Comparative Analysis of Nonstructural Proteins NS3 and NS5A of Hepatitis C Virus Obtained from patients with and witout Hepatocellular Carcinoma

兵庫県及び山形県から得られた検体を用いて, 原発性肝細胞癌 (HCC) 患者及び非癌患者のC型肝炎ウイルス (HCV) の非構造タンパク質NS3とNS5Aの変異について比較解析し, HCC発症におけるこれらの変異の意義を検討した。NS3のアミノ末端領域のアミノ酸配列にはかなりの多様性がみられ, HCV-1b株はその二次構造によってグループAとグループBの2群に大別された。兵庫県, 山形県いずれにおいても, 非癌患者由来株では約半数がグループAであり, 残りはグループBあるいはそれに類似する変異株であった。一方, HCC患者由来株では, グループAは10～20%のみで, 残りの80～90%はグループBあるいはそれに類似する変異株であった。これらのことから, グループAは低発癌性ウイルス株, グループBは高発癌性ウイルス株である可能性が示唆された。NS5Aについては, 兵庫県, 山形県ともに, HCC患者由来株では, 非癌患者由来株に比べて, インターフェロン感受性決定領域 (ISDR) に4ケ所以上のアミノ酸変異を有するものが多くみられた。以上の成績は, NS3アミノ末端領域の二次構造解析によって高発癌性ウイルス株を同定し, HCC発症危険性を予測し得る可能を示すとともに, 高発癌性ウイルス株の多くはインターフェロン感受性であり, HCC発症予防にインターフェロンが有効であることの理論的根拠を示すものである。 / The nonstructural proteins 3 and 5A (NS3 and NS5A, respectively) of hepatitis C virus subtype 1b (HCV-1b) obtained from patients with and without hepatocellular carcinoma (HCC) in Hyogo and Yamagata Prefectures were analyzed to see any possible correlation between mutations of the viral proteins and development of HCC. A considerable degree of variation was observed with the amino acid sequence of an amino-terminal portion of NS3. On the basis of secondary structure of amino-terminal 180 residues of NS3, HCV-1b isolates were classified into two major groups; group A and group B. In both Hyogo and Yamagata Prefectures, about half of the HCV-1b isolates from patients without HCC belonged to group A while the remaining half belonged to group B and a group B-like variant group. On the other hand, 80-90% of the isolates from patients with HCC belonged to group B and the variant group. The distribution patterns of those groups were significantly different between patients with HCC and those without HCC (P<0.001). Thus, group B isolates were highly associated with HCC and this secondary structure analysis would be useful to predict high risk for development of HCC in HCV-1b-infected patients. As for NS5A, HCV-1b isolates obtained from patients with HCC showed more mutations in interferon (IFN) sensitivity-determining region (ISDR). This result suggests the possibility that HCV-1b isolates that are highly associated with HCC are more sensitive to IFN treatment, claiming the effectiveness of IFN to prevent development of HCC

Intracellular neutralization of viral infection in polarized epithelial cells by neonatal Fc receptor (FcRn)-mediated IgG transport

IgG was traditionally thought to neutralize virions by blocking their attachment to or penetration into mucosal epithelial cells, a common site of exposure to viruses. However, we describe an intracellular neutralizing action for an influenza hemagglutinin-specific monoclonal antibody, Y8-10C2 (Y8), which has neutralizing activity only at an acidic pH. When Y8 was applied to the basolateral surface of Madin–Darby canine kidney cells expressing the rat neonatal Fc receptor for IgG (FcRn), it significantly reduced viral replication following apical exposure of the cell monolayer to influenza virus. Virus neutralization by Y8 mAb was dependent on FcRn expression and its transport of IgG. As both FcRn and Y8 mAb bind their partners only at acidic pH, the Y8 mAb is proposed to carry out its antiviral activity intracellularly. Furthermore, the virus, Y8 mAb, and FcRn colocalized within endosomes, possibly inhibiting the fusion of viral envelopes with endosomal membranes during primary uncoating, and preventing the accumulation of the neutralized viral nucleoprotein antigen in the nucleus. Prophylactic administration of Y8 mAb before viral challenge in WT mice, but not FcRn-KO mice, conferred protection from lethality, prevented weight loss, resulted in a significant reduction in pulmonary virus titers, and largely reduced virus-induced lung pathology. Thus, this study reveals an intracellular mechanism for viral neutralization in polarized epithelial cells that is dependent on FcRn-mediated transport of neutralizing IgG

