沙眼衣原体主要外膜蛋白多表位基因的免疫原性研究

目的 构建包含沙眼衣原体(Chlamydia trachomatis,Ct)主要外膜蛋白(major outermembrsne protein,MOMP)多表位基因的真核表达质粒PcDNA3.1-Ct MOMP168,检测其诱导BALB/c小鼠产生Ct MOMP特异性体液和细胞免疫反应的水平.方法 构建包含Ct MOMP多表位基因的重组质粒PcDNA3.1-Ct MOMP168并以肌肉注射方式免疫BALB/c小鼠,同时设置pcDNA3.1组和PBS组对照(每组12只,每只100μg/次).采用酶联免疫吸附试验(ELISA)、乳酸脱氢酶释放法及细胞内细胞因子染色-流式细胞术(ICS-FACS)分别检测免疫小鼠血清中Ct MOMP特异性抗体的滴度及其抗体亚型、脾脏中细胞毒性T淋巴细胞(CTL)的杀伤活性及特异性分泌细胞因子,如γ-干扰素-(IFN-γ)、白细胞介素-4(IL-4)、白细胞介素-10(IL-10)等的CD3+T细胞的水平.结果 同pcDNA3.1(A490=0.180±0.025)和PBS免疫(A490=0.110±0.015)相比,PcDNA3.1-Ct MOMP168免疫可刺激小鼠产生高效价Ct特异性IgG抗体(A490=0.973±0.136;血清滴度1:1000),且抗体亚型以IgG2a为主,差异有统计学意义(F=106.884,P<0.05);pcDNA3.1-Ct MOMP168免疫组小鼠脾细胞中CTL杀伤活性(41.71%±8.34%)高于pcDNA3.1组(18.40%±3.45%)和PBS组(14.50%±2.42%),差异有统计学意义(F=22.315,P<0.05;效靶比为50:1);pcDNA3.1-Ct MOMP168免疫组小鼠的脾细胞中Ct MOMP特异性CD3+IFN-γ+T细胞的水平(1.15%±0.16%)高于pcDNA3.1组(0.12%±0.08%)和PBS组(0.09%±0.03%),差异有统计学意义(F=99.638,P<0.05),而PcDNA3.1-CtMOMP168组IL-4+CD3+T细胞(0.13%±0.08%)和IL-10+CD3+T细胞(0.14%±0.08%)的水平与pcDNA3.1(0.07%±0.05%,0.13%±0.06%)和PBS(0.08%±0.04%,0.07%±0.04%)组比较差异无统计学意义(F值分别为0.886、1.112,P值均>0.05).结论pcDNA3.1-Ct MOMP168重组质粒能诱导BALB/c小鼠产生Ct MOMP特异性的体液免疫和细胞免疫应答.     

B族链球菌多糖—蛋白质结合物黏膜免疫小鼠血清IgG研究

目的探讨B族链球菌荚膜多糖-破伤风类毒素结合物（GBS-CPS-TT）,经不同免疫途径免疫小鼠的可行性及免疫剂量。方法经滴鼻、灌胃、皮下注射途径免疫小鼠,各组均免疫3次,每次免疫间隔时间2周,免疫后1周时采集血清,采用间接酶联免疫吸附实验（ELISA）检测血清中特异性IgG的抗体滴度。应用统计软件SPSS13.0对数据进行统计学分析,组间均数比较采用方差分析。结果 GBS-CPS-TT经滴鼻免疫、灌胃免疫和皮下注射均可以诱导产生特异性血清IgG抗体。IgG抗体滴度分别为：滴鼻5μg组（2.671±0.207）;滴鼻10μg组（2.911±0.263）;皮下注射5μg组（2.866±0.251）;灌胃10μg组（2.851±0.224）。滴鼻10μg组诱导产生的IgG抗体滴度明显高于滴鼻5μg组（P〈0.01）。结论 GBS-CPS-TT经滴鼻免疫小鼠后可产生系统性免疫应答。     

