Munehide Kano

Cytotoxic T Lymphocyte-based Control of Simian Immunodeficiency Virus Replication in a Preclinical AIDS Vaccine Trial

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.

Rapid Appearance of Secondary Immune Responses and Protection from Acute CD4 Depletion after a Highly Pathogenic Immunodeficiency Virus Challenge in Macaques Vaccinated with a DNA Prime/Sendai Virus Vector Boost Regimen

ABSTRACT Heterologous prime/boost regimens are AIDS vaccine candidates because of their potential for inducing cellular immune responses. Here, we have developed a prime/boost regimen leading to rapid control of highly pathogenic immunodeficiency virus infection in macaques. The strategy, priming by an env and nefdeletion-containing simian-human immunodeficiency virus (SHIV) proviral DNA followed by a single booster with a Gag-expressing Sendai virus (SeV-Gag), efficiently induced virus-specific T cells, which were maintained for more than 3 months until challenge. While all naive control macaques showed acute CD4+ T-cell depletion at week 2 after an intravenous SHIV89.6PD challenge, all the macaques vaccinated with the prime/boost regimen were protected from depletion and showed greatly reduced peak viral loads compared with controls. Vaccination with the DNA alone or SeV-Gag alone was not enough to confer the consistent protection from the depletion, although it led to efficient secondary CD8+ T-cell responses at week 2 after challenge. At week 1, a difference in the secondary responses between the protected and the unprotected macaques was clear; rapid augmentation of virus-specific CD8+ T cells was detected in the former but not in the latter. Thus, our results indicate the importance of rapid secondary responses for reduction in the peak viral loads and protection from acute CD4+ T-cell depletion.

Protective Efficacy of an AIDS Vaccine, a Single DNA Priming Followed by a Single Booster with a Recombinant Replication-Defective Sendai Virus Vector, in a Macaque AIDS Model

ABSTRACT We previously demonstrated the excellent protective efficacy of DNA priming followed by Gag-expressing Sendai virus (SeV) boosting (DNA prime/SeV-Gag boost vaccine) against a pathogenic simian-human immunodeficiency virus (SHIV89.6PD) infection in macaques. Here we show that we established a practical, safer AIDS vaccine protocol, a single DNA priming followed by a single booster with a recently developed replication-defective F deletion SeV-expressing Gag, and show its protective efficacy against SHIV89.6PD infections.

Mediation of renal cyst formation by hepatocyte growth factor

Hepatocyte growth factor (HGF) is a potent mitogen for renal tubular cells, and HGF and its receptor (c-met proto-oncogene product, Met) can induce lumen formation in epithelial cells. We measured the concentration of HGF in cyst fluids from patients with renal cystic diseases. The concentration of HGF was high in proximal cyst fluid (mean 2.45 vs 0.42 ng/mL in distal cysts). Cyclic AMP concentration was higher in distal than in proximal cysts (663 vs 6.0 pmol/L). mRNA of HGF and Met were co-expressed in cyst walls from cases with polycystic kidney disease. These findings suggest that HGF is a growth factor that may mediate human renal cyst genesis.

Influence of Glycosylation on the Efficacy of an Env-Based Vaccine against Simian Immunodeficiency Virus SIVmac239 in a Macaque AIDS Model

ABSTRACT The envelope glycoprotein (Env) of human immunodeficiency viruses (HIVs) and simian immunodeficiency viruses (SIVs) is heavily glycosylated, and this feature has been speculated to be a reason for the insufficient immune control of these viruses by their hosts. In a macaque AIDS model, we demonstrated that quintuple deglycosylation in Env altered a pathogenic virus, SIVmac239, into a novel attenuated mutant virus (Δ5G). In Δ5G-infected animals, strong protective immunity against SIVmac239 was elicited. These HIV and SIV studies suggested that an understanding of the role of glycosylation is critical in defining not only the virological properties but also the immunogenicity of Env, suggesting that glycosylation in Env could be modified for the development of effective vaccines. To examine the effect of deglycosylation, we constructed prime-boost vaccines consisting of Env from SIVmac239 and Δ5G and compared their immunogenicities and vaccine efficacies by challenge infection with SIVmac239. Vaccination-induced immune responses differed between the two vaccine groups. Both Env-specific cellular and humoral responses were higher in wild-type (wt)-Env-immunized animals than in Δ5G Env-immunized animals. Following the challenge, viral loads in SIVmac239 Env (wt-Env)-immunized animals were significantly lower than in vector controls, with controlled viral replication in the chronic phase. Unexpectedly, viral loads in Δ5G Env-immunized animals were indistinguishable from those in vector controls. This study demonstrated that the prime-boost Env vaccine was effective against homologous SIVmac239 challenge. Changes in glycosylation affected both cell-mediated and humoral immune responses and vaccine efficacy.

Primary replication of a recombinant Sendai virus vector in macaques

An efficient antigen expression system using a recombinant Sendai virus (SeV) has been established recently and its potential to induce resistance against immunodeficiency virus infections in macaques has been shown. SeV replication has been well characterized in mice, the natural host, but not in primates, including humans. Here, primary SeV replication was investigated in macaques. After intranasal immunization with a recombinant SeV expressing simian immunodeficiency virus Gag protein, SeV-Gag, robust gag expression was observed in the nasal mucosa and much lower but significant levels of gag expression were observed in the local retropharyngeal and submandibular lymph nodes (LN). Expression peaked within a week and lasted at least up to 13 days after immunization. SeV-Gag was isolated from nasal swabs consistently at day 4 but not at all at day 13. Gag expression was undetectable in the lung as well as in remote lymphoid tissues, such as the thymus, spleen and inguinal LN, indicating that the spread of the virus was more restricted in macaques than in mice. SeV-specific T cells were detectable in SeV-immunized macaques at day 7. Finally, no naive macaques showed significant levels of anti-SeV antibodies in the plasma, even after living in a cage together with an acutely SeV-infected macaque for 5 weeks, indicating that SeV transmission from SeV-infected macaques to naive ones was inefficient. None of the SeV-immunized macaques displayed appreciable clinical manifestations. These results support the idea that this system may be used safely in primates, including humans.

Induction of protective immunity against pathogenic simian immunodeficiency virus by a foreign receptor-dependent replication of an engineered avirulent 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.

Comparison between Sendai virus and adenovirus vectors to transduce HIV‐1 genes into human dendritic cells

Immuno‐genetherapy using dendritic cells (DCs) can be applied to human immunodeficiency virus type 1 (HIV‐1) infection. Sendai virus (SeV) has unique features such as cytoplasmic replication and high protein expression as a vector for genetic manipulation. In this study, we compared the efficiency of inducing green fluorescent protein (GFP) and HIV‐1 gene expression in human monocyte‐derived DCs between SeV and adenovirus (AdV). Human monocyte‐derived DCs infected with SeV showed the maximum gene expression 24 hr after infection at a multiplicity of infection (MOI) of 2. Although SeV vector showed higher cytopathic effect on DCs than AdV, SeV vector induced maximum gene expression earlier and at much lower MOI. In terms of cell surface phenotype, both SeV and AdV vectors induced DC maturation. DCs infected with SeV as well as AdV elicited HIV‐1 specific T‐cell responses detected by interferon γ (IFN‐γ) enzyme‐linked immunospot (Elispot). Our data suggest that SeV could be one of the reliable vectors for immuno‐genetherapy for HIV‐1 infected patients. J. Med. Virol. 80:373–382, 2008.