Lack of Protection following Passive Transfer of Polyclonal Highly Functional Low-Dose Non-Neutralizing Antibodies

Recent immune correlates analysis from the RV144 vaccine trial has renewed interest in the role of non-neutralizing antibodies in mediating protection from infection. While neutralizing antibodies have proven difficult to induce through vaccination, extra-neutralizing antibodies, such as those that mediate antibody-dependent cellular cytotoxicity (ADCC), are associated with long-term control of infection. However, while several non-neutralizing monoclonal antibodies have been tested for their protective efficacy in vivo, no studies to date have tested the protective activity of naturally produced polyclonal antibodies from individuals harboring potent ADCC activity. Because ADCC-inducing antibodies are highly enriched in elite controllers (EC), we passively transferred highly functional non-neutralizing polyclonal antibodies, purified from an EC, to assess the potential impact of polyclonal non-neutralizing antibodies on a stringent SHIV-SF162P3 challenge in rhesus monkeys. Passive transfer of a low-dose of ADCC inducing antibodies did not protect from infection following SHIV-SF162P3 challenge. Passively administered antibody titers and gp120-specific, but not gp41-specific, ADCC and antibody induced phagocytosis (ADCP) were detected in the majority of the monkeys, but did not correlate with post infection viral control. Thus these data raise the possibility that gp120-specific ADCC activity alone may not be sufficient to control viremia post infection but that other specificities or Fc-effector profiles, alone or in combination, may have an impact on viral control and should be tested in future passive transfer experiments

Role of complement and antibodies in controlling infection with pathogenic simian immunodeficiency virus (SIV) in macaques vaccinated with replication-deficient viral vectors

<p>Abstract</p> <p>Background</p> <p>We investigated the interplay between complement and antibodies upon priming with single-cycle replicating viral vectors (SCIV) encoding SIV antigens combined with Adeno5-SIV or SCIV pseudotyped with murine leukemia virus envelope boosting strategies. The vaccine was applied via spray-immunization to the tonsils of rhesus macaques and compared with systemic regimens.</p> <p>Results</p> <p>Independent of the application regimen or route, viral loads were significantly reduced after challenge with SIVmac239 (p < 0.03) compared to controls. Considerable amounts of neutralizing antibodies were induced in systemic immunized monkeys. Most of the sera harvested during peak viremia exhibited a trend with an inverse correlation between complement C3-deposition on viral particles and plasma viral load within the different vaccination groups. In contrast, the amount of the observed complement-mediated lysis did not correlate with the reduction of SIV titres.</p> <p>Conclusion</p> <p>The heterologous prime-boost strategy with replication-deficient viral vectors administered exclusively via the tonsils did not induce any neutralizing antibodies before challenge. However, after challenge, comparable SIV-specific humoral immune responses were observed in all vaccinated animals. Immunization with single cycle immunodeficiency viruses mounts humoral immune responses comparable to live-attenuated immunodeficiency virus vaccines.</p

Vaccination against Heterologous R5 Clade C SHIV: Prevention of Infection and Correlates of Protection

A safe, efficacious vaccine is required to stop the AIDS pandemic. Disappointing results from the STEP trial implied a need to include humoral anti-HIV-1 responses, a notion supported by RV144 trial data even though correlates of protection are unknown. We vaccinated rhesus macaques with recombinant simian immunodeficiency virus (SIV) Gag-Pol particles, HIV-1 Tat and trimeric clade C (HIV-C) gp160, which induced cross-neutralizing antibodies (nAbs) and robust cellular immune responses. After five low-dose mucosal challenges with a simian-human immunodeficiency virus (SHIV) that encoded a heterologous R5 HIV-C envelope (22.1% divergence from the gp160 immunogen), 94% of controls became viremic, whereas one third of vaccinees remained virus-free. Upon high-dose SHIV rechallenge, all controls became infected, whereas some vaccinees remained aviremic. Peak viremia was inversely correlated with both cellular immunity (p<0.001) and cross-nAb titers (p<0.001). These data simultaneously linked cellular as well as humoral immune responses with the degree of protection for the first time