序贯联合应用HIV-1 gp160腺病毒载体疫苗的体液免疫效果研究

目的探讨序贯联合应用Ad5-HIVgp160疫苗与其他艾滋病疫苗所诱导的特异性体液免疫反应效果。方法用Ad5-HIVgp160疫苗、其他腺病毒载体和腺病毒相关载体疫苗以不同免疫程序分别免疫豚鼠,通过ELISA和中和实验方法检测其诱导的免疫反应,比较疫苗联合免疫组豚鼠与疫苗单独免疫组豚鼠诱导的体液免疫反应的强度。结果不同免疫程序的豚鼠均能检测到针对HIV gp120的HIV结合抗体和针对HIV敏感株sf162的中和活性;联合疫苗组诱导的gp120特异性抗体滴度和病毒中和活性高于单独免疫组,但无显著性差异。结论 Ad5-HIVgp160疫苗单独或与其他疫苗联合使用均能诱导高水平的体液免疫反应,能激发针对HIV敏感株产生中和活性的抗体。     

成团泛菌脂多糖对乙型肝炎表面抗体的调节作用

目的 研究革、三阴性非致病菌成团泛菌脂多糖（Pantoea agglomerans lipopolysaccharide，LPSp）作为佐剂对重组乙型肝炎表面抗原（HBsAg）及乙型肝炎疫苗诱导小鼠产生抗-HBs抗体的影响。方法 采用纯系（Balb／c）小鼠作为免疫对象，经背部皮下免疫2次，ELISA法检测血清乙型肝炎表面抗体（抗-HBs）滴度。结果 HBsAg加LPSp组，其抗-HBs总抗体滴度较单独注射HBsAg明显增高（P〈0．05）；乙肝疫苗加LPSp组的抗-HBs IgG滴度虽在免疫中期与单独注射乙肝疫苗组无明显差别，但其抗体滴度要高于乙肝疫苗组；加LPSp组抗体持续时间可更长。结论 LPSp对小鼠抗-HBs抗体的产生具有增强作用；在诱导小鼠抗-HBs抗体产生方面，LPSp与疫苗中的铝佐剂具有协同效应，提示LPSp是一种很有潜力的HBsAg佐剂。     

人IL-12基因表达对含信号肽mtb8.4基因疫苗的免疫效应

[目的]观察结核分枝杆菌含信号肽Mtb8.4(MS)基因疫苗与人白细胞介素12(hIL-12)表达质粒联合免疫小鼠所诱导的细胞免疫应答。[方法]15只C57BL/6N小鼠随机分为MS基因疫苗 hIL-12质粒组(联合免疫组)、MS基因疫苗组、卡介苗(BCG)组、空载体组和PBS组,基因疫苗、空载体和PBS经肌内注射法免疫各组小鼠,每隔3周免疫1次,共免疫3次,BCG组经尾部皮下注射1×106 CFU BCG免疫1次。采用酶联免疫吸附法(ELISA)检测小鼠脾细胞培养上清中细胞因子水平;采用乳酸脱氢酶(LDH)释放法检测免疫小鼠细胞毒性T细胞(CTL)杀伤活性。[结果]联合免疫组能诱导较强的抗原特异性Th1型细胞免疫应答,免疫小鼠脾细胞培养上清液IFN-γ和IL-2水平(分别为(1546.18±36.23)pg/ml、(681.64±40.88)pg/ml),显著高于MS基因疫苗组,与BCG组相当,IL-4分泌减少,特异性CTL杀伤活性增强。[结论]hIL-12表达质粒与MS基因疫苗联合免疫后,能够增强MS基因疫苗所诱导的细胞免疫应答。     

