Immunization of woodchucks (Marmota monax) with hepatitis delta virus DNA vaccine

We investigated the DNA immunization approach in order to induce a protective immune response against hepatitis delta virus (HDV) superinfection of chronically woodchuck hepatitis virus (WHV) infected woodchucks. The animals were immunized with an expression vector encoding HDAg by gene gun. T cell and humoral immune responses induced by this protocol were determined and compared with those induced by HDAg immunization using a CpG oligonucleotide as an adjuvant. After immunization the woodchucks were challenged with 10⁶ genome equivalents of HDV. The protein immunization with HDAg induced good humoral and T helper cell responses in the woodchucks, but did not protect them from HDV superinfection. The DNA immunized woodchucks were also not protected from HDV superinfection, however, the course of infection was modified: HDV viremia occurred later, the typical fluctuation of the HDV RNA titer with several peaks was absent, and antibodies to HDV were not detectable.     

戊型肝炎病毒嵌合基因疫苗的初步研究

目的　构建含戊型肝炎病毒 (HEV)开放读码框架 (ORF2 )和ORF3嵌合基因的重组质粒 ,并探讨其作为DNA疫苗的可能性。方法　利用PCR方法获得约 0 8kb的戊型肝炎ORF2片段和ORF3的完整片段 ,同时将 2段片段克隆到同一真核表达载体 pcDNA3中 ,构建出含有ORF2和ORF3嵌合基因的质粒 pcHEV2 3;该重组质粒作为DNA疫苗免疫BALB/c小鼠 ,观察其在小鼠体内诱发的体液免疫应答和细胞免疫应答。结果　质粒DpcHEV2 3免疫组小鼠特异性抗体水平明显高于对照组。T细胞增殖反应显著强于空质粒对照组 (P <0 0 5 ) ,与生理盐水对照组比较有显著性差异 (P <0 0 1)。结论　重组质粒 pcHEV2 3可诱发小鼠产生特异性体液和细胞免疫应答 ,可为戊型肝炎DNA疫苗的研制提供依据。     

Search for hepatitis delta virus (HDV) infection in hepatitis C patients in north-eastern Poland. Comparison with anti-HDV prevalence in chronic hepatitis B

Hepatitis C virus and hepatitis D virus have been shown to suppress HBsAg synthesis. Thus it is possible that HDV infection occurs despite the lack of detectable HBsAg. The aim of our study was to (a) determine the prevalence of HDV infection in patients with chronic hepatitis C (b) compare it with the prevalence of HDV infection in HBsAg positive patients with hepatitis B. The study group consisted of 51 chronic hepatitis C patients, 30 HIV infected drug addicts (27 of them were also positive for anti-HCV) and 102 hepatitis B patients. The participants were tested for anti-HDV, anti-HCV and HBsAg. All anti-HCV positive patients were negative for anti-HDV. Four individuals with anti-HDV belonged to hepatitis B group and constituted 3.9% of all HBsAg positive subjects. We conclude that (a) there is currently no evidence of HDV infection among HCV infected patients in our region (b) hepatitis delta infection is rare in north-eastern Poland.     

Eighth major clade for hepatitis delta virus

Hepatitis delta virus is the only representative of the Deltavirus genus, which consists of 7 differentiated major clades. In this study, an eighth clade was identified from 3 distinct strains. Deltavirus genetic variability should be considered for diagnostic purposes. Clinical consequences of the diversity have yet to be evaluated.     

DNA芯片检测乙型肝炎病毒基因型

目的 制备检测HBV基因型的DNA芯片并进行杂交验证.方法 设计多条HBV分型寡核苷酸探针,固定于醛基基片的特定位置,制备成芯片,采用酶呈色技术(BCIP/NBT显色)检测杂交信号,进行杂交验证分析.结果 106例临床样本均测出基因型,其中B型37例,C型69例;对所有样本进行测序分析,结果与芯片杂交一致,杂交的灵敏度和重复性等指标均佳.结论 DNA芯片检测HBV基因型,操作简便易行,技术要求不高,适于临床推广应用.     