B Cell Depletion in HIV-1 Subtype A Infected Ugandan Adults: Relationship to CD4 T Cell Count, Viral Load and Humoral Immune Responses

To better understand the nature of B cell dysfunctions in subjects infected with HIV-1 subtype A, a rural cohort of 50 treatment-naïve Ugandan patients chronically infected with HIV-1 subtype A was studied, and the relationship between B cell depletion and HIV disease was assessed. B cell absolute counts were found to be significantly lower in HIV-1+ patients, when compared to community matched negative controls (p<0.0001). HIV-1-infected patients displayed variable functional and binding antibody titers that showed no correlation with viral load or CD4+ T cell count. However, B cell absolute counts were found to correlate inversely with neutralizing antibody (NAb) titers against subtype A (p = 0.05) and subtype CRF02_AG (p = 0.02) viruses. A positive correlation was observed between subtype A gp120 binding antibody titers and NAb breadth (p = 0.02) and mean titer against the 10 viruses (p = 0.0002). In addition, HIV-1 subtype A sera showed preferential neutralization of the 5 subtype A or CRF02_AG pseudoviruses, as compared with 5 pseudoviruses from subtypes B, C or D (p<0.001). These data demonstrate that in patients with chronic HIV-1 subtype A infection, significant B cell depletion can be observed, the degree of which does not appear to be associated with a decrease in functional antibodies. These findings also highlight the potential importance of subtype in the specificity of cross-clade neutralization in HIV-1 infection

HYPOMETHYLATION OF THE P53-TARGET ZMAT3 IS ASSOCIATED WITH SENESCENCE OF SUBCUTANEOUS ADIPOCYTE PRECURSOR CELLS IN INDIVIDUALS WITH A FAMILY HISTORY OF TYPE 2 DIABETES

Senescence of adipose precursor cells (APCs) impairs adipogenesis, contributes to the age-related subcutaneous adipose tissue (SAT) dysfunction, and increases risk of type 2 diabetes (T2D). First-degree relatives of T2D individuals (FDRs) feature restricted adipogenesis, reflecting the detrimental effects of APC senescence earlier in life and rendering FDRs more vulnerable to T2D. Epigenetics may contribute to these abnormalities but the underlying mechanisms remain unclear. In previous methylome comparison in APCs from FDRs and individuals with no diabetes familiarity (CTRLs), ZMAT3 emerged as one of the top-ranked senescence-related genes featuring hypomethylation in FDRs and associated with T2D risk. Here, we investigated whether and how DNA methylation changes at ZMAT3 promote early APC senescence. APCs from FDR individuals revealed increases in multiple senescence markers compared to CTRLs. Senescence in these cells was accompanied by ZMAT3 hypomethylation, which caused ZMAT3 upregulation. Demethylation at this gene in CTRL APCs led to increased ZMAT3 expression and premature senescence, which were reverted by ZMAT3 siRNA. Furthermore, ZMAT3 overexpression in APCs determined senescence and activation of the p53/p21 pathway, as observed in FDR APCs. Adipogenesis was also inhibited in ZMAT3-overexpressing APCs. In FDR APCs, rescue of ZMAT3 methylation through senolytic exposure simultaneously downregulated ZMAT3 expression and improved adipogenesis. Interestingly, in human SAT, ageing and T2D were associated with significantly increased expression of both ZMAT3 and the P53 senescence marker. Thus, DNA hypomethylation causes ZMAT3 upregulation in FDR APCs accompanied by acquisition of the senescence phenotype and impaired adipogenesis, which may contribute to FDRs predisposition for T2D

Comparative Analysis of Nonstructural Proteins NS3 and NS5A of Hepatitis C Virus Obtained from Patients with and without Hepatocellular Carcinoma

博士（医学）神戸大