丙型肝炎病毒E区基因及其疫苗研究的现状

丙型肝炎病毒Ｅ区基因及其疫苗研究的现状李庆生，陶其敏１９８９年Ｃｈｏｏ等成功地克隆了丙型肝炎病毒（ＨＣＶ）ｃＤＮＡ，并建立了检测ＨＣＶ感染的方法。随着对献血员及血制品进行抗－ＨＣＶ筛查，目前输血后丙型肝炎（ＨＣ）发病率已明显下降，但仍无有效的方法预防...     

乙型与丙型肝炎病毒DNA疫苗研究概况

基因疫苗不仅能诱导宿主CD4^ T细胞介导的体液免疫应答，而且在机体内表达的蛋白还能诱导CD8^ 细胞介导的细胞免疫应答，给乙型肝炎病毒（HBV）和丙型肝炎病毒（HCV）治疗和预防性疫苗的研制带来了新希望。本文综述了近年有关HBV和HCVDNA疫苗的研究进展。     

丙型肝炎疫苗的研究进展

基因疫苗又称核酸疫苗,包括DNA疫苗和RNA疫苗,目前以前者研究为多。基因疫苗不仅能诱导机体CD4~+T细胞介导的免疫反应,且还能诱导机体CD8~+T细胞介导的特异性细胞免疫反应,在丙型肝炎预防和治疗性中具有广阔的应用前景。本文就近年来有关丙型肝炎病毒DNA疫苗的进展作了综述。     

丙型肝炎病毒E2包膜糖蛋白的研究近况

丙型肝炎病毒E2包膜糖蛋白含有重要的受体结合位点和抗原表位,在病毒感染及诱导宿主免疫反应中发挥重要作用,也是目前疫苗研究的热点。E2分子功能特性的研究对理解HCV与宿主相互作用机制,研究新的预防治疗措施有重要意义。本文就E2蛋白与病毒的入胞作用,机体免疫应答及疫苗研制等方面的研究进展作了综述。     

丙型肝炎病毒E2包膜糖蛋白的研究近况

丙型肝炎病毒E2包膜糖蛋白含有重要的受体结合位点和抗原表位，在病毒感染及诱导宿主免疫反应中发挥重要作用，也是目前疫苗研究的热点。E2分子功能特性的研究对理解HCV与宿主相互作用机制，研究新的预防治疗措施有重要意义。本文就E2蛋白与病毒的人胞作用，机体免疫应答及疫苗研制等方面的研究进展作了综述。     

丙型肝炎中和抗体研究进展 FREE

丙型肝炎病毒（hepatitis C virus, HCV）是1989年由美国学者Choo等首次从受感染的黑猩猩血液标本中分离。HCV呈全球性流行,根据世界卫生组织 (WHO) 的统计,HCV在人群中的感染率约为3%,全球约有1.8亿人感染HCV,每年新发丙型肝炎病例约300~500万例。我国进行的全国HCV血清流行病学调查显示［1］,我国一般人群抗 HCV阳性率为3.2%,约有4 000万人感染HCV;70%以上的感染者将发展成肝纤维化、肝硬化,甚至肝细胞癌。常规的干扰素（IFN）单用或者IFN联合利巴韦林治疗的总体应答率比较低,治疗费用颇高,且具有明显的毒副作用［2］。据分析,在未来的10~20年中,将会出现一个因HCV感染而导致的肝硬化与肝癌高峰期。HCV感染后缺乏有效的保护性免疫,因此,HCV中和抗体一直是近年来的研究热点［3］。近年来HCV体外培养系统的建立为HCV中和抗体相关研究提供了强有力的工具,使人们对HCV的认识提升到新的层次,为从根本上认识HCV和彻底防治丙型肝炎奠定了基础。本文就近年来在丙型肝炎中和抗体及机体抗HCV感染免疫方面的研究作一简要综述。     

输血后丙型肝炎E1,E2区抗体的动态研究

用大肠杆菌表达的Ｅ１和Ｅ２区抗原检测７例输血后病例的系列血清，发现ＥＩ、Ｅ２抗体检出率较低，分别为１９％和１７％；Ｅ１、Ｅ２抗体在一些病例中具有早期诊断意义；不同病例膜区抗体动态变化不同；膜区抗体阳性血样中有３０％ＨＣＶＲＮＡ呈阳性。     