麻疹母传抗体水平及6～8月龄接种麻疹疫苗的免疫效果监测

[目的]了解8月龄以下婴儿麻疹母传抗体水平及6～8月龄婴儿接种麻疹疫苗的免疫效果和安全性,探讨控制小月龄婴儿麻疹的免疫策略。[方法]在南昌市的12个县(区)随机抽取0～8月龄婴儿各70余名,监测麻疹母传抗体;对6～8月婴儿初免麻疹疫苗后免疫效果及安全性观察,采用酶联免疫吸附试验检测免前和免后1个月的麻疹IgG抗体滴度。[结果]8月龄以下婴儿母传抗体几何平均滴度为1︰209,各月龄组抗体几何平均滴度均未达保护水平;6月龄、7月龄、8月龄初免后GMT分别为1︰1407、1︰1897、1︰2410,初免前后GMT差异有统计学意义(P﹤0.05),初免后3组的阳性率均≥90%,免疫成功率﹥85%,接种MV未出现异常反应,提示6月龄的MV初免程序是可行的;但6、7、8月龄3组间免疫成功率差异有统计学意义(P﹤0.05),免前母传抗体阴性和阳性的免疫成功率差异有统计学意义(P﹤0.05),提示婴儿母传抗体的存在对MV的免疫成功率有干扰作用。[结论]婴儿麻疹母传抗体滴度均较低,无有效保护作用;6足月后接种麻疹疫苗安全有效。建议进行育龄妇女孕前接种MV和提前婴儿MV初免时间的成本效益研究,制定安全有效的免疫策略,减少小月龄婴儿麻疹发病率。     

卡氏肺孢子虫p55基因片段DNA疫苗的免疫原性研究

[目的]构建卡氏肺孢子虫抗原p55基因片段DNA疫苗,并研究该疫苗的体外表达及在小鼠体内的免疫原性.[方法]构建真核表达质粒pcDNA3.1(+)-582,用免疫印渍技术检测目的蛋白的免疫原性.将雌性BALB/c小鼠分为3组,以肌肉注射方式分别注射重组表达质粒pcDNA3.1(+)-582(免疫组)、pcDNA3.1(+)及PBS(对照组).ELISA法检测小鼠血清中特异性抗体水平,MTT法检测免疫小鼠的淋巴细胞增殖能力.[结果]成功构建了重组真核表达质粒pcDNA3.1(+)-582,免疫印渍技术证实目的蛋白与免疫后的小鼠阳性血清能发生特异性反应;ELISA结果显示免疫后pcDNA3.1(+)-582组小鼠抗体水平呈增高的趋势,pcDNA3.1(+)-582组小鼠淋巴细胞的增殖指数明显高于pcDNA3.1(+)组及PBS组.免疫组与对照组相比较差异显著,差异有统计学意义(P<0.05).[结论]卡氏肺孢子虫抗原p55基因片段DNA疫苗构建成功,免疫小鼠后能诱导产生一定的体液免疫和细胞免疫.     

成团泛菌脂多糖对乙型肝炎表面抗体的调节作用

目的研究革兰阴性非致病菌成团泛菌脂多糖（Pantoea agglomerans lipop01ysaccharide，LPSp）作为佐剂对重组乙型肝炎表面抗原（HBsAg）及乙型肝炎疫苗诱导小鼠产生抗-HBs抗体的影响。方法采用纯系（Balb／C）小鼠作为免疫对象，经背部皮下免疫2次，ELISA法检测血清乙型肝炎表面抗体（抗-HBs）滴度。结果HbsAg加LPSp组，其抗-HBs总抗体滴度较单独注射HbsAg明显增高（P〈0．05）；乙肝疫苗加LPSp组的抗-HBs IgG滴度虽在免疫中期与单独注射乙肝疫苗组无明显差别，但其抗体滴度要高于乙肝疫苗组；加LPSp组抗体持续时间可更长。结论LPSp对小鼠抗-HBs抗体的产生具有增强作用；在诱导小鼠抗-HBs抗体产生方面，LPSp与疫苗中的铝佐剂具有协同效应，提示LPSp是一种很有潜力的HbsAg佐剂。     

2013-2016年天津市狂犬病暴露后免疫效果分析

目的 了解天津市狂犬病暴露后免疫者血清中和抗体水平,为指导狂犬病预防控制工作提供科学依据。方法 对2013-2016年在天津市疾病预防控制中心动物致伤门诊全程注射狂犬病疫苗并于免疫后14～30天采血进行快速荧光灶抑制试验者进行统计学分析。结果 不同月份接种狂犬病疫苗,中和抗体水平趋于平稳,在10IU/ml上下波动;未成年组头面部暴露比例最高,青壮年组下肢暴露比例最高,老年组上肢暴露比例最高;91例暴露后免疫者血清中和抗体几何平均滴度为9714IU/ml,阳转率为100%;女性中和抗体水平明显高于男性(t=2482,P=0015);复种组中和抗体水平远高于初种组(疫苗组及疫苗+蛋白组)(F=5356,P=0006);不同年龄组、不同暴露部位、不同暴露等级及接种不同疫苗中和抗体水平无统计学差异(P皆＞005)。结论 狂犬病暴露后免疫效果不受季节、年龄、暴露部位、暴露等级及接种疫苗种类影响,女性免疫效果优于男性,复种组免疫效果明显优于初种组。     

