, these Abs were associated with vigorous Δ5G-specific virion capture ability in only two Δ5G-infected animals that exhibited a functional nAb response. Thus, whereas the deglycosylation mutant infection elicited early virion capture and subsequent nAbs, the responses differed among animals, suggesting the existence of host factors that may influence the functional humoral responses against human immunodeficiency virus/SIV.

Auto-antibodies appear in the sera of rhesus macaques following SIV infection. The present study was conducted to examine the role of viral load, anti-viral chemotherapy and stage of disease on the titers of such auto-antibodies and the spectrum of auto-antigens that become the target of such auto-immune responses. In addition, the role of regulatory T cells (Tregs) was also examined. Results of these studies showed that the highest auto-antibody titers were noted in animals with lower relative plasma viral loads with a wider spectrum of auto-antigens that are the target of such responses as compared with lower auto-antibody titers in animals with relatively higher plasma viral loads and a narrower spectrum of auto-antigens. Short term anti-viral chemotherapy did not influence the titers of auto-antibodies. While there was a gradual decrease in the frequency and absolute number of Tregs, the levels of Tregs was inversely correlated with viral load and lower autoantibody titers. The mechanisms for these differences remain unknown and suggest complex relationships exist between levels of immuno-suppression, auto-immune response, homeostatic proliferation and the spectrum of auto-antigens that become the target of such auto-immune responses.

Differences in clinical outcome of simian immunodeficiency virus (SIV) infection in disease-resistant African sooty mangabeys (SM) and disease-susceptible Asian rhesus macaques (RM) prompted us to examine the role of regulatory T cells (Tregs) in these two animal models. Results from a cross-sectional study revealed maintenance of the frequency and absolute number of peripheral Tregs in chronically SIV-infected SM while a significant loss occurred in chronically SIV-infected RM compared to uninfected animals. A longitudinal study of experimentally SIV-infected animals revealed a transient increase in the frequency of Tregs from baseline values following acute infection in RM, but no change in the frequency of Tregs occurred in SM during this period. Further examination revealed a strong correlation between plasma viral load (VL) and the level of Tregs in SIV-infected RM but not SM. A correlation was also noted in SIV-infected RM that control VL spontaneously or in response to antiretroviral chemotherapy. In addition, immunofluorescent cell count assays showed that while Treg-depleted peripheral blood mononuclear cells from RM led to a significant enhancement of CD4+and CD8+T-cell responses to select pools of SIV peptides, there was no detectable T-cell response to the same pool of SIV peptides in Treg-depleted cells from SIV-infected SM. Our data collectively suggest that while Tregs do appear to play a role in the control of viremia and the magnitude of the SIV-specific immune response in RM, their role in disease resistance in SM remains unclear.

Virus-specific cellular immune responses play an important role in the control of immunodeficiency virus replication. However, preclinical trials of vaccines that induce virus-specific cellular immune responses have failed to contain simian immunodeficiency virus (SIV) replication in macaques. A defective provirus DNA vaccine system that efficiently induces virus-specific CD8+ T-cell responses has previously been developed. The vaccinated macaques showed reduced viral loads, but failed to contain SIVmac239 replication. In this study, macaques that showed partial control of SIV replication were followed up to see if or how they lost this control in the chronic phase. Two of them showed increased viral loads about 4 or 8 months after challenge and finally developed AIDS. Analysis of SIV-specific T-cell levels by detection of SIV-specific gamma interferon (IFN-γ) production revealed that these two macaques maintained SIV-specific CD8+ T cells, even after loss of control, but lost SIV-specific CD4+ T cells when plasma viral loads increased. The remaining macaque kept viral loads at low levels and maintained SIV-specific CD4+ T cells, as well as CD8+ T cells, for more than 3 years. Additional analysis using macaques vaccinated with a Gag-expressing Sendai virus vector also found loss of viraemia control, with loss of SIV-specific CD4+ T cells in the chronic phase of SIV infection. Thus, SIV-specific CD4+ T cells that were able to produce IFN-γ in response to SIV antigens were preserved by the vaccine-based partial control of primary SIV replication, but were lost with abrogation of control in the chronic phase.

