Development of Vero Cell-Derived Inactivated Japanese Encephalitis Vaccine

We have established a manufacturing system for a Vero cell-derived inactivated Japanese encephalitis vaccine at a 500l scale. The production system involves expansion of Vero cells using microcarrier, followed by virus infection. Except for an additional purification step, the downstream purification processes are similar to those used for the current mouse brain-derived vaccine; cell removal, concentration and removal of low-molecular weight impurities by membrane filtration, formalin-inactivation, sucrose density gradient ultracentrifugation, and Sulfate-Cellulofine column chromatography are conducted. The antigen obtained from the manufacturing system was highly purified and its physico-chemical and immunological properties were comparable with those of antigen derived from mouse brains. Our system is very simple and could be easily scaled-up to allow vaccine production at a several thousand litre scale.     

Association of IL-10 receptor 2 (IL10RB) SNP with systemic sclerosis

Interleukin-10 (IL-10) signaling has been suggested to play a role in systemic sclerosis (SSc). IL10RB codes for IL-10 receptor 2 (IL-10R2), a component shared in receptor complexes for IL-10, IL-22, IL-26 and interferon (IFN)-λ. In this study, we examined association of IL10RB polymorphism with susceptibility to SSc. Genotype A/A at rs2834167 (47K/K) was significantly increased in diffuse cutaneous SSc (dcSSc) (41.3% in dcSSc, 20.9% in controls, P = 0.0018, odds ratio = 2.67). A SNP in the 5′ flanking region of IL10RB, rs999788, also showed association with dcSSc; however, this association was shown to be secondarily caused by linkage disequilibrium with rs2834167. Significant association was not observed in limited cutaneous SSc (lcSSc). Presence of anti-topoisomerase I antibody was also associated with rs2834167A/A genotype (P = 0.0019). Serum IL-10 level was significantly associated with the number of rs2834167A allele (P = 0.007). These findings suggested that signaling through IL-10R2 may play a causative role in dcSSc.     

Different immunodominance of HIV-1-specific CTL epitopes among three subtypes of HLA-A*26 associated with slow progression to AIDS

It is speculated that HLA-A^∗26-restricted HIV-1-specific CTLs can control HIV-1, since HLA-A^∗26 is associated with a slow progression to AIDS. In three major HLA-A^∗26 subtypes, HLA-A^∗2601-restricted, and HLA-A^∗2603-restricted HIV-1 epitopes have been identified, but HLA-A^∗2602-restricted ones have not. We here identified HLA-A^∗2602-restricted HIV-1 epitopes by using reverse immunogenetics and compared the immunodominance of the epitopes among the three subtypes. Out of 110 HIV-1 peptides carrying HLA-A^∗26 anchor residues, only the Gag169-177 peptide, which had been previously identified as an HLA-A^∗2601- and HLA-A^∗2603-restricted immunodominant epitope, induced Gag169-177-specific CD8⁺ T cells from only two of six HLA-A^∗2602⁺ HIV-1-infected individuals. No difference in affinity of this epitope peptide was found among these three HLA-A^∗26 subtypes, indicating that Gag169-177 was effectively presented by HLA-A^∗2602 but recognized as a subdominant epitope in HIV-1-infected HLA-A^∗2602⁺ individuals. These findings indicate different immunodominance of Gag169-177 epitope among 3 HLA-A^∗26 subtypes.     

TGF-β type I receptor Alk5 regulates tooth initiation and mandible patterning in a type II receptor-independent manner

TGF-β superfamily members signal through a heteromeric receptor complex to regulate craniofacial development. TGF-β type II receptor appears to bind only TGF-β, whereas TGF-β type I receptor (ALK5) also binds to ligands in addition to TGF-β. Our previous work has shown that conditional inactivation of Tgfbr2 in the neural crest cells of mice leads to severe craniofacial bone defects. In this study, we examine and compare the defects of TGF-β type II receptor (Wnt1-Cre;Tgfbr2^fl/fl) and TGF-β type I receptor/Alk5 (Wnt1-Cre;Alk5^fl/fl) conditional knockout mice. Loss of Alk5 in the neural crest tissue resulted in phenotypes not seen in the Tgfbr2 mutant, including delayed tooth initiation and development, defects in early mandible patterning and altered expression of key patterning genes including Msx1, Bmp4, Bmp2, Pax9, Alx4, Lhx6/7 and Gsc. Alk5 controls the survival of CNC cells by regulating expression of Gsc and other genes in the proximal aboral region of the developing mandible. We conclude that ALK5 regulates tooth initiation and early mandible patterning through a pathway independent of Tgfbr2. There is an intrinsic requirement for Alk5 signal in regulating the fate of CNC cells during tooth and mandible development.     

