Acquired Immunodeficiency Syndrome: Hayami M

Takemura T, Hayami M. · Institute for Virus Research, Kyoto University, Kyoto, 606-8507, Japan. · Front Biosci. · Pubmed #14766387 No free full text.

Abstract: SIVmnd was isolated from mandrills in Gabon in 1989 soon after the existence of simian counterparts of HIV such as SIVmac and SIVagm was known. Since then the SIVmnd has been long considered as an independent SIV lineage and the natural host is the mandrill. However this initial finding turned out to be more complex by the recent finding of new SIV isolated from mandrills living in northern mandrill habitat, and other SIV isolated from drills and other species. One fact which made these findings complicated was the fact that the SIVmnd made a tight cluster with SIVlhoest and SIVsun from genus Cercopithecus, which is different from genus Mandrillus. A second fact is that the second SIVmnd isolates are different in the genomic structure from the former SIVmnd and almost similar to SIVdrl from drills, and phylogenetically closely related with each other. At present, the former SIVmnd isolate is termed SIVmnd-1 and the second SIVmnd isolate is called SIVmnd-2. Interestingly SIVmnd-2/SIVdrl has the same mosaic structure containing the vpx gene which is absent in SIVmnd-1. The mosaic structure was probably due to a recombination between SIVmnd-1 and SIVrcm from red capped mangabey (or similar viruses) having the vpx gene. However this recombination event is not recent, and SIVmnd-1, SIVmnd-2 and SIVdrl have been likely maintained for a long period of time in each species. In this article, we speculate on the origin and evolution of these SIVs.

Fukazawa Y, Miyake A, Ibuki K, Inaba K, Saito N, Motohara M, Horiuchi R, Himeno A, Matsuda K, Matsuyama M, Takahashi H, Hayami M, Igarashi T, Miura T. · Laboratory of Primate Model, Experimental Research Center for Infectious Diseases, Institute for Virus Research, Kyoto University, 53 Shogoinkawaramachi, Sakyo-ku, Kyoto 606-8507, Japan. · J Virol. · Pubmed #18400862 links to  free full text

Abstract: To analyze the relationship between acute virus-induced injury and the subsequent disease phenotype, we compared the virus replication and CD4(+) T-cell profiles for monkeys infected with isogenic highly pathogenic (KS661) and moderately pathogenic (#64) simian-human immunodeficiency viruses (SHIVs). Intrarectal infusion of SHIV-KS661 resulted in rapid, systemic, and massive virus replication, while SHIV-#64 replicated more slowly and reached lower titers. Whereas KS661 systemically depleted CD4(+) T cells, #64 caused significant CD4(+) T-cell depletion only in the small intestine. We conclude that SHIV, regardless of pathogenicity, can cause injury to the small intestine and leads to CD4(+) T-cell depletion in infected animals during acute infection.

Kuwata T, Kodama M, Sato A, Suzuki H, Miyazaki Y, Miura T, Hayami M. · Laboratory of Primate Model, Experimental Research Center for Infectious Disease, Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan. · AIDS Res Hum Retroviruses. · Pubmed #17411370 No free full text.

Abstract: Monocytes are known as an alternative target for HIV/SIV infection, but the contribution of monocytes to viral spread in a host is unclear. In this study, CD14 monocytes were monitored in 6 macaques until six weeks postinfection (wpi) with SIVmac239 to evaluate their contribution to viral load. The monocyte count in blood significantly increased with peak viremia at 2 wpi and the expression level of CD14 on monocytes significantly decreased at 1-2 wpi, though the number of CD4(+) T cells was stable in these macaques. The number of CD14 monocytes and the expression level of CD14 on monocytes at 2 wpi were also significantly related to the extent of viremia in plasma. An increased number of monocytes at 2 wpi was associated with a lower postacute viral load, suggesting that monocytes have a role in suppressing the virus. The lower expression level of CD14 in monocytes at 2 wpi was associated with a higher viral load and greater degree of infection of monocytes. This correlation suggests that monocytes with a low level of CD14 may be more susceptible to SIV and may enhance viral replication. The analysis of monocytes in persistently infected macaques revealed that the expression level of CD14 was also significantly low during persistent infection compared with naïve macaques, though the monocyte count was within the normal range. Monocytes may suppress viruses, perhaps by their immune function, during acute infection. However, infection of monocytes may increase the viral load and spread viruses in a host.

Ishimatsu M, Suzuki H, Akiyama H, Miura T, Hayami M, Ido E. · Laboratory for Viral Replication, Center for Emerging Virus Research, Institute for Virus Research, Kyoto University, 53 Shogoin-Kawaracho, Kyoto 606-8507, Japan. · Microbes Infect. · Pubmed #17350308 No free full text.