Highly active antiretroviral therapy (HAART) has been successful to reduce progression of acquired immunodeficiency syndrome (AIDS). Nevertheless, recent autopsy analysis of the brain from patients with human immunodeficiency virus (HIV)-1 infection reported same or even increasing numbers of AIDS encephalopathy. This insufficient effect of HAART for central nervous system (CNS) complication might be explained by independent pathogenetic processes in lymph node and CNS. We inoculated macaques with three Simian immunodeficiency virus (SIV) strains and investigated relationship between degree of the lymph node pathology and that of AIDS-related brain pathology. Animals infected with T-cell-tropic viruses SIVmac239 and SHIV-RT developed typical AIDS pathology in the lymph node 46 to 156 weeks after infection. The cerebral cortex of these animals showed focal or diffuse gliosis, and electron microscopic analysis demonstrated degenerative changes, such as accumulation of dense lamellar bodies in the dendrites and swelling of astrocytic processes. However, there was no evidence of microglial nodules or multinucleated giant cells in the white mater. The animals infected with macrophage-tropic SIV239env/MERT did not develop lymph node pathology of AIDS in the same or longer period of infection. The white mater of the animal, however, showed microglial nodules with multinucleated giant cells, a pathological hallmark of AIDS encephalopathy. SIV immunoreactivity was demonstrated in these giant cells as well as macrophage/microglia cells. On the other hand, there was no abnormality in the cerebral cortex. These findings suggest that there are two independent pathogenetic processes in AIDS encephalopathy: immune response against virus infected macrophage/microglial cells in the white mater without immunodeficiency and cortical degeneration caused in the late stage of AIDS.

The pathogenesis of AIDS virus infection in a nonhuman primate AIDS model was studied by comparing plasma viral loads, CD4 + T-cell subpopulations in peripheral blood mononuclear cells, and simian immunodeficiency virus (SIV) infection in lymph nodes for rhesus macaques infected with a pathogenic molecularly cloned SIVmac239 strain and those infected with its nef deletion mutant (Δnef). In agreement with many reports, whereas SIVmac239 infection induced AIDS and depletion of memory CD4 + T cells in 2 to 3 years postinfection (p.i.), Δnef infection did not induce any manifestation associated with AIDS up to 6.5 years p.i. To explore the difference in SIV infection in lymphoid tissues, we biopsied lymph nodes at 2, 8, 72, and 82 weeks p.i. and analyzed them by pathological techniques. Maximal numbers of SIV-infected cells (SIV Gag + , Env + , and RNA + ) were detected at 2 weeks p.i. in both the SIVmac239-infected animals and the Δnef-infected animals. In the SIVmac239-infected animals, most of the infected cells were localized in the T-cell-rich paracortex, whereas in the Δnef-infected animals, most were localized in B-cell-rich follicles and in the border region between the paracortex and the follicles. Analyses by double staining of CD68 + macrophages and SIV Gag + cells and by double staining of CD3 + T cells and SIV Env + cells revealed that SIV-infected cells were identified as CD4 + T cells in either the SIVmac239 or the Δnef infection. Whereas the many functions of Nef protein were reported from in vitro studies, our finding of SIVmac239 replication in the T-cell-rich paracortex in the lymph nodes supports the reported roles of Nef protein in T-cell activation and enhancement of viral infectivity. Furthermore, the abundance of SIVmac239 infection and the paucity of Δnef infection in the T-cell-rich paracortex accounted for the differences in viral replication and pathogenicity between SIVmac239 and the Δnef mutant. Thus, our in vivo study indicated that the nef gene enhances SIV replication by robust productive infection in memory CD4 + T cells in the T-cell-rich region in lymphoid tissues.