乙肝疫苗联合免疫球蛋白阻断母婴传播效果

目的 观察比较乙肝疫苗(Hep-B)联合乙肝免疫球蛋白(HBIG)阻断乙肝母婴传播的效果,为制订阻断乙肝母婴传播策略提供依据.方法 选择上海市宝山区6家综合性医院妇产科产前检查中检出的HBsAg阳性和HBsAg/HBeAg双阳性的本区户籍产妇85人,分为3组.其中观察Ⅰ组(22人)新生儿出生24 h内和15 d各接种1针HBIG;观察Ⅱ组孕产妇(37人)于28,32,36孕周各接种1针HBIG,婴儿出生24 h内和15 d各接种1针HBIG;对照组(26人)不接种HBIG.HBIG均为200 U/针.3个组的新生儿均按正常免疫程序于0,1,6个月时各接种1针10μg重组(酵母)Hep-B疫苗.1年后采集幼儿静脉血,采用ELISA法检测HBsAg和抗-HBs.结果 观察Ⅰ组、Ⅱ组和对照组婴儿出生后12个月时,HBsAg阳性率分别为17.24%,2.70%和23.04%,抗-HBs阳性率分别为86.36%,97.30%和76.92%;观察Ⅱ组母婴传播阻断率和免疫保护率均高于观察Ⅰ组和对照组,差异有统计学意义(P<0.05).结论 HBIG与重组(酵母)Hep-B疫苗联合应用免疫效果好,安全可行,且观察Ⅱ组的效果优于观察Ⅰ组.     

Epidemiology of hepatitis delta virus (HDV) infection

Markers of hepatitis delta virus (HDV) infection have been detected all over the five continents. Geographical prevalence varied heavily: HDV infection is very rare in Far East Asia, but extremely frequent in Arabian countries, in Romania and in certain Indian populations of South America. In Europe and in the USA the infection is widely spread among high risk groups such as intravenous drug abusers and haemophiliacs,I Corresponding author.     

Prevalence of hepatitis B markers in patients with hepatitis C infection in north-eastern Poland: Risk factors and vaccine use

We evaluated the prevalence of hepatitis B virus (HBV) markers and established HBV vaccination status among 111 patients with hepatitis C virus (HCV) infection. A history of HBV immunisation was recorded in 30 patients (27.0%) and only 17/30 (66.7%) had anti-HBs level 10 mIU/ml. All patients were HBsAg-negative and 22.2% of nonvaccinated subjects had evidence of HBV infection as determined by anti-HBc presence. Among patients with anti-HBc in 7/18 cases (38.9%) anti-HBc was the only marker of HBV infection (without anti-HBs). The prevalence of anti-HBc was significantly higher among patients who reported a history of acute hepatitis. In conclusion the prevalence of HBV markers in patients with HCV infection in north-eastern Poland is similar to the prevalence in general population, which suggests no increased risk for nosocomial HBV infection among those individuals. HCV infection seems to favour unusual serological pattern of HBV infection with anti-HBc as the only marker. HBV vaccine use is low among patients with HCV infection in north-eastern Poland.     

The successful immune response against hepatitis C nonstructural protein 5A (NS5A) requires heterologous DNA/protein immunization

The aim of this study was to evaluate the immunogenicity of NS5A protein of human hepatitis C virus (HCV) when delivered as naked DNA (NS5A DNA), or recombinant protein (rNS5A). DBA/2J mice received NS5A DNA, rNS5A, or NS5A DNA/rNS5A in different prime-boost combinations with a peptidoglycan Immunomax^®. The weakest response was induced after rNS5A prime and NS5A DNA boost; rNS5A alone induced an immune response with a strong Th2-component; and NS5A DNA alone, a relatively weak secretion of IL-2 and IFN-γ. The most efficient was co-injection of NS5A DNA and rNS5A, which induced a significant increase in CD4⁺ and CD8⁺ T-cell counts, anti-NS5A antibodies, specific T-cell proliferation, and proinflammatory cytokine production in vitro against a broad spectrum of NS5A epitopes. Administration of the mixture of adjuvanted DNA and protein immunogens can be selected as the best regimen for further preclinical HCV-vaccine trials.     