Abstract: We generated a novel SHIV (termed SHIV-pr) that possesses the HIV-1-derived protease (PR) gene in the corresponding position in the SIVmac genome. SHIV-pr is replication-competent in human and monkey CD4(+) T lymphoid cell lines as well as rhesus macaque PBMCs. The viral growth of SHIV-pr was completely blocked in the presence of a peptide-analog PR inhibitor at the tissue culture level. When SHIV-pr was intravenously inoculated into two rhesus macaques, it resulted in a weak but long-lasting persistent infection in one monkey, whereas the infection of another was only temporary. To enhance the viral growth competence by adaptation, we then passaged the virus in vivo from a monkey up to the fourth generation. The initial peak values of plasma viral loads as well as the setpoint values increased generation by generation and reached those of a parental virus SIVmac. When a medication using the content of Kaletra capsule (a mixture of two PR inhibitors, lopinavir and ritonavir) was orally given to three SHIV-pr-infected monkeys for 4 weeks, plasma viral loads dropped to near or below the detection limit and quickly rebounded after the cessation of medication. The results suggest that SHIV-pr can be used to evaluate PR inhibitors using monkeys.

Shimizu Y, Inaba K, Kaneyasu K, Ibuki K, Himeno A, Okoba M, Goto Y, Hayami M, Miura T, Haga T. · Department of Veterinary Microbiology, University of Miyazaki, Miyazaki 889-2192, Japan. · Virology. · Pubmed #17157892 No free full text.

Abstract: Regulated-on-activation-normal-T-cell-expressed-and-secreted (RANTES), a CC-chemokine, enhances antigen-specific T helper (Th) type-1 responses against HIV-1. To evaluate the adjuvant effects of RANTES against HIV vaccine candidate in SHIV-macaque models, we genetically engineered a live-attenuated SHIV to express the RANTES gene (SHIV-RANTES) and characterized the virus's properties in vivo. After the vaccination, the plasma viral loads were same in the SHIV-RANTES-inoculated monkeys and the parental nef-deleted SHIV (SHIV-NI)-inoculated monkeys. SHIV-RANTES provided some immunity in monkeys by remarkably increasing the antigen-specific CD4+ Th cell-proliferative response and by inducing an antigen-specific IFN-gamma ELISpot response. The magnitude of the immunity in SHIV-RANTES-immunized animals, however, failed to afford greater protection against a heterologous pathogenic SHIV (SHIV-C2/1) challenge compared to control SHIV-NI-immunized animals. SHIV-RANTES immunized monkeys, elicited robust cellular CD4+ Th responses and IFN-gamma ELISpot responses after SHIV-C2/1 challenge. These findings suggest that the chemokine RANTES can augment vaccine-elicited, HIV-specific CD4+ T cell responses.

Motohara M, Ibuki K, Miyake A, Fukazawa Y, Inaba K, Suzuki H, Masuda K, Minato N, Kawamoto H, Nakasone T, Honda M, Hayami M, Miura T. · Laboratory of Primate Model, Experimental Research Center for Infectious Diseases, Institute for Virus Research, Kyoto University, 53 Shogoinkawara-Machi, Sakyo-Ku, Kyoto 606-8507, Japan. · Microbes Infect. · Pubmed #16702011 No free full text.

Abstract: One of the mechanisms by which HIV infection induces the depletion of CD4+ T cells has been suggested to be impairment of T-cell development in the thymus, although there is no direct evidence that this occurs. To examine this possibility, we compared T-cell maturation in the intrathymic progenitors between macaques infected with an acute pathogenic chimeric simian-human immunodeficiency virus (SHIV), which causes profound and irreversible CD4+ T-cell depletion, and macaques infected with a less pathogenic SHIV, which causes only a transient CD4+ T-cell decline. Within 27 days post-inoculation (dpi), the two virus infections caused similar increases in plasma viral loads and similar decreases in CD4+ T-cell counts. However, in the thymus, the acute pathogenic SHIV resulted in increased thymic involution, atrophy and the depletion of immature T cells including CD4(+)CD8(+) double-positive (DP) cells, whereas the less pathogenic SHIV did not have these effects. Ex vivo differentiation of CD3(-)CD4(-)CD8(-) triple-negative (TN) intrathymic progenitors to DP cells was assessed by a monkey-mouse xenogenic fetal thymus organ culture system. Differentiation was impaired in the TN intrathymic progenitors of the acute pathogenic SHIV-infected monkeys, while differentiation was not impaired in the TN intrathymic progenitors of the less pathogenic SHIV-infected monkeys. These differences suggest that dysfunction of thymic maturation makes an important contribution to the irreversible depletion of circulating CD4+ T cells in vivo.

