Tomoyuki Miura

Natural Infection of Wild-Born Mandrills (Mandrillus sphinx) with Two Different Types of Simian Immunodeficiency Virus

We found a novel primate lentivirus in mandrill (Mandrillus sphinx). To clarify the evolutionary relationships and transmission patterns of human/simian immunodeficiency virus (HIV/SIV), we screened blood samples from 30 wild-born healthy Cameroonian mandrills. Five (16.7%) of them were seropositive for SIV. Three SIV strains were isolated from the five seropositive mandrills by cocultivation of their peripheral blood mononuclear cells (PBMCs) with PBMCs of rhesus macaques, a human T cell line (M8166), and/or a cynomolgus macaque T cell line (HSC-F). One of the newly isolated SIV strains was intravenously inoculated into two rhesus macaques and resulted in chronic infection. In the SIV-infected macaques at 45 weeks after inoculation, we observed a mild decline in the number of peripheral CD4(+) lymphocytes, lymphadenopathy, and blastic follicular dendritic cells with mild follicular hyperplasia in the peripheral lymph nodes. A phylogenetic analysis based on the pol sequence showed that the newly found SIVs from Cameroonian mandrills did not cluster with SIVmndGB1, which is the former representative strain of SIVmnd. The SIVmnds from Cameroon formed a new, independent lineage that branched before the root of the HIV-1/SIVcpz lineage with 996 of 1000 bootstrap replications. They clustered host specifically, and exhibited about 16.9% diversity at the level of nucleotide sequence among Cameroonian SIVmnd strains. These results indicate that the SIVmnds isolated in Cameroon are a novel type of SIVmnd and have infected Cameroonian mandrills for a long time. We therefore designated the Cameroonian SIVmnd as SIVmnd type 2 and redesignated SIVmndGB1 as SIVmnd type 1. To date, M. sphinx is the only primate species other than humans that is naturally infected with two different types of SIV.

Protective effects of nef-deleted SHIV or that having IFN-γ against disease induced with a pathogenic virus early after vaccination

Summary.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-γ inserted into the deleted nef region (SHIV IFN-γ). 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-γ-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-γ 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.

Augmentation of antigen-specific cytokine responses in the early phase of vaccination with a live-attenuated simian/human immunodeficiency chimeric virus expressing IFN-γ

Summary.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-γ (SHIV-IFN-γ) was constructed and the effect of co-expression of IFN-γ 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 24u2009h, and the production of IL-2, IL-4, IL-6, IL-10, IL-12, TNF-α and IFN-γ was determined by ELISA and flow cytometry. All of the vaccinated macaques showed increases in cytokine production. However, the production of IFN-γ (Th1-type cytokine) was more rapidly induced by SHIV-IFN-γ vaccination, and IFN-γ-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-γ with SHIV can modulate the antiviral immune responses into the Th1 type response, which would probably provide more protective immunity.

Construction and in vitro properties of chimeric simian and human immunodeficiency virus with the human TNF-alpha gene.

Tumor necrosis factor‐alpha (TNF‐α) 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‐α 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‐α was detected in the culture supernatant by ELISA. The maximum expression level of TNF‐α 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‐α in AIDS etiology.

Infection of macaques with chimeric simian and human immunodeficiency viruses containing Env from subtype F

Summaryu2002Chimeric 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-1NL432 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.

Comparative histopathological studies in the early stages of acute pathogenic and nonpathogenic SHIV-infected lymphoid organs.

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.

Characterization of simian and human immunodeficiency chimeric viruses re-isolated from vaccinated macaque monkeys after challenge infection

Summaryu2002Monkeys that have been vaccinated with nef-deleted SHIVs were either fully or partially protected against challenge with acute pathogenic SHIV-89.6u2009P. Viruses isolated from these vaccinated monkeys were all found to be the 89.6u2009P 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 89.6u2009P and re-isolated viruses from unvaccinated 89.6u2009P-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 89.6u2009P and re-isolated viruses from unvaccinated 89.6u2009P-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 89.6u2009P 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.

Apoptosis induced by in vitro infection with simian-human immunodeficiency chimeric virus in macaque and human peripheral blood mononuclear cells.

We investigated apoptosis induced by in vitro infection with the chimeric virus of simian immunodeficiency virus and human immunodeficiency virus (SHIV). Macaque and human peripheral blood mononuclear cells (PBMCs) were infected with pathogenic SHIV-89.6p (89.6p) or nonpathogenic SHIV-NM-3rN (NM-3rN). In macaque PBMCs, the extent of virus production and apoptosis induction in CD4(+) cells was much greater in 89.6p infection than in NM-3rN infection. The result was consistent with our previous study of in vivo SHIV infection. In human PBMCs, 89.6p replicated and induced apoptosis more extensively than did NM-3rN, when the cells were infected with the same infectious doses of the viruses. However, in cells infected with a high dose of NM-3rN, the levels of virus production and apoptosis induction were comparable to those in 89.6p infection. There was no significant difference in the extent of apoptosis induction between 89.6p and NM-3rN infection when growth curves of the two viruses matched. Thus, apoptosis induction by SHIV might depend quantitatively on the amount of virus production rather than on the strains of the virus. Moreover, the correlation between the extent of apoptosis induction and virus pathogenicity in macaque PBMCs has also been found in SHIV-infected macaques. This suggests that the profiles of SHIV infection in vitro reflect the in vivo phenomena. Therefore, the in vitro evaluation of apoptosis induction by SHIV could be useful as a safety test for the development of live-attenuated vaccines.