呼吸道合胞病毒疫苗研究进展

呼吸道合胞病毒(RSV)是引起严重下呼吸道感染的重要病原体,尽管经历了半个多世纪的努力,至今仍未有安全有效的RSV疫苗上市。近年来在RSV F蛋白结构生物学方面的研究进展为新一代RSV疫苗的开发提供了新方向,同时更多的采用不同技术、或针对不同人群的RSV侯选疫苗也在迅速发展,尤其是针对婴幼儿及老年人的RSV侯选苗已有60多种在研究中,大部分已处于临床前研究阶段,18种侯选苗已进入临床试验。我们简要介绍RSV疫苗的最新研究进展。     

呼吸道传染病治疗中抗体药物的研发进展

呼吸系统疾病影响着全世界数百万人,主要病变发生在气管、支气管、肺部及胸腔,病变轻者多咳嗽、胸痛,重者呼吸困难、缺氧甚至呼吸衰竭,可造成多种并发症,导致患者严重残疾甚至死亡。治疗性抗体的临床使用为肺癌、哮喘以及各类呼吸道传染病等的治疗开辟了新途径。目前已有数十种抗体（antibodies,Abs）获得市场批准,而且还有更多的抗体药物正在临床开发中。这些Abs中的大多数是针对哮喘、肺癌、慢性阻塞性肺病、特发性肺纤维化以及呼吸道传染病等疾病。其中,呼吸道传染病的爆发具有传播迅猛、传染性强的特点,常引发全球关注,如当下肆虐全球的新型冠状病毒肺炎。针对呼吸道传染病的多种Abs为其临床治疗提供了新策略。基于此,综述了已获准和正在临床开发的适用于治疗呼吸道传染病的Abs,通过综述抗体治疗的分子机制、优势和发展趋势,以期为呼吸道传染病治疗中抗体药物的研发提供参考。     

呼吸道合胞病毒诱导细胞凋亡与自噬

呼吸道合胞病毒感染与细胞凋亡、自噬的关系错综复杂。研究发现呼吸道合胞病毒感染细胞后,既能产生促细胞凋亡作用,也能产生抗细胞凋亡作用,还能诱导细胞发生自噬。研究这些过程机理,能帮助我们更好地认识呼吸道合胞病毒感染发病机制,为预防和治疗呼吸道合胞病毒感染提供一些新的方向。     

呼吸道合胞病毒疫苗研究进展

呼吸道合胞病毒(RSV)是引起婴幼儿支气管炎和肺炎的主要原因,并可致免疫缺陷病人显著发病和死亡,RSV疫苗已被WHO列为全球最优先发展的疫苗之一。经过几十年的研究,虽然取得了显著进展,但尚未有RSV疫苗上市。目前RSV疫苗的研究主要集中于亚单位疫苗、减毒活疫苗和DNA疫苗等,其中亚单位疫苗和减毒活疫苗被认为最有前途,已分别进行了的临床试验。     

NK cell immunophenotypic and genotypic analysis of infants with severe respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infection in infants. Reduced numbers of NK cells have been reported in infants with severe RSV infection; however, the precise role of NK cells during acute RSV infection is unclear. In this study the NK and T cell phenotypes, LILRB1 gene polymorphisms and KIR genotypes of infants hospitalized with RSV infection were analyzed. Compared to controls, infants with acute RSV infection showed a higher proportion of LILRB1+ T cells; in addition, a subgroup of infants with RSV infection showed an increase in LILRB1+ NK cells. No differences in NKG2C, NKG2A, or CD161 expression between RSV infected infants and controls were observed. LILRB1 genotype distribution of the rs3760860 A>G, and rs3760861 A>G single nucleotide polymorphisms differed between infants with RSV infection and healthy donors, whereas no differences in any of the KIR genes were observed. Our results suggest that LILRB1 participates in the pathogenesis of RSV infection. Further studies are needed to define the role of LILRB1+ NK in response to RSV and to confirm an association between LILRB1 polymorphisms and the risk of severe RSV infection.     