Virus-like particle vaccine with B-cell epitope from porcine epidemic diarrhea virus (PEDV) incorporated into hepatitis B virus core capsid provides clinical alleviation against PEDV in neonatal piglets through lactogenic immunity

Porcine epidemic diarrhea virus (PEDV) has had a negative economic impact on the global swine industry for decades since its first emergence in the 1970s in Europe. In 2013, PEDV emerged for the first time in the United States, causing immense economic losses to the swine industry. Efforts to protect U.S. swine herds from PEDV infection and limit PEDV transmission through vaccination had only limited success so far. Following the previous success in our virus-like particle (VLP) based vaccine in mouse model, in this study we determined the immunogenicity and protective efficacy of a VLP-based vaccine containing B-cell epitope ⁷⁴⁸YSNIGVCK⁷⁵⁵ from the spike protein of PEDV incorporated into the hepatitis B virus core capsid (HBcAg), in a comprehensive pregnant gilt vaccination and piglet challenge model. The results showed that the vaccine was able to induce significantly higher virus neutralization response in gilt milk, and provide alleviation of clinical signs for piglets experimentally infected with PEDV. Piglets from pregnant gilt that was vaccinated with the VLP vaccine had faster recovery from the clinical disease, less small intestinal lesions, and higher survival rate at 10 days post-challenge (DPC).     

乙肝疫苗免疫规划对乙肝流行影响

目的 了解乙型肝炎(HB)疫苗纳入免疫规划后,江苏省HB的流行病学特征及影响因素.方法 根据《2006年全国人群乙肝等有关疾病血清学调查总体方案》,对3 906人进行现场和血清流行病学调查,并对结果进行描述流行病学分析.结果 全省HBsAg、抗-HBs、抗-HBc阳性率和乙肝病毒(HBV)感染率分别为4.99%,58.70%,25.91%和25.93%.<15岁人群乙肝疫苗(HepB)接种率达96.99%;HBsAg、抗-HBc阳性率及HBV感染率均比其他年龄组低,抗-HBs阳性率则较高,首针及时接种率为70.75%.城市HBsAg阳性率为5.19%,农村为4.80%.家庭中HBsAg阳性者关系中,夫妻占41.67%.结论 HepB纳入免疫规划后,江苏省乙肝感染率大幅下降,<15岁人群更为明显,感染高峰后移;<15岁人群首针及时接种率仍处于较低水平.     

Profiles of B and T cell immune responses elicited by different forms of the hepatitis B virus surface antigen

Gene-based hepatitis B virus (HBV) vaccines have been proposed as a novel approach to improve the immunogenicity toward non-responders and to allow for protection against potential viral escape mutants. Furthermore, there is significant interest in using DNA or viral vector vaccines to serve as therapeutic agents to treat chronic HBV infections that are resistant to existing drug therapies. However, the key protective antigen of HBV, the surface protein (HBsAg), can be expressed in three different sizes due to its multiple translational initiation sites: small, middle, and large forms of HBsAg. It is not clear whether the immunogenicity of these HBsAg is same, especially their ability to elicit HBsAg-specific B cell and T cell immune responses in addition to the traditional serum HBsAg-specific antibody responses. In the current study, the immunogenicity of three forms of HBsAg DNA vaccines was analyzed individually in a mouse model. Our results indicated that different forms of the HBsAg have unique immunogenicity profiles and this information is useful for the selection of optimal gene-based HBV vaccines for further improved prophylactic and therapeutic applications.     

乙肝免疫球蛋白联合乙肝疫苗阻断HBV母婴传播的相关性研究

目的:探讨乙肝免疫球蛋白(HBIG)联合乙肝疫苗阻断乙肝病毒(HBV)母婴传播失败原因。方法:选择HBsAg阳性、HBeAg阳性、HBV-DNA阳性孕妇218例,检测孕妇分娩前HBVDNA,新生儿(出生24h内且未进行阻断前)、7月龄及1岁时婴儿的HBsAg、抗HBs;所有新生儿出生后24h内在三角肌注射HBIG200IU,同时在大腿前部外侧肌内注射基因工程乙肝疫苗10μg,2周再注射同等剂量的HBIG,1、6月时分别注射同等剂量的乙肝疫苗。结果:孕妇分娩前血清HBVDNA含量>1×106copies/ml组7月龄、1岁时HBsAg阳性率分别为18.12%、19.38%,HBVDNA含量<1×106copies/mi组为7.50%、7.25%(P<0.05)。注射HBIG及乙肝疫苗后,宫内感染组7月龄、1岁时HBsAg阳性率分别为75.00%、74.19%,非宫内感染组为3.76%、4.19%(P<0.01)。结论:宫内感染及孕妇分娩前血清HBVDNA含量高是HBV母婴阻断失败的主要原因。采取综合措施可提高母婴阻断效果。     