微小隐孢子虫pcDNA3.0-23重组质粒免疫效果评价

目的 比较微小隐孢子虫表面抗原CP23真核表达载体pcDNA3.0-23经不同免疫途径产生的免疫效果。方法 提取微小隐孢子虫基因组DNA,PCR扩增CP23基因片段,克隆至真核表达载体pcDNA3.0,构建pcD-NA3.0-23重组质粒,分别通过肌肉注射和滴鼻(粘膜)免疫2种途径免疫BALB/c小鼠,免疫3次,2周后检测抗CP23特异性抗体IgG滴度、小鼠脾脏、血清中CD4⁺和CD8⁺T细胞、细胞因子γ干扰素(IFN-γ);用微小隐孢子虫攻击感染被免疫小鼠,收集小鼠粪便,计算小鼠排出的卵囊量。结果 肌注组与滴鼻组小鼠血清抗CP23特异性抗体IgG滴度随免疫次数增加均明显升高,高于对照组及空质粒组(P<0.05),肌注组高于滴鼻组(P<0.05);肌注组与滴鼻组小鼠的CD4⁺T细胞、CD4⁺/CD8⁺比值均高于磷酸缓冲液(PBS)对照组及pcDNA3.0空质粒组(P<0.05),但2种免疫途径的差异无统计学意义(P>0.05);肌注组和滴鼻组脾细胞培养上清中IFN-γ明显高于对照组及空质粒组(P<0.05);微小隐孢子虫攻击小鼠后,2种免疫途径的小鼠排出卵囊量明显少于对照组,且排出时间缩短,2种途径的差异无统计学意义。结论 pcDNA3.0-23重组质粒作为基因疫苗,可产生较好的细胞及体液免疫反应;不同的免疫途径可产生不同的免疫反应。     

Immunogenicity of a DNA vaccine for coxsackievirus B3 in mice: protective effects of capsid proteins against viral challenge

Coxsackievirus (CVB) 3 induces viral myocarditis and ultimately dilated cardiomyopathy (DCM). However, there is no vaccine in clinical use. We constructed recombinant CVB3 plasmids using a highly effective mammalian expression vector and evaluated their immunogenicity in vivo on the basis of survival rate. Four recombinant plasmids were constructed, which encode CVB3 capsid proteins (VP1 or VP3) or VP1 partial proteins (VP1-1 or VP1-2), and used to immunize BALB/c mice by electroporation. Although VP1, VP3, VP1-1, and VP1-2 induced specific antibodies against the corresponding proteins in mice, neutralizing antibodies were not present in the sera. These recombinant plasmids, except VP1-1 (12.5%), dramatically increased the survival rate in mice at 46 days after challenge (42.9-75.0%, p<0.05). VP3 (75.0%) protected mice against viral infection and the middle regions of VP1 (VP1-2, 50.5%) conferred a protective effect like that conferred by VP1 (42.9%), suggesting that the epitopes in VP3 as well as in the middle of VP1 protect against CVB3 infection in vivo. In conclusion, some recombinant CVB3 plasmids used in this study reduced the destruction of myocytes and improved the survival rates in mice immunized and challenged compared with the control. Thus, pCA-VP3 as well as pCA-VP1 are good candidates for a CVB3 DNA vaccine.     