Local interleukin-10 production during respiratory syncytial virus bronchiolitis is associated with post-bronchiolitis wheeze

Background

Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis in infants. Following RSV bronchiolitis, 50% of children develop post-bronchiolitis wheeze (PBW). Animal studies have suggested that interleukin (IL)-10 plays a critical role in the pathogenesis of RSV bronchiolitis and subsequent airway hyperresponsiveness. Previously, we showed that ex vivo monocyte IL-10 production is a predictor of PBW. Additionally, heterozygosity of the single-nucleotide polymorphism (SNP) rs1800872 in the IL10 promoter region was associated with protection against RSV bronchiolitis.

Methods

This study aimed to determine the in vivo role of IL-10 in RSV pathogenesis and recurrent wheeze in a new cohort of 235 infants hospitalized for RSV bronchiolitis. IL-10 levels in nasopharyngeal aspirates (NPAs) were measured at the time of hospitalization and the IL10 SNP rs1800872 genotype was determined. Follow-up data were available for 185 children (79%).

Results

Local IL-10 levels during RSV infection turned out to be higher in infants that later developed physician diagnosed PBW as compared to infants without PBW in the first year after RSV infection (958 vs 692 pg/ml, p = 0.02). The IL10 promoter SNP rs1800872 was not associated with IL-10 concentration in NPAs.

Conclusion

The relationship between high local IL-10 levels during the initial RSV infection and physician diagnosed PBW provides further evidence of the importance of the IL-10 response during RSV bronchiolitis.     

表达猪繁殖与呼吸综合征病毒GP3基因的重组伪狂犬病病毒的构建

猪繁殖与呼吸综合征 (porcinereproductiveandrespiratorysyndrome ,PRRS)是引起怀孕母猪早产、流产、死胎及仔猪呼吸系统疾病的一种新发现的病毒性传染病[1] .该病毒的基因组为单股正链RNA ,约15kb ,含有 8个开放阅读框架 (ORFs) ,ORF1编码病毒非结构蛋白 (依赖RNA的RNA聚合酶 ) ,ORF2 ORF7编码病毒的结构蛋白 .其中ORF3含有 2 6 5个氨基酸 ,编码的GP3蛋白为高度糖基化的结构蛋白 ,有 7个糖基化位点 ,具有免疫原性[2 ,3 ] .目前 ,用于预防PRRS的疫苗主要是弱毒苗和灭活苗 ,虽然都有一定的免疫效果 ,但由于PRRS抗体依赖性…     

表达猪繁殖与呼吸综合征病毒修饰型GP5m蛋白的重组伪狂犬病毒的构建及其在小鼠的免疫反应

猪伪狂犬病毒（PRV）是一种良好的兽用活病毒疫苗载体。但以PRV基因缺失疫苗株TK^-/gE^-/LacZ⁺为载体表达PRRSV GP5的重组病毒TK^-/gE^-/GP5⁺免疫实验动物后难以激发抗PRRSV的中和抗体。为了进一步增强这种重组病毒的免疫效力,用具有更好免疫原性的修饰的ORF5基因（ORF5m）代替天然ORF5基因,构建了表达PRRSV的修饰型GP5m蛋白的重组伪狂犬病毒TK^-/gE^-/GP5m⁺。经PCR、Southern blot、Western blot 证实构建正确,并能表达具有活性的GP5m蛋白。将TK^-/gE^-/GP5m⁺与TK^-/gE^-/GP5⁺分别免疫Balb/c小鼠,结果TK^-/gE^-/GP5m⁺免疫小鼠不仅产生了较高水平的抗PRRSV的中和抗体(3/6只达到了1∶16),而且在诱导PRRSV特异性细胞免疫方面也显著优于TK^-/gE^-/GP5⁺,表明TK^-/gE^-/GP5m⁺是一种极有希望的PRRSV和PRV二价基因工程候选疫苗。     