丙型肝炎病毒核酸疫苗的研究进展

目前,对丙型肝炎病毒(HCV)所致肝病尚无特效药物及疫苗进行治疗和预防。核酸疫苗技术的出现,为丙型肝炎的防治提供了一个新的机遇。本文就HCV基因免疫中表达区段的选取对基因免疫的影响,接种的剂量和途径,多价DNA疫苗的研制以及是否采用发挥“佐剂样作用”的细胞因子共表达技术等方面作一综述。     

乙型与丙型肝炎病毒DNA疫苗研究概况

基因疫苗不仅能诱导宿主CD4~+T细胞介导的体液免疫应答,而且在机体内表达的蛋白还能诱导CD8~+细胞介导的细胞免疫应答,给乙型肝炎病毒(HBV)和丙型肝炎病毒(HCV)治疗和预防性疫苗的研制带来了新希望。本文综述了近年有关HBV和HCV DNA疫苗的研究进展。     

HSV-2DNA疫苗诱导小鼠免疫应答研究

目的 探讨含有单纯疱疹病毒Ⅱ型(HSV-2)糖蛋白D全基因序列的重组质粒作为DNA疫苗的可能性。方法 采用PCR方法获得HSV-2gD片段，构建含HSV-gD片段的重组质粒pcDNA3-gD。重组质粒作为DNA疫苗免疫小鼠，观察其对小鼠免疫效果及保护作用。结果 重组质粒pcCDNA3-gD免疫小鼠后可产生特异性抗体，可保护小鼠免受HSV-2的致死性攻击，存活率达75％。结论 重组质粒pcDNA3-gD可诱导小鼠产生特异性免疫反应，可以作为HSV的候选DNA疫苗。     

Evaluation of protection induced by a dengue virus serotype 2 envelope domain III protein scaffold/DNA vaccine in non-human primates

We describe the preclinical development of a dengue virus vaccine targeting the dengue virus serotype 2 (DENV2) envelope domain III (EDIII). This study provides proof-of-principle that a dengue EDIII protein scaffold/DNA vaccine can protect against dengue challenge. The dengue vaccine (EDIII-E2) is composed of both a protein particle and a DNA expression plasmid delivered simultaneously via intramuscular injection (protein) and gene gun (DNA) into rhesus macaques. The protein component can contain a maximum of 60 copies of EDIII presented on a multimeric scaffold of Geobacillus stearothermophilus E2 proteins. The DNA component is composed of the EDIII portion of the envelope gene cloned into an expression plasmid. The EDIII-E2 vaccine elicited robust antibody responses to DENV2, with neutralizing antibody responses detectable following the first boost and reaching titers of greater than 1:100,000 following the second and final boost. Vaccinated and naïve groups of macaques were challenged with DENV2. All vaccinated macaques were protected from detectable viremia by infectious assay, while naïve animals had detectable viremia for 2–7 days post-challenge. All naïve macaques had detectable viral RNA from day 2–10 post-challenge. In the EDIII-E2 group, three macaques were negative for viral RNA and three were found to have detectable viral RNA post challenge. Viremia onset was delayed and the duration was shortened relative to naïve controls. The presence of viral RNA post-challenge corresponded to a 10–30-fold boost in neutralization titers 28 days post challenge, whereas no boost was observed in the fully protected animals. Based on these results, we determine that pre-challenge 50% neutralization titers of >1:6000 correlated with sterilizing protection against DENV2 challenge in EDIII-E2 vaccinated macaques. Identification of the critical correlate of protection for the EDIII-E2 platform in the robust non-human primate model lays the groundwork for further development of a tetravalent EDIII-E2 dengue vaccine.     