Miyake A, Ibuki K, Enose Y, Suzuki H, Horiuchi R, Motohara M, Saito N, Nakasone T, Honda M, Watanabe T, Miura T, Hayami M. · Institute for Virus Research, Laboratory of Primate Model, Experimental Research Center for Infectious Disease, Kyoto University, Sakyo-ku, Japan. · J Gen Virol. · Pubmed #16603534 links to  free full text

Abstract: A better understanding of virological events during the early phase of human immunodeficiency virus 1 (HIV-1) infection is important for development of effective antiviral vaccines. In this study, by using quantitative PCR and an infectious plaque assay, virus distribution and replication were examined in various internal organs of rhesus macaques for almost 1 month after intrarectal inoculation of a pathogenic simian immunodeficiency virus/HIV chimeric virus (SHIV-C2/1-KS661c). At 3 days post-inoculation (p.i.), proviral DNA was detected in the rectum, thymus and axillary lymph node. In lymphoid tissues, infectious virus was first detected at 6 days p.i. and a high level of proviral DNA and infectious virus were both detected at 13 days p.i. By 27 days p.i., levels of infectious virus decreased dramatically, although proviral DNA load remained unaltered. In the intestinal tract, levels of infectious virus detected were much lower than in lymphoid tissues, whereas proviral DNA was detected at the same level as in lymphoid tissues throughout the infection. In the thymus and jejunum, CD4CD8 double-positive T cells were depleted earlier than CD4 single-positive cells. These results show that the virus spread quickly to systemic tissues after mucosal transmission. Thereafter, infectious virus was actively produced in the lymphoid tissues, but levels decreased significantly after the peak of viraemia. In contrast, in the intestinal tract, infectious virus was produced at low levels from the beginning of infection. Moreover, virus pathogenesis differed in CD4 single-positive and CD4CD8 double-positive T cells.

Kaneyasu K, Kita M, Ohkura S, Yamamoto T, Ibuki K, Enose Y, Sato A, Kodama M, Miura T, Hayami M. · Institute for Virus Research, Kyoto University, Kyoto, Kyoto 606-8507, Japan. · Microbiol Immunol. · Pubmed #16365534 links to  free full text

Abstract: We previously reported that a nef-deleted SHIV (SHIV-NI) is nonpathogenic and gave macaques protection from challenge infection with pathogenic SHIV-C2/1. To investigate whether IFN-gamma augments the immune response induced by this vaccination, we examined the antiviral and adjuvant effect of recombinant human IFN-gamma (rIFN-gamma) in vaccinated and unvaccinated monkeys. Nine monkeys were vaccinated with nef-deleted nonpathogenic SHIV-NI. Four of them were administered with rIFN-gamma and the other five monkeys were administered with placebo. After the challenge with pathogenic SHIV-C2/1, CD4(+) T-cell counts were maintained similarly in monkeys of both groups, while those of the unvaccinated monkeys decreased dramatically at 2 weeks after challenge. However, the peaks of plasma viral load were reduced to 100-fold in SHIV-NI vaccinated monkeys combined with rIFN-gamma compared with those in SHIV-NI vaccinated monkeys without rIFN-gamma. The peaks of plasma viral load were inversely correlated with the number of SIV Gag-specific IFN-gamma-producing cells. In SHIV-NI-vaccinated monkeys with rIFN-gamma, the number of SIV Gag-specific IFN-gamma-producing cells of PBMCs increased 2-fold compared with those in SHIV-NI-vaccinated monkeys without rIFN-gamma, and the NK activity and MIP-1alpha production of PBMCs were also enhanced. Thus, vaccination of SHIV-NI in combination with rIFN-gamma was more effective in modulating the antiviral immune system into a Th1 type response than SHIV-NI vaccination alone. These results suggest that IFN-gamma augmented the anti-viral effect by enhancing innate immunity and shifting the immune response to Th1.

Shimizu Y, Miyazaki Y, Ibuki K, Suzuki H, Kaneyasu K, Goto Y, Hayami M, Miura T, Haga T. · Department of Veterinary Microbiology, University of Miyazaki, Miyazaki 889-2192, Japan. · Virology. · Pubmed #16169034 No free full text.

Abstract: TNF-alpha has been implicated in the pathogenesis of, and the immune response against, HIV-1 infection. To clarify the roles of TNF-alpha against HIV-1-related virus infection in an SHIV-macaque model, we genetically engineered an SHIV to express the TNF-alpha gene (SHIV-TNF) and characterized the virus's properties in vivo. After the acute viremic stage, the plasma viral loads declined earlier in the SHIV-TNF-inoculated monkeys than in the parental SHIV (SHIV-NI)-inoculated monkeys. SHIV-TNF induced cell death in the lymph nodes without depletion of circulating CD4(+) T cells. SHIV-TNF provided some immunity in monkeys by increasing the production of the chemokine RANTES and by inducing an antigen-specific proliferation of lymphocytes. The monkeys immunized with SHIV-TNF were partly protected against a pathogenic SHIV (SHIV-C2/1) challenge. These findings suggest that TNF-alpha contributes to the induction of an effective immune response against HIV-1 rather than to the progression of disease at the early stage of infection.