Genotypic linkages of VP4, VP6, VP7, NSP4, NSP5 genes of rotaviruses circulating among children with acute gastroenteritis in Thailand

Rotavirus is the main cause of acute viral gastroenteritis in infants and young children worldwide. Surveillance of group A rotavirus has been conducted in Chiang Mai, Thailand since 1987 up to 2004 and those studies revealed that group A rotavirus was responsible for about 20-61% of diarrheal diseases in hospitalized cases. In this study, we reported the continuing surveillance of group A rotavirus in 2005 and found that group A rotavirus was detected in 43 out of 147 (29.3%) stool samples. Five different G and P genotype combinations were detected, G1P[8] (27 strains), G2P[4] (12 strains), G9P[8] (2 strains), G3P[8] (1 strain), and G3P[10] (1 strain). In addition, analysis of their genotypic linkages of G (VP7), P (VP4), I (VP6), E (NSP4), and H (NSP5) genotypes demonstrated that the rotaviruses circulating in Chiang Mai, Thailand carried 3 unique linkage patterns. The G1P[8], G3P[8], and G9P[8] strains carried their VP6, NSP4, NSP5 genotypes of I1, E1, H1, respectively. The G2P[4] strains were linked with I2, E2, H2 genotypes, while an uncommon G3P[10] genotype carried unique genotypes of I8, E3 and H6. These findings provide the overall picture of genotypic linkage data of rotavirus strains circulating in Chiang Mai, Thailand.     

Generation of protective immune responses against coxsackievirus B3 challenge by DNA prime-protein boost vaccination

Coxsackievirus B3 (CVB3) causes viral myocarditis and can ultimately result in dilated cardiomyopathy. However, there is no vaccine available for clinical use. In this study, we assessed the protection provided by three immunization strategies against CVB3 infection. Vaccination was performed with a DNA vaccine expressing the cloned capsid gene VP1 or a vaccine developed from purified VP1 protein. Third, a strategy of vaccination was attempted with the DNA vaccine followed by two boosts with the recombinant protein vaccine (DNA prime-protein boost vaccine). Followed immunization, mice were challenged with CVB3 infection. Improved induction of CVB3-specific antibodies and neutralizing antibodies were found in mice immunized by the DNA prime-protein boost regimen. Furthermore, virus-specific cytotoxic activity of spleen cells derived from DNA prime-protein boost vaccinated mice was elicited. In addition, the DNA prime-protein boost vaccine resulted in protection of 75% of mice from lethal CVB3 challenge and a significant reduction of viral load in sera of immunized mice after acute CVB3 infection. There was a significant reduction in myonecrosis and infiltrating myocardial immune cells indicating reduced severity of myocarditis in surviving mice. These findings demonstrated that a DNA prime-protein boost immunization strategy, but not a DNA vaccine or protein vaccine alone, was effective in eliciting both humoral and cell-mediated immune responses against CVB3 infection in mice and might be a promising vaccine candidate.     

Attenuation of coxsackievirus B3 by VP2 mutation and its application as a vaccine against virus-induced myocarditis and pancreatitis

Coxsackievirus B3 (CVB3) is a common agent of viral myocarditis, a major cause of sudden cardiac death, and ultimately dilated cardiomyopathy. However, there is no vaccine in clinical use. In this study, we identified the conserved amino acid sequences in the C-terminal region of the VP2 of the coxsackievirus B group and some echoviruses. The mutant virus, YYFF, with phenylalanines substituted for two tyrosines in these conserved sequences was highly attenuated in vivo and could induce a high neutralizing antibody titer and a cytotoxic T-lymphocyte response against CVB3. Thereby, mutant-virus-immunized mice showed a 100% survival rate and protection against inflammation of the heart and pancreas after lethal dose challenge. Thus, this mutant virus is a good candidate for an attenuated CVB3 vaccine.     