Using tetanus toxoid (TT) and influenza (Flu) immunization of rhesus macaques as a model, the effect of IL-2 and IL-15 on the generation and maintenance of antigen specific memory T cells was evaluated following primary and secondary immunization. Daily cytokine administration expanded primarily effector but not memory cells, while spacing cytokine administration to q3-7 days markedly enhanced TT and Flu specific memory responses. Following primary immunization, TT specific CD4 and influenza matrix protein (Flu-MP) specific CD8 effector responses were enhanced by IL-2 administration but CD8 specific memory responses were no different from cytokine non-treated monkeys. In contrast, expansion of Flu specific CD8 cells with IL-15 was only modest but resulted in significantly elevated levels of memory cells at 6 months. IL-15 also significantly enhanced early and late TT specific CD4 responses. The highest levels of primary effector and memory T cells were observed following alternate administration of both IL-2 and IL-15. Following booster immunization, however, only IL-15 appeared able to enhance CD8 T cell responses while IL-2 or IL2/IL-15 administration were less effective.

By using the dominant simian immunodeficiency virus (SIV) Gag Mamu-A01 restricted major histocompatibility complex (MHC) class I epitope p11CM, we demonstrate antibody-mediated enhanced MHC class I cross presentation of SIV Gag. In vitro restimulation of peripheral blood mononuclear cells from SIV-infected rhesus macaques with recombinant full-length SIV Gag p55 plus p55 affinity-purified immunoglobulin G (p55 Gag/p55-IgG) led to the generation of markedly higher frequencies of p11CM specific precursor cytotoxic T lymphocytes (p-CTLs) compared with restimulation with (i) SIV Gag p55 alone or (ii) optimal concentrations of the p11CM peptide alone. These results, along with the finding that CD4 depletion abrogated the enhancement, suggest a prominent role for CD4+T cells. Testing for p-CTLs against other Mamu-A01-restricted SIV Gag epitopes suggested that this mechanism favored recognition of the dominant p11CM peptide, potentially further skewing of the CTL response. The p-CTL enhancing effect was also decreased or abrogated by pepsin digestion of the p55-specific IgG or by the addition of monoclonal antibodies to Fc receptor (FcR) II/III, suggesting that the effect was dependent on FcR-mediated uptake of the immune-complexed antigen. Finally, incubation of antigen-presenting cells with SIV Gag p55 immune complexes in the presence of lactacystin or of bafilomycin indicated that the mechanism of antibody-mediated enhancement of cross presentation required both the proteasomal and the endosomal pathways. These data demonstrate for the first time the cross presentation of antigens via immune complexes in lentiviral infection and indicate a heretofore-unrecognized role for antibodies in modulating the magnitude and potentially also the breadth of MHC class I-restricted antigen processing and presentation and CTL responses.

Adoptive transfer of autologous preinfection-collected peripheral blood mononuclear cells (PBMCs) or activated CD4+ T cells was performed in simian immunodeficiency virus (SIVmac239)–infected monkeys following short-term antiviral therapy with PMPA (9-R-[2-phosphonylmethoxypropyl] adenine). Short-term chemotherapy alone led to a transient decrease in plasma and cellular proviral DNA loads and transient rescue of gag/pol and env cytotoxic T-lymphocyte precursors (pCTLs). However, cessation of therapy allowed for SIV infection to resume its clinical course. PMPA chemotherapy coupled with infusions of either autologous pre-SIV infection–collected PBMCs or activated CD4+ T cells led to extended control of plasma and cellular proviral DNA loads after infusion, in spite of the fact that the transfused cells were not primed against SIV. However, qualitatively different antiviral defenses were induced by infusion of unfractionated and unmanipulated PBMCs versus purified and activated CD4+ T cells: PBMC infusions significantly favored development of SIVenv-specific pCTLs, neutralizing antibodies, and secretion of soluble noncytotoxic suppressor factors of SIV replication. In contrast, activated CD4+ T cells predominantly promoted CTL responses to SIVgag/pol and SIVenv. In addition, infusion of influenza-primed activated CD4+ T cells markedly enhanced influenza-specific pCTL responses, whereas infusion of similarly influenza-primed unfractionated PBMCs enhanced such pCTL responses only modestly, suggesting that the predominant immune defect after SIV infection lies in the T helper cell compartment rather than the effector cell compartment. Thus, adoptive immunotherapy with autologous “SIV naı̈ve” CD4+ lymphocytes was sufficient to rescue cell-mediated immune responses and induce long-term anti-SIV control and immune responses in the absence of continued antiviral chemotherapy.