Miyake A, Ibuki K, Suzuki H, Horiuchi R, Saito N, Motohara M, Hayami M, Miura T. · Institute for Virus Research, Kyoto University, Kyoto, Japan. · J Med Primatol. · Pubmed #16128924 No free full text.

Abstract: Children infected with human immunodeficiency virus type 1 often have higher viral loads and progress to acquired immunodeficiency syndrome more rapidly than adults. In our previous study of simian-human immunodeficiency virus (SHIV)-infected adult monkeys, immature CD4CD8 double-positive T cells in the thymus and jejunum decreased faster than mature CD4 single-positive T cells. Here, we examined the effect of virus replication on immature T cells from the same SHIV-inoculated newborn monkeys having more immature T cells than adults. The infectious viruses were more abundantly detected in the thymus than in other tissues at both 13 and 26 days post-infection (dpi). However, mature CD4(+) T cells in the thymus declined after 13 dpi and immature CD3(-) CD4 single-positive T cells remained at 26 dpi. These results suggested that many immature CD4(+) T cells in the thymus of newborns support the production of infectious viruses even after the depletion of mature CD4(+) T cells.

Suzuki H, Motohara M, Miyake A, Ibuki K, Fukazawa Y, Inaba K, Masuda K, Minato N, Kawamoto H, Hayami M, Miura T. · Laboratory of Primate Model, Institute for Virus Research, Kyoto University, Japan. · Microbiol Immunol. · Pubmed #16034211 links to  free full text

Abstract: We intrarectally infected newborn macaques with a pathogenic simian/human immunodeficiency virus (SHIV) that induced rapid and profound CD4 (+) T cell depletion, and examined the early effects of this SHIV on the thymus. After intrarectal infection, viral loads were much higher in the thymus than in other lymphoid tissues in newborns. In contrast, no clear difference was seen in the viral loads of different tissues in adults. Histological and immunohistochemical observations showed severe thymic involution. Depletion of CD4 (+) thymocytes began in the medulla at 2 weeks post infection and spread over the whole thymus. After in vivo infection, the CD2 (+) subpopulation, which represents a relatively later stage of T cell progenitors, was selectively reduced and development of thymocytes from CD3 (-) CD4 (-) CD8 (-) cells to CD4 (+) CD8 (+) cells was impaired. These results suggest that profound and irreversible loss of CD4 (+) cells that are observed in the peripheral blood of SHIV-infected monkeys are due to destruction of the thymus and impaired thymopoiesis as a result of SHIV infection in the thymus.

Takemura T, Ekwalanga M, Bikandou B, Ido E, Yamaguchi-Kabata Y, Ohkura S, Harada H, Takehisa J, Ichimura H, Parra HJ, Nende M, Mubwo E, Sepole M, Hayami M, Miura T. · Institute for Virus Research, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. · J Gen Virol. · Pubmed #15958675 links to  free full text

Abstract: In order to understand primate lentivirus evolution, characterization of additional simian immunodeficiency virus (SIV) strains is essential. Here, an SIV from a black mangabey (Lophocebus aterrimus) originating from the Democratic Republic of Congo was analysed phylogenetically. The monkey had cross-reactive antibodies against human immunodeficiency virus type 1 (HIV-1) and HIV-2. The viral pol region sequence was amplified by nested PCR and sequence analysis confirmed that it was related to known SIV sequences. This is the first report to characterize genetically an SIV from the monkey genus Lophocebus. Phylogenetic analysis of the pol region revealed that this novel SIV, designated SIVbkm, fell into the SIVsyk and SIVgsn virus group, containing viruses isolated from the genus Cercopithecus, and suggests that cross-species transmission has occurred between species of the genera Lophocebus and Cercopithecus.

Takahashi M, Ido E, Uesaka H, Fukushima T, Ibuki K, Miura T, Hayami M, Takahashi H. · Department of Microbiology and Immunology, Nippon Medical School, Tokyo, Japan. · Arch Virol. · Pubmed #15841338 No free full text.