Intragastric administration of attenuated Salmonella typhimurium harbouring transmissible gastroenteritis virus (TGEV) DNA vaccine induced specific antibody production

Attenuated Salmonella typhimurium was selected as a transgenic vehicle for the development of live mucosal vaccines against transmissible gastroenteritis virus (TGEV). A 2.2 kb DNA fragment, encoding for N-terminal domain glycoprotein S of TGEV, was amplified by RT-PCR and cloned into eukaryotic expression vector pVAX1. The recombinant plasmid pVAX-S was transformed by electroporation into attenuated S. typhimurium SL7207, the expression and translation of the pVAX-S delivered by recombinant S. typhimurium SL7207 (pVAX-S) was detected in vitro and in vivo respectively. BALB/c mice were inoculated orally with SL7207 (pVAX-S) at different dosages, the bacterium was safe to mice at dosage of 2 × 10⁹ CFU and eventually eliminated from the spleen and liver at week 4 post-immunization. Mice immunized with different dosages of SL7207 (pVAX-S) elicited specific anti-TGEV local mucosal and humoral responses as measured by indirect ELISA assay. Moreover, the immunogenicity of the DNA vaccine was highly dependent on the dosage of the attenuated bacteria used for oral administration, 10⁹ CFU dosage group showed higher antibody response than 10⁸ CFU and 10⁷ CFU dosages groups during week 4–8 post-immunization. The results indicated that attenuated S. typhimurium could be used as a delivery vector for oral immunization of TGEV DNA vaccine.     

An active DNA vaccine against infectious pancreatic necrosis virus (IPNV) with a different mode of action than fish rhabdovirus DNA vaccines

Although there are some commercial vaccines available against infectious pancreatic necrosis virus (IPNV), the disease still continues to be a major problem for aquaculture development worldwide. In the current work, we constructed a DNA vaccine against IPNV (pIPNV-PP) by cloning the long open reading frame of the polyprotein encoded by the viral RNA segment A. In vitro, the vaccine is properly translated giving the functional IPNV polyprotein since preVP2, VP2 and VP3 proteins were detected because of the VP4-protease cleavage. EPC cells transfected with the vaccine plasmid expressed the viral proteins and induced the expression of type I interferon (IFN)-induced Mx genes. Furthermore, IPNV synthesized proteins seemed to assemble in virus-like particles as evidenced by electron microscopy. In vivo, rainbow trout specimens were intramuscularly injected with the vaccine and expression of immune-relevant genes, the presence of neutralizing antibodies and effect on viral load was determined. The pIPNV-PP vaccine was expressed at the injection site and up-regulated MHC Iα, MHC IIα, type-I interferon (IFN), Mx, CD4 and CD8α gene expression in the muscle, head kidney or spleen, although to a much lower extent than the up-regulations observed in response to an effective DNA vaccine against viral hemorrhagic septicaemia virus (VHSV). However, the IPNV vaccine was also very effective in terms of acquired immunity since it elicited neutralizing antibodies (in 6 out of 8 trout fingerlings) and decreased 665-fold the viral load after IPNV infection. The effectiveness of this new IPNV DNA vaccine and its possible mechanism of action are discussed and compared to other viral vaccines.     

Development of a live attenuated duck hepatitis A virus type 3 vaccine (strain SD70)

Duck hepatitis A virus type 3 (DHAV-3) is an important pathogen that causes substantial losses in the Chinese duck industry. DHAV-3 is highly fatal to ducklings and there is no licensed vaccine in China available to reduce DHAV-3 infection. Our goal was to develop a live attenuated vaccine candidate against DHAV-3. A field isolated strain, SD, was attenuated by serially passaging in specific-pathogen-free (SPF) chicken embryos, and it lost its pathogenicity after 40 passages. The 70th passaged strain (SD70), which achieved good growth capacity in chicken embryos with a viral titer of 10^7.5 ELD₅₀/mL, was chosen to be the live attenuated vaccine candidate. The SD70 strain did not cause clinical signs of disease or mortality in 1-day-old ducklings and showed no virulence reversion after seven rounds of in vivo back passages. The minimum effective dose of SD70 was determined to be 10^2.5 ELD₅₀ via the vaccination route of subcutaneous inoculation. A single dose of the SD70 provided good protection to susceptible ducklings against the lethal DHAV-3 strain. Compared with the genomic sequence of the parent SD strain, the SD70 had 12 amino acid substitutions, some of which may play a role in virulence attenuation. This study demonstrated that the attenuated SD70 strain is a promising vaccine candidate for the prevention of DHAV-3 infection in China. It exhibited safety, good stability and excellent protection.     