口服IL 12重组双歧杆菌对柯萨奇B3病毒诱导BLAB/c小鼠心肌炎的影响

目的构建白细胞介素（IL） 12基因重组双歧杆菌(pBBADs IL 12转化双歧杆菌),观察pBBADs IL 12转化双歧杆菌是否对柯萨奇B3病毒(CVB3)诱导的小鼠心肌炎具有治疗作用。方法构建小鼠IL 12(mIL 12)基因的pBBADs IL 12表达载体,转化双歧杆菌,体外通过酶联免疫吸附试验及Western免疫印迹验证重组双歧杆菌工程菌在L 阿拉伯糖诱导下mIL 12的表达。选取BLAB/c小鼠30只,腹腔内注射CVB3感染剂量,14 d后形成病毒性心肌炎,将感染的小鼠随机分成IL 12组、绿荧光蛋白(GFP)组及生理盐水组。IL 12组给予口服pBBADs IL 12转化双歧杆菌;GFP组给予口服pBBADs GFP转化双歧杆菌;生理盐水组给予腹腔注射无菌PBS（1次/d）。所有小鼠均在治疗14 d后取心脏标本观察心肌病理变化,检测心肌病毒滴度,荧光定量PCR分析Th1细胞因子水平。结果治疗14 d后,HE染色显示IL 12组小鼠心肌炎症程度较GFP组和生理盐水组明显减轻;IL 12组小鼠心脏炎症病变百分比为（18±5）%,心肌的病毒滴度为（2.89±0.18）pfu/g,显著低于GFP组［分别为（31±6）%和（4.83±0.14）pfu/g］及生理盐水组［分别为（32±9）%和（4.80±0.15）pfu/g］,差异有统计学意义(均P＜0.01）;IL 12组小鼠心脏γ 干扰素［（2.27±0.15）pg/mL］和肿瘤坏死因子 α［（3.05±0.17）pg/mL］水平显著高于GFP组［分别为（1.32±0.11）pg/mL和（2.37±0.16）pg/mL］及生理盐水组［分别为（1.38±0.11）pg/mL和(2.37±0.12)pg/mL］,差异有统计学意义（均P<0.01）。结论此次研究成功构建了一种新型表达mIL 12的双歧杆菌载体,口服IL 12转化双歧杆菌对CVB3病毒诱导的小鼠心肌炎具有较好疗效。     

重组乳酸杆菌中A组轮状病毒VP7基因表达

目的扩增A组轮状病毒VP7基因,构建pEDM27/5-VP7重组质粒,转化乳酸杆菌并分析VP7蛋白的表达及活性。方法通过逆转录聚合酶链反应(RT-PCR)获得目的基因VP7,纯化后与pEDM27/5载体进行连接,转化乳酸杆菌,乳糖进行诱导表达,分别于4,8,12 h后,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白印迹技术(Western blot)对产物进行分析测定。结果A组轮状病毒经RT-PCR扩增后,1%琼脂糖电泳检测可见一条约1.0 kb的特异性条带,与预期目的基因(VP7基因)大小相符;SDS-PAGE和Western-blot杂交分析发现,经乳糖诱导4,8,12 h后分别可见一条分子量约为28 kD蛋白带表达,经扫描分析,诱导后表达蛋白分别约占菌体总蛋白的2.30%,5.12%,5.38%,且随着时间变化而持续表达增加,并能与抗A组轮状病毒VP7蛋白特异结合。结论重组乳酸杆菌持续表达免疫活性A组轮状病毒VP7蛋白,为进一步研究乳酸杆菌作为轮状病毒基因工程疫苗的表达载体提供了基础依据。     

Genomic analysis of group A rotavirus G12P[8] including a new Japanese strain revealed evidence for intergenotypic recombination in VP7 and VP4 genes

Group A rotavirus is a leading cause of severe acute gastroenteritis worldwide. In this study, the first complete coding sequences of 11 RNA segments of human group A rotavirus G12P[8] in Japan were determined by an unbiased viral metagenomics. Its genomic constellation (VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 genes) was identified as G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. When performing the genetic analysis, we discovered an intergenotypic recombination event in the pig group A rotavirus G12P[8] strain BUW-14-A008. The novel recombination was found between two different genotypes G12 and G3 in the VP7 gene, and P[8] and P[13] in the VP4 gene.     