Abstract: CD4-bearing T cells are the primary targets for human immunodeficiency virus type 1(HIV-1)/simian immunodeficiency virus (SIV) infection. However, it is unclear whether the susceptibility of CD4-bearing T cells including CD4 single positive and CD4/8 double positive T cells to HIV/SIV infection is the same or not. In this study, we compared the susceptibility to SIV infection between CD4(+) and CD4(+)8(+) T cells, using Herpesvirus saimiri (HVS)-transformed CD4(+) and CD4(+)8(+) T cells established from peripheral blood mononuclear cells (PBMC) of rhesus macaques. Although there was little difference between the two CD4-bearing T cell population in the expression level of CD4 molecules and chemokine receptors such as CXCR4 and CCR5, SIV replicated more efficiently in CD4(+)8(+) T cells than in CD4(+) T cells. Moreover, we found that reverse transcription initiated more efficiently in CD4(+)8(+) T cells than in CD4(+) T cells and that the cell lysates from CD4(+) T cells impaired the RT activity more strongly than that from CD4(+)8(+) T cells. These findings suggest that intracellular environment in CD4(+) 8(+) T cells is better for reverse transcription and that the infection of those CD4(+)8(+) T cells might play critical and different roles in HIV-1/SIV infection and dissemination.

Enose Y, Kita M, Yamamoto T, Suzuki H, Miyake A, Horiuchi R, Ibuki K, Kaneyasu K, Kuwata T, Takahashi E, Sakai K, Shinohara K, Miura T, Hayami M. · Institute for Virus Research, Kyoto University, Kyoto, Japan. · Arch Virol. · Pubmed #15593414 No free full text.

Abstract: To clarify the involvement of primitive non-specific immune responses in the protective effects of a live, attenuated virus, each two rhesus macaques were intravenously immunized with an attenuated chimeric simian and human immunodeficiency virus (SHIV) in which the nef gene was deleted (SHIV-NI) or a SHIV having human IFN-gamma inserted into the deleted nef region (SHIV IFN-gamma). These immunized monkeys were intravenously challenged with a heterologous pathogenic SHIV (SHIV-C2/1) at four weeks post immunization (wpi). After vaccination, one of each SHIV-NI- or SHIV IFN-gamma-immunized monkeys showed a low level of SIV Gag-specific lymphocyte proliferative response but did not have neutralizing antibodies to both the parental and challenge viruses. After the challenge, the plasma viral RNA loads of the challenge virus were suppressed in all the immunized monkeys and the severe CD4+ T cell loss observed in the unimmunized monkeys was not found. Thus, both SHIV IFN-gamma and SHIV-NI infections could prevent from disease progression by a pathogenic virus early after immunization, suggesting that primitive non-specific immune response elicited by attenuated virus infection, in addition to highly acquired virus-specific immunity, contributes to the protective effect against a pathogenic virus.

Miyake A, Akagi T, Enose Y, Ueno M, Kawamura M, Horiuchi R, Hiraishi K, Adachi M, Serizawa T, Narayan O, Akashi M, Baba M, Hayami M. · Laboratory of Primate Model, Experimental Research Center for Infectious Disease, Institute for Virus Research, Kyoto University, Kyoto, Japan. · J Med Virol. · Pubmed #15170630 No free full text.

Abstract: We have previously reported that concanavalin A-immobilized polystyrene nanospheres (Con A-NS) could efficiently capture HIV-1 particles and that intranasal immunization with inactivated HIV-1-capturing nanospheres (HIV-NS) induced vaginal anti-HIV-1 IgA antibody response in mice. In this study, to evaluate the protective effect of immunization, each three macaques was intranasally immunized with Con A-NS or inactivated simian/human immunodeficiency virus KU-2-capturing nanospheres (SHIV-NS) and then intravaginally challenged with a pathogenic virus, SHIV KU-2. After a series of six immunizations, vaginal anti-HIV-1 gp120 IgA and IgG antibodies were detected in all SHIV-NS-immunized macaques. After intravaginal challenge, one of the three macaques in each of the Con A-NS- and SHIV-NS-immunized groups was infected. Plasma viral RNA load of infected macaque in SHIV-NS-immunized macaques was substantially less than that in unimmunized control macaque and reached below the detectable level. However, it could not be determined whether intranasal immunization with SHIV-NS is effective in giving complete protection against intravaginal challenge. To explore the effect of the SHIV-NS vaccine, the remaining non-infected macaques were rechallenged intravenously with SHIV KU-2. After intravenous challenge, all macaques became infected. However, SHIV-NS-immunized macaques had lower viral RNA loads and higher CD4(+) T cell counts than unimmunized control macaques. Plasma anti-HIV-1 gp120 IgA and IgG antibodies were induced more rapidly in the SHIV-NS-immunized macaques than in the controls. The rapid antibody responses having neutralizing activity might contribute to the clearance of the challenge virus. Thus, SHIV-NS-immunized macaques exhibited partial protection to vaginal and systemic challenges with SHIV KU-2.

Miyake A, Enose Y, Ohkura S, Suzuki H, Kuwata T, Shimada T, Kato S, Narayan O, Hayami M. · Institute for Virus Research, Kyoto University, Kyoto, Japan. · Arch Virol. · Pubmed #15098109 No free full text.

Abstract: 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.