We previously generated a mutant of simian immunodeficiency virus (SIV) lacking 5 of a total of 22 N-glycans in its external envelope protein gp120 with no impairment in viral replication capability and infectivity in tissue culture cells. Here, we infected rhesus macaques with this mutant and found that it also replicated robustly in the acute phase but was tightly, though not completely, contained in the chronic phase. Thus, a critical requirement for the N-glycans for the full extent of chronic infection was demonstrated. No evidence indicating reversion to a wild type was obtained during the observation period of more than 40 weeks. Monkeys infected with the mutant were found to tolerate a challenge infection with wild-type SIV very well. Analyses of host responses following challenge revealed no neutralizing antibodies against the challenge virus but strong secondary responses of cytotoxic T lymphocytes against multiple antigens, including Gag-Pol, Nef, and Env. Thus, the quintuple deglycosylation mutant appeared to represent a novel class of SIV live attenuated vaccine.

Genetic variations, occurring during the infection in the AIDS animal model using a molecular clone virus, often consist of single nucleotide substitutions distributed sparsely in the viral genome. A highly sensitive method was developed to detect such mutations, consisting of denaturing gradient gel electrophoresis (DGGE) and direct sequencing. The genetic variation in the env of a macrophage tropic simian immunodeficiency virus (SIV) in a macaque infected chronically was examined by this technique and compared with that of a T cell tropic SIV in an animal infected chronically. A whole env sequence was amplified by 11 sets of PCR for DNA ranging from 300 to 420 bp. The amplified DNA was subjected to mutational screening by DGGE and to subsequent direct sequencing. Imaging analysis of separated DNA bands by DGGE provided information on the proportion of each variant DNA. The validity of this technique was confirmed by sequencing of variant DNAs cloned by limiting dilutions. Thus, this technique is suitable for analysis of genetic variations in the AIDS animal model using a molecular clone virus.

In AIDS vaccine strategies, live attenuated vaccines can confer good resistance against pathogenic virus infections but have the potential risk of inducing disease, whereas safer replication-negative strategies such as DNA vaccinations have so far failed to prevent the disease onset. Here, we developed a novel DNA vaccine strategy to induce restricted replication of an avirulent virus and evaluated it in a simian immunodeficiency virus (SIV) infection model. We generated a chimeric SIV, FMSIV, by replacing SIV env with ecotropic Friend murine leukemia virus (FMLV) env to confine its replication to FMLV receptor (mCAT1)-expressing cells. In primate cells lacking mCAT1, FMSIV did not replicate unless mCAT1 was introduced exogenously. Vaccination to macaques with both the FMSIV DNA and the mCAT1-expression plasmid DNA induced SIV Gag-specific cellular immune responses and resistance against pathogenic SIV(mac239) challenge more efficiently than the replication-negative control vaccination with the FMSIV DNA alone. This strategy may be useful for development of safe and effective vaccines against various kinds of pathogenic viruses.

A nonnucleoside reverse transcriptase (RT) inhibitor, GW420867, was tested for postexposure prophylaxis (PEP) in rhesus macaques experimentally infected with 100 50% tissue culture infective doses of a chimeric simian/human immunodeficiency virus (SHIV) containing the RT gene of HIV-1 (SHIV-RT). Animals were either mock treated, or treated for 4 weeks starting at 8 or 24 h postinfection (p.i.) with GW420867. While such therapy led to undetectable plasma viremia in three of six monkeys, a transient plasma viremia was noted in the other three treated animals at 2 to 4 weeks following cessation of therapy. Following this transient viremia all drug-treated animals showed low or undetectable levels of plasma viremia up to the last sample examined at 90 weeks p.i. Despite low and/or undetectable viremia, virus-specific cytotoxic T lymphocyte and viral Env-specific proliferative responses were seen in the peripheral blood mononuclear cells of both mock- and drug-treated animals as early as 3 weeks p.i. Such virus-specific cellular responses, however, were better maintained in the drug-treated animals than the mock-treated animals. In contrast to the virus-specific cellular response, the magnitude and kinetics of virus specific humoral responses appeared to correlate with the detection of viremia. These data support the view that a short-term PEP with GW420867 permits the generation and maintenance of long-lasting virus-specific cell-mediated immune responses while markedly reducing viral loads to undetectable levels for a prolonged period of time (90 weeks) and leads to long-term disease protection. This model provides a unique means to define mechanisms and correlates of disease protection.