G1P[8] Rotavirus in children with severe diarrhea in the post-vaccine introduction era in Brazil: Evidence of reassortments and structural modifications of the antigenic VP7 and VP4 regions

Worldwide rotaviruses A (RVA) are responsible for approximately 215,000 deaths annually among children aged <5 years. RVA G1P[8] remains associated with >50% of gastroenteritis cases in this age group. The aim of this study was to assess the genetic variability of G1P[8] strains detected in children with severe diarrhea in Belém, Pará, Brazil, during the post-rotavirus vaccine introduction era. Phylogenetic analysis clustered the VP4 and VP7 genes of 40 samples selected between 2009 and 2011 into lineages found to be different from the Rotarix® vaccine strain. A detailed investigation of their complete genotype constellations identified 2 reassortant viruses (5%), resulting from reassortments between the genogroups Wa-like and DS-1-like (G1-P[8]-I1-R2-C1-M1-A1-N1-T2-E1-H1) and Wa-like and AU-1-like (G1-P[8]-I1-R3-C1-M1-A1-N1-T1-E1-H1) genotype constellations. A comparison of the amino acid residues presents in the antigenic epitopes of VP7 and VP4, showed differences in the electrostatic charges distribution, between wild type Brazilian strains and the Rotarix® and RotaTeq® vaccine strains. These findings reflect the structural analyses of the antigenic regions of VP7 and VP4 of the RVA G1P[8] in children with gastroenteritis in Northern Brazil raising the hypothesis that structural modifications at these sites over time may account for the emergence of new strains that could possibly pose a challenge to current vaccines.     

Construction and characterization of human rotavirus recombinant VP8* subunit parenteral vaccine candidates

Two currently licensed live oral rotavirus vaccines (Rotarix^® and RotaTeq^®) are highly efficacious against severe rotavirus diarrhea. However, the efficacy of such vaccines in selected low-income African and Asian countries is much lower than that in middle or high-income countries. Additionally, these two vaccines have recently been associated with rare case of intussusception in vaccinated infants. We developed a novel recombinant subunit parenteral rotavirus vaccine which may be more effective in low-income countries and also avert the potential problem of intussusception. Truncated recombinant VP8* (ΔVP8*) protein of human rotavirus strain Wa P[8], DS-1 P[4] or 1076 P[6] expressed in Escherichia coli was highly soluble and was generated in high yield. Guinea pigs hyperimmunized intramuscularly with each of the ΔVP8* proteins (i.e., P[8], P[4] or P[6]) developed high levels of homotypic as well as variable levels of heterotypic neutralizing antibodies. Moreover, the selected ΔVP8* proteins when administered to mice at a clinically relevant dosage, route and schedule, elicited high levels of serum anti-VP8* IgG and/or neutralizing antibodies. Our data indicated that the ΔVP8* proteins may be a plausible additional candidate as new parenteral rotavirus vaccines.     

The VP1 S154D mutation of type Asia1 foot-and-mouth disease virus enhances viral replication and pathogenicity

One of the proteins encoded by the foot-and-mouth disease virus (FMDV), the VP1 protein, a capsid protein, plays an important role in integrin receptor attachment and humoral immunity-mediated host responses. The integrin receptor recognition motif and an important antigenic epitope exist within the G–H loop, which is comprised of amino acids 134–160 of the VP1 protein. FMDV strain, Asia1/HN/CHA/06, isolated from a pig, was passaged four times in suckling mice and sequenced. Sequencing analyses showed that there was a mutation of the integrin receptor recognition motif Arg-Gly-Asp/Arg-Asp-Asp (RGD/RDD, VP1 143–145) and a VP1 154 serine/Asp (VP1 S154D) mutation in the G–H loop of the VP1 protein. The influence of the RGD/RDD mutation on Asia1 FMDV disease phenotype has been previously studied. In this study, to determine the influence of the VP1 S154D mutation on FMDV Asia1 replication and pathogenicity, two recombinant FMDVs with different residues only at the VP1 154 site were rescued by reverse genetics techniques and their infectious potential in host cells and pathogenicity in pigs were compared. Our data indicates that the VP1 S154D mutation increases the replication level of FMDV Asia1/HN/CHA/06 in BHK-21, IB-RS-2, and PK-15 cells and enhances pathogenicity in pigs. Through the transient transfection-infection assay to compare integrin receptor usage of two recombinant viruses, the result shows that the VP1 S154D mutation markedly increases the ability of type Asia1 FMDV to use the integrin receptors α_υβ₆ and α_υβ₈ from pig. This study identifies a key research target for illuminating the role of residues located at G–H loop in FMDV pathogenicity.