Iida T, Kuwata T, Ui M, Suzuki H, Miura T, Ibuki K, Takahashi H, Yamamoto T, Imanishi J, Hayami M, Kita M. · Department of Microbiology, Kyoto Prefectural University of Medicine, Kyoto, Japan. · Arch Virol. · Pubmed #15045561 No free full text.

Abstract: A nef-deleted SHIV-NM-3rN (SHIV-NI) was previously shown to be nonpathogenic and to induce protective immunity. In the present study, a SHIV-NI expressing human interferon-gamma (SHIV-IFN-gamma) was constructed and the effect of co-expression of IFN-gamma on virus replication and immunopotentiation was investigated in macaques that were vaccinated with both viruses, by comparing cytokine responses during the first 4 weeks after vaccination. Peripheral blood mononuclear cells (PBMC) isolated from vaccinated macaques were stimulated with inactivated viral particles for 24 h, and the production of IL-2, IL-4, IL-6, IL-10, IL-12, TNF-alpha and IFN-gamma was determined by ELISA and flow cytometry. All of the vaccinated macaques showed increases in cytokine production. However, the production of IFN-gamma (Th1-type cytokine) was more rapidly induced by SHIV-IFN-gamma vaccination, and IFN-gamma-producing cells appeared to be still increasing at 4 weeks after vaccination, although the difference of virus replication during the time was not significant in contrast to in vitro replication in cultured PBMC. These results suggest that co-expression of IFN-gamma with SHIV can modulate the antiviral immune responses into the Th1 type response, which would probably provide more protective immunity.

Yoshimura K, Ido E, Akiyama H, Kimura T, Aoki M, Suzuki H, Mitsuya H, Hayami M, Matsushita S. · Division of Clinical Retrovirology and Infectious Diseases, Center for AIDS Research, Kumamoto University, 2-2-1 Honjo, Kumamoto, 860-0811, Japan. · J Virol Methods. · Pubmed #12951220 No free full text.

Abstract: The objective of this study was to assess the impact of highly active antiretroviral therapy (HAART) by an oral route on the peripheral blood CD8 subset in the monkeys infected persistently with a pathogenic strain, SHIV(89.6P). Two rhesus macaques were inoculated intravenously with SHIV(89.6P), then treated with the combination of AZT, 3TC and Lopinavir/Ritonavir (LPV/RTV) as recommended in humans by the oral route with confectionery continued for 28 days. In one of two chronically infected macaques, MM260, the viral load was maintained in the range of 10(4)-10(5) copies/ml before HAART. The plasma viral load and proviral DNA decreased dramatically during the treatment, and cessation of this therapy the viral load rebounded to the pre-treatment level but the proviral DNA rebound was delayed. The other monkey, MM242, had low viral loads (1.2x10(3)-<5x10(2) copies/ml) both before and after HAART. CD4(+) and CD8(+) T cell counts and proviral DNA level were not significantly changed after the treatment. The percentages of CD8(+)CD45RA(-)Ki67(+)cells increased during (MM260) or after (MM242) HAART and the subset was maintained at a high percentage until 18 weeks post HAART in MM242. These findings suggest that this primate model might serve an important role in testing the virological and immunological efficacy of novel therapeutic strategies combined with HAART.

Akahata W, Ido E, Akiyama H, Uesaka H, Enose Y, Horiuchi R, Kuwata T, Goto T, Takahashi H, Hayami M. · Laboratory of Viral Pathogenesis, Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan. · J Gen Virol. · Pubmed #12867656 links to  free full text

Abstract: A DNA vaccination regime was investigated previously in rhesus macaques using a full-genome human immunodeficiency virus type 1 (HIV-1) plasmid, which, due to mutations in the nucleocapsid (NC) proteins, produced only non-infectious HIV-1 particles (Akahata et al., Virology 275, 116-124, 2000). In that study, four monkeys were injected intramuscularly 14 times with the plasmid. All of them showed immunological responses against HIV-1 and partial protection from challenge with a simian immunodeficiency virus/HIV (SHIV) chimeric virus. To improve this DNA vaccination regime, the plasmid used for vaccination was changed. In the present study, four macaques were injected intramuscularly eight times with a full-genome SHIV plasmid that produces non-infectious SHIV particles. CTL activities were higher than those observed in monkeys vaccinated previously with the HIV-1 plasmid. In all macaques vaccinated, peak plasma virus loads after homologous challenge with SHIV were two to three orders of magnitude lower than those of the naive controls, and virus loads fell below the level of detection at 6 weeks post-challenge. This suggested that the vaccination regime in this study was partially effective and better than the previous regime.