呼吸道病毒核酸快速检测方案的建立与应用

本研究旨在建立一套便携、准确、操作简便的呼吸道病毒核酸快速检测方案。通过实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction,qRT-PCR)验证免提取的呼吸道病毒处理试剂(extraction-free respiratory virus treatment reagent,RTU)对病毒核酸处理的效果以及超快速荧光定量PCR仪(FQ-8A)对核酸扩增的效果;将RTU和FQ-8A结合构建呼吸道病毒核酸快速检测方案,通过荧光定量PCR仪中Ct值判断阳性检出率,以验证该方案检测临床样本时的准确性。结果表明,RTU与全自动核酸提取仪在提取效果上灵敏度相当;RTU在提取不同病毒类型样本时,与其他3种提取方法效果相当,但RTU提取时间少于5 min;FQ-8A检测呼吸道合胞病毒(respiratory syncytial virus,RSV)及腺病毒(adenovirus,ADV)与对照仪器ABI-7500具有良好一致性,kappa系数分别为0.938(P<0.001)和0.887(P<0.001),但FQ-8A耗时更短,扩增时间仅在0.5 h左右;RTU和FQ-8A相结合的快检方案与常规检测方案具有高度一致的检出率,其灵敏度为91.70%,特异度为100%,kappa系数为0.944(P<0.001)。总之,通过RTU与FQ-8A的结合构建了一套可在35 min内完成全部流程的呼吸道病毒核酸快速检测方案。该方案准确性高、操作简便,可为呼吸道病毒快速诊断和治疗提供重要支持。     

采用基因微阵列技术对常见呼吸道病毒进行检测的初步研究

建立一种高通量的基因微阵列检测技术,对常见呼吸道病毒感染进行监控.根据公开发表的8个病毒科38种常见呼吸道病毒的序列,计算其保守区域,设计病毒的特异性检测探针,制备呼吸道病毒检测基因微阵列.利用随机引物PCR方法标记样品中的病毒靶序列,标记产物与基因微阵列上的探针杂交,清洗、扫描后进行结果分析.采用流感病毒、麻疹病毒、腮腺炎病毒和风疹病毒作为报告病毒,并对80例上呼吸道感染患者的咽拭子标本进行验证测试.初步结果表明,该呼吸道病毒微阵列基因芯片检测是可行的,在利用基因微阵列技术对病毒监控方面进行了有益的尝试,得到了有经验的信息.     

住院肺炎患儿偏肺病毒和呼吸道合胞病毒感染研究

为探讨沈阳地区肺炎患儿中偏肺病毒(hMPV)和呼吸道合胞病毒(RSV)感染情况,用反转录聚合酶链反应(RT-PCR)法对部分因肺部感染住院患儿进行了病原学研究。结果显示,hMPV感染率为9%;而RSV感染率为46%,其中A型占40%,B型占60%。研究结果表明,在婴幼儿肺炎患儿中RSV感染率高于hMPV感染率,两者所致肺炎在发病年龄、性别及临床症状上无显著区别。     

A nested real-time PCR assay has an increased sensitivity suitable for detection of viruses in aerosol studies

Aims:  Influenza is commonly spread by infectious aerosols; however, detection of viruses in aerosols is not sensitive enough to confirm the characteristics of virus aerosols. The aim of this study was to develop an assay for respiratory viruses sufficiently sensitive to be used in epidemiological studies.
Method:  A two-step, nested real-time PCR assay was developed for MS2 bacteriophage, and for influenza A and B, parainfluenza 1 and human respiratory syncytial virus. Outer primer pairs were designed to nest each existing real-time PCR assay. The sensitivities of the nested real-time PCR assays were compared to those of existing real-time PCR assays. Both assays were applied in an aerosol study to compare their detection limits in air samples.
Conclusions:  The nested real-time PCR assays were found to be several logs more sensitive than the real-time PCR assays, with lower levels of virus detected at lower Ct values. The nested real-time PCR assay successfully detected MS2 in air samples, whereas the real-time assay did not.
Significance and Impact of the Study:  The sensitive assays for respiratory viruses will permit further research using air samples from naturally generated virus aerosols. This will inform current knowledge regarding the risks associated with the spread of viruses through aerosol transmission.     