Identification of CD4+ and CD8+ T cell epitopes of woodchuck hepatitis virus core and surface antigens in BALB/c mice

A therapeutic vaccine against chronic hepatitis B virus (HBV) infection requires the development of a strong and multispecific Th1 cell immune response. Woodchucks chronically infected with the woodchuck hepatitis virus (WHV) closely resemble HBV infection and represent the best animal model for this hepadnavirus-induced disease. Using the BIMAS “HLA Peptide Binding Predictions” program, we have identified and further characterized novel H-2^d-restricted CD8+ epitopes within the WHV core (peptides C#12–21, C#18–32, C#19–27, C#61–69) and surface antigens (peptides preS2#10–18, preS2#27–35, S#76–84, S#133–140 and S#257–265), respectively. These peptides bind to H-2^d with high efficiency and upon immunization of mice with peptide and Freund's adjuvant they induce the development of IFN-γ producing T cells. More importantly, WHV core peptides C#19–27 and C#61–69 and WHV surface peptides S#133–140 and S#257–265 were also recognized by CD8+ T cells after immunization of mice with DNA/PEI nanoparticles. Direct stimulation of splenocytes obtained from such DNA-immunized mice with peptides C#18–32, S#76–84, and S#257–265 resulted in significant production of IFN-γ. Thus, we have identified T cell determinants in mice from WHV core and surface antigens that have important value for designing and evaluating an effective vaccine against hepadnavirus infection.     

重庆市北碚区居民丙肝知识知晓率及相关行为现状调查

目的了解重庆市北碚区居民丙肝知识知晓率、高危行为现状及两者之间的关系,为构建提高居民丙肝安全行为的干预方式提供科学依据。方法2018—2019年期间根据《重庆市丙肝防治知识知晓率基线调查方案》要求采取分层随机抽样法抽取重庆市北碚区居民开展丙肝知识、高危行为问卷调查,采用SPSS 20.0软件对数据进行分析,以P<0.05为差异有统计学意义。结果共调查200名居民,丙肝防治知识问题回答对6道及以上51人(25.5%),全部答对仅4人(2.0%),全部答错103人(51.5%)。丙肝防治知识知晓率与文化程度有关(P<0.05),与人群类别、家庭人均月收入、年龄、性别等因素不相关(P均>0.05)。丙肝防治知识知晓与不知晓的高危行为发生率比较,差异无统计学意义(χ²=0.966,P=0.367)。结论该地区人群丙肝防治知识知晓率还处于较低水平,建议在今后的工作中应针对丙肝制定详细宣教计划,组织开展各种形式的宣教活动,有效引导公众提高丙肝防治意识。     

Understanding the molecular mechanism(s) of hepatitis C virus (HCV) induced interferon resistance

Hepatitis C virus (HCV) is one of the foremost causes of chronic liver disease affecting over 300 million globally. HCV contains a positive-stranded RNA of ∼9600 nt and is surrounded by the 5′ and 3′untranslated regions (UTR). The only successful treatment regimen includes interferon (IFN) and ribavirin. Like many other viruses, HCV has also evolved various mechanisms to circumvent the IFN response by blocking (1) downstream signaling actions via STAT1, STAT2, IRF9 and JAK-STAT pathways and (2) repertoire of IFN Stimulatory Genes (ISGs). Several studies have identified complex host demographic and genetic factors as well as viral genetic heterogeneity associated with outcomes of IFN therapy. The genetic predispositions of over 2000 ISGS may render the patients to become resistant, thus identification of such parameters within a subset of population are necessary for management corollary. The ability of various HCV genotypes to diminish IFN antiviral responses plays critical role in the establishment of chronic infection at the acute stage of infection, thus highlighting importance of the resistance in HCV treated groups. The recently defined role of viral protein such as C, E2, NS3/NS4 and NS5A proteins in inducing the IFN resistance are discussed in this article. How the viral and host genetic composition and epistatic connectivity among polymorphic genomic sites synchronizes the evolutionary IFN resistance trend remains under investigation. However, these signals may have the potential to be employed for accurate prediction of therapeutic outcomes. In this review article, we accentuate the significance of host and viral components in IFN resistance with the aim to determine the successful outcome in patients.