Akiyama H, Ido E, Akahata W, Kuwata T, Miura T, Hayami M. · Institute for Virus Research, Laboratory of Viral Pathogenesis, Kyoto University, 53 Shogoin-kawaharacho, Sakyo-ku, Kyoto 606-8507, Japan. · J Gen Virol. · Pubmed #12810859 links to  free full text

Abstract: A new simian/human immunodeficiency virus (SHIV) chimera with the reverse transcriptase (RT)-encoding region of pol, in addition to the 3' region encoding vpr, vpu, tat, rev, env and nef of HIV-1, on an SIV(mac) (SIV from a macaque monkey) background was constructed. This new SHIV chimera, named SHIVrt/3rn, could replicate in monkey peripheral blood mononuclear cells (PBMCs) as well as in the human and monkey CD4(+) T-cell lines M8166 and HSC-F. Since SHIVrt/3rn contains the RT gene of HIV-1, replication of the virus in M8166 cells was inhibited by an HIV-1-specific non-nucleoside RT inhibitor, MKC-442, with a sensitivity similar to that of HIV-1. To investigate the replication competence of SHIVrt/3rn in vivo, two rhesus monkeys were inoculated intravenously with the virus. At 2 to 4 weeks post-inoculation (p.i.), plasma viral RNA loads of both monkeys showed a peak value of more than 10(4) copies ml(-1). Infectious virus was isolated from the PBMCs of one monkey at 2 and 3 weeks p.i. and from the other at 4 weeks p.i. Moreover, proviral DNA was detected constantly throughout the observation period, starting from 3 weeks p.i. An antibody response, detected first at 3 weeks p.i., was maintained at high titres. These results indicate that SHIVrt/3rn can infect and replicate in vivo. SHIVrt/3rn, having part of HIV-1 pol in addition to the 3' part of the HIV-1 genome is genetically more close to HIV-1 than any of the other monkey-infecting SHIVs reported previously.

Shimada T, Suzuki H, Motohara M, Kuwata T, Ibuki K, Ui M, Iida T, Fukumoto M, Miura T, Hayami M. · Department of Pathology, Kyoto City Hospital, 604-8845, Kyoto, Japan. · Virology. · Pubmed #12642106 No free full text.

Abstract: To clarify the early pathological events in simian and human immunodeficiency chimeric virus (SHIV)-infected lymphoid organs, we examined rhesus macaques infected with an acute pathogenic SHIV (SHIV89.6P) or a nonpathogenic SHIV (NM-3rN) by sequential biopsies and serial necropsies. In the SHIV89.6P-infected monkeys, acute thymic involution as shown by increased cortical tingible-body macrophages and by neutrophilic infiltrates without follicular aggregation in the medulla began within 14 days postinoculation (dpi). Cells that were strongly positive for the virus were identified in the thymic medulla. SHIV89.6P-infected lymph nodes showed severe paracortical lymphadenitis with scattered virus-positive cells at 14 dpi and they developed paracortical depletion without the obvious follicular involution. In contrast, NM-3rN-infected monkeys showed no signs of thymic dysinvolution and the lymph nodes exhibited only follicular hyperplasia. NM-3rN-infected monkeys showed much fewer virus-positive cells in these lymphoid tissues than did SHIV89.6P-infected monkeys during the same period. These differences clearly reflect the difference in the virulence of these SHIVs.

Haga T, Shimizu Y, Okoba M, Kumabe S, Goto Y, Shinjo T, Ichimura H, Kuwata T, Hayami M, Miura T. · Department of Veterinary Microbiology, Miyazaki University, Kibanadai Nishi, Miyazaki 889-2192, Japan. · Microbiol Immunol. · Pubmed #12597359 links to  free full text

Abstract: Tumor necrosis factor-alpha (TNF-alpha) has been reported to be involved in the development and progression of acquired immunodeficiency syndrome (AIDS). To study the role of this cytokine in AIDS pathogenesis, we constructed a chimeric simian and human immunodeficiency virus (SHIV) having the human TNF-alpha gene (SHIV-TNF) and characterized its properties in vitro. SHIV-TNF replicated both in M8166, a human T cell line, and in monkey peripheral blood mononuclear cells (PBMCs). Along with SHIV-TNF replication, TNF-alpha was detected in the culture supernatant by ELISA. The maximum expression level of TNF-alpha reached 120 ng/ml in M8166 cells, and 2.5 ng/ml in monkey PBMCs. The expressed TNF was biologically active, as shown by a cytotoxic assay using TNF-sensitive L929 mouse fibroblasts. This activity was detected at least until 10 passages of SHIV-TNF (74 days after the initial infection). In monkey PBMCs, SHIV-TNF replicated much better than the parental SHIV-NI. Flow cytometric analysis showed that the death of monkey PBMCs infected with SHIV-TNF was severer than that caused by the parental SHIV-NI. These results suggest that SHIV-TNF would be useful for inducing the disease in a monkey model, which may contribute to a better understanding of the role of TNF-alpha in AIDS etiology.