Babesia rodhaini: the protective effect of pyruvate kinase deficiency in mice

Despite the evidence suggesting that mouse pyruvate kinase (PK) deficiency provides protection against malaria in rodents, there has been no investigation of a parallel protective effect against babesiosis caused by Babesia rodhaini. Here, we examined whether a PK-deficient co-isogenic mouse strain (CBA-Pk-1^slc) was protected against B. rodhaini infection. We demonstrated that deficiency in pyruvate kinase correlated with a significant protective effect, with survival rates of 50%, 58% and 56% in groups inoculated with 10, 10³ and 10⁵ parasitized erythrocytes, respectively. In contrast, control CBA (CBA-Pk-1⁺) mice exhibited 100% lethality, regardless of the infectious dose. In addition, CBA-Pk-1^slc mice showed decreased levels of parasitemia when compared to CBA-Pk-1⁺ mice, in groups given 10, 10³ or 10⁵ parasitized erythrocytes. These results indicate that similar to PK deficiency in rodents, PK deficiency in mice affects the in vivo growth of B. rodhaini and protects the mice from lethal babesiosis.     

Localization of polypyrimidine-tract-binding protein is involved in the regulation of albumin synthesis by branched-chain amino acids in HepG2 cells

Long-term supplementation of branched-chain amino acids (BCAA) improves hypoalbuminemia in patients with cirrhosis. Our previous findings have suggested that the binding of polypyrimidine-tract-binding protein (PTB) to rat albumin mRNA attenuates its translation. The aim of the present study was to investigate the role of PTB in the regulation of albumin synthesis by BCAA in human hepatoma cells. HepG2 cells were cultured in a medium containing no amino acids (AA-free medium), a medium containing only 1 amino acid (a BCAA: valine, leucine or isoleucine) or a medium containing all 20 amino acids (AA-complete medium). HepG2 cells cultured in AA-complete medium secreted much more albumin than cells cultured in AA-free medium, with no difference in albumin mRNA levels. In cells cultured in AA-free medium, nuclear export of PTB was observed, and the level of the albumin mRNA-PTB complex was greater than in cells cultured in AA-complete medium. Addition of amino acids stimulated nuclear import of PTB. However, addition of amino acids with rapamycin inhibited the nuclear import of PTB. The addition of leucine, but not of valine or isoleucine, to AA-free medium increased albumin secretion and stimulated the nuclear import of PTB. These data indicate that the mammalian target of rapamycin is involved in the regulation of PTB localization and that leucine promotes albumin synthesis by inhibiting the formation of the albumin mRNA-PTB complex.     

HLA-A*2402-restricted HIV-1-specific cytotoxic T lymphocytes and escape mutation after ART with structured treatment interruptions

Although a limited duration of immune activation of structured treatment interruptions (STIs) has been reported, the immune escape mechanism during STIs remains obscure. We therefore investigated the role of three immunodominant cytotoxic T lymphocyte (epitopes) in 12 HLA-A*2402-positive patients participating longitudinally during the clinical study of early antiretroviral treatment (ART) with five series of structured treatment interruptions (STIs). The frequency of HLA-A*2402-restricted CTLs varied widely and a sustained CTL response was rarely noted. However, a Y-to-F substitution at the second position in an immunodominant CTL epitope Nef138-10 (Nef138-2F), which was previously demonstrated as escape mutation, was frequently detected in seven patients primarily and emerged in the remaining five patients thereafter, and the existence of escape mutations was correlated with high pVL levels early in the clinical course. These findings suggest that escape mutation in the immunodominant CTL epitope may be one of the mechanisms to limit HIV-1-specific immune control in STIs.     

Engineering of a mammalian O-glycosylation pathway in the yeast Saccharomyces cerevisiae: production of O-fucosylated epidermal growth factor domains

Development of a heterologous system for the production of homogeneoussugar structures has the potential to elucidate structure–functionrelationships of glycoproteins. In the current study, we usedan artificial O-glycosylation pathway to produce an O-fucosylatedepidermal growth factor (EGF) domain in Saccharomyces cerevisiae.The in vivo O-fucosylation system was constructed via expressionof genes that encode protein O-fucosyltransferase 1 and theEGF domain, along with genes whose protein products convertcytoplasmic GDP-mannose to GDP-fucose. This system allowed identificationof an endogenous ability of S. cerevisiae to transport GDP-fucose.Moreover, expression of EGF domain mutants in this system revealedthe different contribution of three disulfide bonds to in vivoO-fucosylation. In addition, lectin blotting revealed differencesin the ability of fucose-specific lectin to bind the O-fucosylatedstructure of EGF domains from human factors VII and IX. Furtherintroduction of the human fringe gene into yeast equipped withthe in vivo O-fucosylation system facilitated the addition ofN-acetylglucosamine to the EGF domain from factor IX but notfrom factor VII. The results suggest that engineering of anO-fucosylation system in yeast provides a powerful tool forproducing proteins with homogenous carbohydrate chains. Suchproteins can be used for the analysis of substrate specificityand the production of antibodies that recognize O-glycosylatedEGF domains.