The efficacy of 6-chloro-2',3'-dideoxyguanosine (6-Cl-ddG) was investigated in vivo by using a male ARC/AIDS rhesus macaque infected with simian immunodeficiency virus (SIVmac251/32H). He was administered subcutaneously 6-Cl-ddG (50 mg/kg B.W.) every 8 hr for 14 days when he showed clinical features of recurrent weight loss, severe diarrhea and neuropathy. The number of CD4+, CD8+ cells and total T cells increased rapidly after administration of 6-Cl-ddG and a high level was maintained for 2 months, but the B cell count decreased during the treatment. The antibody titer to SIV did not change significantly during or after the treatment, but the virus load in the plasma measured by RT-PCR dropped to one-third at the start of the 6-Cl-ddG treatment. Within 3 days after the start of 6-Cl-ddG administration, he began to show recovery in clinical signs including weight increase, and disappearance of diarrhea and neuropathy. These findings suggested that 6-Cl-ddG was effective at the stage of ARC/AIDS in a rhesus monkey infected with SIV.

Peripheral blood T lymphocytes of Old World monkeys, rhesus and cynomolgus monkey (Macaca mulattaandMacaca fascicularis,respectively), were successfully immortalized by infection withHerpesvirus Saimirisubtype C. The T cell lines were stably cultured without addition of exogenous IL-2. TheSTP-C488protein, the oncogene product of subtype C strain 488-77, was detected in these cells by Western blotting. They also expressed some markers of activated or matured T cell phenotypes such as CD2+, monkey Pan-T+, CD25+, CD29+, and MHC-II DR+. Interestingly, not only CD4+CD8− or CD4−CD8+ single positive subpopulations but also CD4+CD8+ double positive ones were present in all of them. Furthermore, they were productively infected with both SIVmac and SIVagm. The levels of the viral replication were comparable to those in human T cell lines. Thus, Herpes Virus Saimiri-immortalized Old World monkey T lymphocytes will be suitable for further studies of immune system in Old World monkeys and cell–virus interactions in SIV infection.

Summary We isolated the full length provirus of human T cell leukaemia virus type I (HTLV-I) from MT-2, a lymphoid cell line producing HTLV-I. In three non-lymphoid cell lines (COS7, human osteosarcoma HOS cells, and HeLa) this provirus expressed a trans-acting activity after co-transfection with a recombinant plasmid carrying a bacterial chloramphenicol acetyltransferase gene under the control of a long terminal repeat of HTLV-I provirus. The trans-acting protein p40 was detected by immunoprecipitation in transfected HOS cells. Structural proteins of HTLV-I, the gag and env products, were also formed and processed in the same manner as observed in MT-2 cells. In transfected HeLa cells, the p40 protein was mainly localized in the nucleus, while other structural proteins were detected in the cytoplasm and/or the membrane by indirect immunofluorescence. Syncytium formation was observed in HeLa cells after transfection. These results demonstrated that non-lymphoid cells could produce the major proteins of HTLV-I after DNA transfection of the cloned provirus.

We purified human prostatic acid phosphatase (hPAP) from prostatic tissues by affinity chromatography, DEAE cellulose and gel filtration and also examined physicochemical properties of highly purified PAP. We developed a double-antibody radioimmunoassay for hPAP in serum, with use of antiserum raised in rabbit against highly purified PAP. The antiserum did not cross react with acid phosphatase from platelets and red blood cells. Experimental detail are outlined to assess the reproducibility and reliability of the method under various conditions. The upper limit of the serum PAP levels in the present assay was set at 3.0 ng/ml by 162 determinations of samples. The serum PAP levels of 2 untreated patients with prostatic carcinoma were higher than 3.0 ng/ml and 39 patients with benign prostatic hyperplasia were an average value of 1.9 ng/ml.