Kuwata T, Takemura T, Takehisa J, Miura T, Hayami M. · Institute for Virus Research, Kyoto University, Japan. · Arch Virol. · Pubmed #12111423 No free full text.

Abstract: Chimeric simian and human immunodeficiency viruses (SHIVs) are useful for investigating the pathogenicity of human immunodeficiency virus (HIV-1) and to develop an anti-HIV-1 vaccine. We attempted to construct SHIVs containing Env from various subtypes, because almost all SHIVs which have been reported so far have Env from HIV-1 that belongs to subtype B. Two infectious SHIVs containing Env from two strains of HIV-1, CMR304 and CMR306, which belong to subtype F and A, respectively, were newly obtained. These SHIVs essentially showed a coreceptor usage and a neutralization pattern that were similar to those of the parental HIV-1s. In macaque PBMC, SHIVcmr304 replicated with kinetics similar to that of prototypic SHIV-NM-3rN with HIV-1 NL432 Env, but SHIVcmr306 replicated poorly. Inoculation of four rhesus macaques with SHIVcmr304 resulted in an increase of plasma viral load in all the macaques, though viral RNA copies were 100-fold lower than that in the infection with NM-3rN. This SHIV containing Env from HIV-1 subtype F will be a valuable source for the analysis of HIV-1 subtype F and the evaluation of vaccine candidates as a genetically divergent challenge virus.

Kwofie TB, Miura T, Ibuki K, Enose Y, Suzuki H, Ui M, Kuwata T, Hayami M. · Laboratory of Viral Pathogenesis, Research Center for AIDS, Institute for Virus Research, Kyoto University, Japan. · Arch Virol. · Pubmed #12111421 No free full text.

Abstract: Monkeys that have been vaccinated with nef-deleted SHIVs were either fully or partially protected against challenge with acute pathogenic SHIV-89.6 P. Viruses isolated from these vaccinated monkeys were all found to be the 89.6 P challenge virus using PCR amplification and restriction enzyme analysis of the env region of the viruses. Analysis of the 3'-end of the env region and 5'-half of the nef region using a heteroduplex mobility assay revealed that the parental 89.6 P and re-isolated viruses from unvaccinated 89.6 P-infected monkeys had quite an abundant and similar heterogeneous quasispecies population. In contrast, the viruses isolated from the vaccinated monkeys had different and fewer quasispecies indicating a selective immune pressure in the vaccinated monkeys. The in vitro replication of the viruses isolated from the vaccinated monkeys in human and macaque peripheral blood mononucular cells (PBMCs) as well as in established cell lines such as M8166 and HSC-F cells, were slow and delayed when compared to the parental 89.6 P and re-isolated viruses from unvaccinated 89.6 P-infected monkeys. Further comparison revealed that in HSC-F cells the viruses from vaccinated monkeys again showed delayed and weak CD4(+) cell down-modulation as well as having little or no effect on cell growth or cell viability on HSC-F cells and monkey PBMC. Thus we noticed that these re-isolated 89.6 P viruses from the vaccinated monkeys had changed or had been selected for low pathogenic viruses in the monkeys. This suggests that though the vaccination did not completely prevent the replication of the challenge virus in the monkeys it did contain the challenge virus by suppressing the pathogenic variants. This further enhances the prospects of this nef-deleted SHIV as the bases for effective anti-HIV vaccine candidates.

Miyazaki Y, Kuwata T, Takehisa J, Hayami M. · Laboratory of Viral Pathogenesis, Institute for Virus Research, Kyoto University, 53 Shogoin Kawahara-cho, Kyoto 606-8507, Japan. · AIDS Res Hum Retroviruses. · Pubmed #11958690 No free full text.

Abstract: To investigate the changes of neutralizing antibodies and viruses during simian and human immunodeficiency virus (SHIV) infection, we examined the cross-neutralizing ability of sequential sera from three macaques infected with SHIV, NM-3rN, and analyzed the sensitivity of the reisolate to neutralizing antibodies. Neutralizing activities of macaques' sera against the parental HIV-1 showed a persistent increase. Neutralizing activities were highly strain specific, but the spectrum of the neutralizing activity expanded against various clades of primary HIV-1s at 3 years after infection in one of the three macaques. The reisolate from an NM3-rN-infected macaque at 56 wpi, designated as R4356, was neutralized by sera from this macaque at a much lower titer than NM-3rN, even by the sera collected 2 years after the reisolation. Sera from macaques that were newly infected with R4356 also did not neutralize R4356 despite neutralizing NM-3rN strongly. These results suggested that long-term persistent infection with SHIV induced neutralizing antibodies with a broad spectrum. However, a virus resistant to the neutralizing antibodies emerged in the persistently infected macaque.