Antigenicity of hepatitis C virus envelope proteins expressed in Chinese hamster ovary cells

A putative second envelope glycoprotein (E2) of hepatitis C virus (HCV) was constitutively produced in a Chinese hamster ovary cell line stably transformed with a plasmid expressing E2 protein under the control of an exogenous promoter and a signal sequence. E2 protein that lacked part of the C-terminal hydrophobic region was glycosylated with high-mannose type oligosaccharides and retained in the cells. On the other hand, E2 protein lacking the entire C-terminal hydrophobic region was glycosylated with complex type oligosaccharides (complex form) and excreted into the culture medium. Immunoreactivity of the high-mannose and complex forms of E2 proteins against sera from HCV infected patients were analyzed. We found that the antigenicity of the complex form of E2 protein was greater than that of the high-mannose form of E2 protein. This result indicated that the complex form of the E2 protein is superior for use in diagnosing HCV infection.     

DNA vaccine encoding Middle East respiratory syndrome coronavirus S1 protein induces protective immune responses in mice

The Middle East respiratory syndrome coronavirus (MERS-CoV), is an emerging pathogen that continues to cause outbreaks in the Arabian peninsula and in travelers from this region, raising the concern that a global pandemic could occur. Here, we show that a DNA vaccine encoding the first 725 amino acids (S1) of MERS-CoV spike (S) protein induces antigen-specific humoral and cellular immune responses in mice. With three immunizations, high titers of neutralizing antibodies (up to 1: 10⁴) were generated without adjuvant. DNA vaccination with the MERS-CoV S1 gene markedly increased the frequencies of antigen-specific CD4⁺ and CD8⁺ T cells secreting IFN-γ and other cytokines. Both pcDNA3.1-S1 DNA vaccine immunization and passive transfer of immune serum from pcDNA3.1-S1 vaccinated mice protected Ad5-hDPP4-transduced mice from MERS-CoV challenge. These results demonstrate that a DNA vaccine encoding MERS-CoV S1 protein induces strong protective immune responses against MERS-CoV infection.     

Therapeutic effect of DNA vaccine encoding the 60-kDa-heat shock protein from Paracoccidoides brasiliensis on experimental paracoccidioidomycosis in mice

Paracoccidioidomycosis (PCM) is a systemic mycosis autochthonous to Latin America and endemic to Brazil, which has the majority of the PCM cases. PCM is acquired through the inhalation of propagules of fungi from genus Paracoccidioides spp. and mainly affects the lungs. We have previously shown that P. brasiliensis-infected mice treated with single-dose of recombinant 60-kDa-heat shock protein from P. brasiliensis (rPbHsp60) had a worsening infection in comparison to animals only infected. In this study, we investigate whether the treatment of infected mice with PB_HSP60 gene cloned into a plasmid (pVAX1-PB_HSP60) would result in efficient immune response and better control of the disease. The harmful impact of single-dose therapy with protein was not seen with plasmid preparations. Most importantly, three doses of pVAX1-PB_HSP60 and protein induced a beneficial effect in experimental PCM with a reduction in fungal load and lung injury when compared with infected mice treated with pVAX1 or PBS. The increase of the cytokines IFN-γ, TNF, and IL-17 and the decrease of IL-10 observed after treatment with three doses of pVAX1-PB_HSP60 appears to be responsible for the control of infection. These results open perspectives of the therapeutic use of Hsp60 in PCM.     

A novel chimeric dengue vaccine candidate composed of consensus envelope protein domain III fused to C-terminal-modified NS1 protein

There is an urgent need for a safe and effective vaccine against dengue virus (DENV) which infects about 390 million humans per year. In the present study we combined modifications of two DENV proteins, the nonstructural protein 1 (NS1) and the envelope (E) protein, to produce a DENV vaccine candidate with enhanced features. One of these modified proteins was a C-terminal-deleted fragment of NS1 called ΔC NS1 which we have shown previously to be protective without the potentially harmful effects of cross-reactive epitopes common to surface antigens on platelets and endothelial cells. The other modified protein was an envelope protein domain III (cEDIII) containing a consensus amino acid sequence among the four serotypes of DENV, which induces neutralizing antibody against all four DENV serotypes. The cEDIII and ΔC NS1 were expressed as a fusion protein cEDIII-ΔC NS1 and its protective effects against DENV were evaluated in a mouse model. C3H/HeN mice were immunized three times with cEDIII-ΔC NS1 fusion protein mixed with alum as adjuvant. Sera collected from cEDIII-ΔC NS1-immunized mice neutralized four serotypes of DENV and also caused complement-mediated cytolysis of HMEC-1 cells infected with each of the four different DENV serotypes. Mice immunized with cEDIII-ΔC NS1 and challenged with DENV showed reduced serum virus titer, soluble NS1 and bleeding time, compared with mice infected with DENV alone. The results reveal that antibodies induced by cEDIII-ΔC NS1 not only show anti-viral efficacy by in vitro assays but also provide protective effects against DENV infection in a mouse model. The cEDIII-ΔC NS1 thus represents a novel, effective DENV vaccine candidate.     