In the course of study to obtain murine dendritic cell lines using oncogenic retroviruses, we have established several immortalized cell lines with characteristics different from those of dendritic cells. The transformants were mainly round nonadherent cells, capable of growing in soft agar, and negative for nonspecific esterase activity. Profiles of cell surface antigens were examined by indirect immunofluorescence technique. The cell lines were positive for Fc receptor (2.4G2), J11d (J11d.2), and B220 (RA3-3A1/6.1) antigens and negative (or dull positive in small percentages) for Ia (M5/144.15.2), IL-2 receptor (3C7), Thy-1 (B5-5), Mac-1 (M1/70.-15.11.5), and macrophage (F4/80) antigens. They were negative for both surface and cytoplasmic immunoglobulins. Several clones were established from these transformant cell lines and cell surface antigens were examined. Antigenic profiles of these clones were very similar to those of the parental cell lines. Some of these clones, however, seemed to increase their Ia antigen expression. The results suggest that the transformants originated from early B-lineage cells.

The complete nucleotide sequence of the cya gene from E. coli was determined. The gene encodes a polypeptide consisting of 848 amino acid residues with a calculated molecular weight of 97,542. The deduced protein structure reveals that cyclase is comprised of two domains, an amino-terminal region exhibiting catalytic activity and a carboxy-terminal region possibly carrying regulatory function. The frequent appearance of rare codons in the beginning of the gene as well as the sequence duplication in the promoter-initiator region suggest possible regulation(s) at the translational level. An unknown gene (cyaX) which seems to code for a very hydrophobic protein was found following the cya gene. Sequence analysis suggests that the cyax is a part of the cya operon.

One of the striking features of human immunodeficiency virus, simian immunodeficiency virus (SIV), and other lentiviruses is extensive N glycosylation of the envelope protein. To assess the requirement of each N glycan for viral infectivity, we individually silenced all 23 N glycosylation sites in the gp120 subunit of SIVmac239 envelope protein by mutagenizing the canonical Asn-Xaa-Thr/Ser N glycosylation motif in an infectious molecular clone, attempted to rescue viruses from the clones, and compared the replication capability of the rescued viruses in MT4 cells. The mutation resulted in either the recovery of a fully infectious virus (category I); recovery of a faster-replicating virus, compared with the parental virus (category II); or no virus recovery (category III). These categorically different sites were not distributed randomly but were clustered. The sites of category I were localized largely in the N-terminal half, whereas the sites of categories II and III were localized in the C-terminal region, including the CD4 binding site, and the central part, including the C loop, respectively. To learn how far SIV can tolerate the removal of glycans, multiplex mutagenesis was also attempted. When they were appreciably distant from one another in the primary sequence, up to five sites could be silenced in combination without disturbing infectivity. On the other hand, it was difficult to silence contiguous sites. Thus, it appeared that a certain degree of sugar chain density over the local region had to be preserved. We discuss the potential utility of these variously deglycosylated mutants for clarifying the role of N glycans in SIV replication in vivo, as well as in the host response, and for designing vaccines and the generation of glycoprotein crystals.

Outbreaks of infectious diseases such as HIV and the much televised and attention-getting outbreaks of diseases such as Ebola, Hantaviruses, and the most recent outbreak of SARS have induced a significant new interest in the formulations and more importantly the science of vaccinology, which has previously to a large extent been conducted empirically. Our laboratory has focused on the use of recombinant nonhuman primate cytokines as adjunctive therapies for inducing antigen-specific immune responses in monkeys because most recombinant human cytokines appear to be immunogenic. This article provides a summary of our work with such cytokines, which includes attempts to define optimum dosing schedules that lead to optimal primary and lasting memory antigen-specific immune responses.