重组体pEF—HCF诱发小鼠体液免疫反应的研究

目的：探讨基因疫苗在HCV感染中的预防和治疗价值,方法：将HCV C区基因插入pEF质粒EF－1α启动子下游。构建pEF-HCV C重组质粒,然后经肌肉注射及皮下注射免疫Balb/c小鼠。结果：在接种后3周内在肌注组内检测到抗HCV,但是抗体滴度较低,在加强免疫后7天血清抗－HCV明显增高,而其它几组 未检出抗-HCV。结论：构建的HCVDNA重组体可诱发Balb/c上鼠产生体液免疫应答。     

DNA疫苗抗原提呈机制研究进展

DNA疫苗是基因治疗研究中衍生并发展起来的一个新的研究领域。虽然该疫苗的免疫效应在动物实验及部分临床工作中得到验证,但其免疫效果仍不够理想,免疫效价低于现有疫苗。深入研究DNA疫苗免疫后抗原提呈机制成为近年研究热点内容,此文就近年来这方面的研究进展综述如下。     

流感病毒神经氨酸酶双表达DNA疫苗的构建及在小鼠中的免疫保护研究

目的研究对多亚型流感病毒流行提供预防的DNA疫苗。方法以pIRES为双表达载体,在其两个多克隆位点中分别插入H1N1（A/PR/8/34）和H3N2（A/Guizhou/54/89）的NA DNA片段,构建双表达质粒pN1-IRES-N2及pN2-IRES-N1。以BALB/c小鼠为模型,采用电穿孔法进行免疫,并用致死量流感病毒感染以检测双表达载体的保护力。结果实验表明pN1-IRES-N2能完全保护小鼠抵御致死量H1N1同源病毒的攻击,部分保护小鼠抵御致死量H3N2同源病毒攻击;pN2-IRES-N1能完全保护小鼠抵御致死量H3N2同源病毒攻击,对致死量H1N1同源病毒的攻击提供部分保护。结论双表达DNA疫苗可望开发为一种提供广泛保护的新型流感疫苗。     

Baculoviral COVID-19 Delta DNA vaccine cross-protects against SARS-CoV2 variants in K18-ACE2 transgenic mice

After severe acute respiratory syndrome coronavirus-2 (SARS-CoV2) made the world tremble with a global pandemic, SARS-CoV2 vaccines were developed. However, due to the coronavirus's intrinsic nature, new variants emerged, such as Delta and Omicron, refractory to the vaccines derived using the original Wuhan strain. We developed an HERV-enveloped recombinant baculoviral DNA vaccine against SARS-CoV2 (AcHERV-COVID19S). A non-replicating recombinant baculovirus that delivers the SARS-CoV2 spike gene showed a protective effect against the homologous challenge in a K18-hACE2 Tg mice model; however, it offered only a 50 % survival rate against the SARS-CoV2 Delta variant. Therefore, we further developed the AcHERV-COVID19 Delta vaccine (AcHERV-COVID19D). The AcHERV-COVID19D induced higher neutralizing antibodies against the Delta variant than the prototype or Omicron variant. On the other hand, cellular immunity was similarly high for all three SARS-CoV2 viruses. Cross-protection experiments revealed that mice vaccinated with the AcHERV-COVID19D showed 100 % survival upon challenge with Delta and Omicron variants and 71.4 % survival against prototype SARS-CoV2. These results support the potential of the viral vector vaccine, AcHERV-COVID19D, in preventing the spread of coronavirus variants such as Omicron and SARS-CoV2 variants.