Construction and immunogenicity of recombinant pseudorabies virus expressing the modified GP5m protein of porcine reproduction and respiratory syndrome virus

Pseudorabies virus (PRV),an alpha-herpesvirus,has been developed as a live viral vector for animal vaccines.However,the PRV recombinant virus TK-/gE-/GP5+expressing GP5 of porcine reproductive and respiratory syndrome virus (PRRSV),based on the PRV genetically depleted vaccine strain TK-/gE-/LacZ+,scarcely stimulated the vaccinated animals to produce neutralizing antibodies against PRRSV.To develop a booster-specific immune response of such PRV recombinants,the ORF5m gene (the modified ORF5 gene having better immune responses)was substituted for the ORF5 gene and introduced into PRV TK-/gE-/LacZ+,resulting in a PRV recombinant named TK-/gE-/GPSm+,which expressed the modified GPSm protein.The recombinant virus was confirmed using PCR,Southern blotting and Western blotting.TK-/gE-/GPSm+and TK-/gE-/GP5+expressing the authentic GP5 protein were inoculated into Balb/c mice to evaluate their immune responses.The results indicated that the protecting neutralization antibodies (the 3/6 vaccinated mice obtained 1:16)and cell immune responses induced by TK-/gE-/GPSm+against PRRSV were higher than that induced by TK-/gE-/GP5+.Thus,the development of the new PRV recombinant expressing the modified GP5m protein as a candidate vaccine established the basis for the study of bivalent genetic engineering vaccines against PRRSV and PRV.     

ELISA间接法检测呼吸道合胞病毒和副流感病毒血清IgM和IgA抗体

建立了检测呼吸道合胞病毒(RSV)和副流感病毒(PFV)血清特异性IgM和IgA抗体的间接ELISA方法。在方法统一的基础上比较了检测IgG、IgM和IgA抗体的结果,证明检测血清IgM和IgA可以作为RSV和PFV感染的早期诊断指标。检测了120份临床急性下呼吸道感染患儿的血清,RSV-IgM检出率为33.3％,RSV-IgA为36.7％;PFV-IgM为27.5％,PFV-IgA为31.6％。提出了对RSV和PFV感染以检测特异性IgA替代IgM或两者互补的设想。     

沈阳地区婴幼儿RSV感染的病原学调查研究

查明冬春季沈阳地区婴幼儿RSV感染流行情况.采用APAAP法和IFA法对65例临床诊断为急性病毒性下呼吸道感染的婴幼儿进行了检查.在RSV感染的高发季节由RSV引起婴幼儿的急性下呼吸道感染的阳性率为44.60%(29/65),0～6个月婴幼儿感染率最高,为74.07%(20/27),并有明显的喘息倾向.以上结果表明RSV仍是引起婴幼儿急性下呼吸道感染,特别是肺炎和毛细支气管炎的重要病原体,沈阳地区仍有流行.为防止和控制RSV的流行及为RSV的防治提供了依据.     

呼吸道合胞病毒TaqMan探针实时定量RT-PCR检测方法的建立及应用

目的:建立呼吸道合胞病毒(RSV)核酸特异、快速、敏感的TaqMan探针实时荧光定量PCR检测方法,并对临床样本进行检测。方法:比对编码RSV非编码蛋白的基因序列,选取其保守片段设计引物和探针,建立实时荧光定量RT-PCR检测方法,并与传统RT-PCR方法进行比较,分别对两者的灵敏性、特异性、重复性及临床样本检验的适用性进行评价。结果:所建立的实时荧光定量RT-PCR检测方法可用于RSV的特异性检测。相对于传统RT-PCR方法100拷贝/反应的检测灵敏度,实时荧光定量RT-PCR的检测灵敏度达到10拷贝/反应,检测范围为1010~101拷贝/反应,且具有良好的特异性和重复性。从169份临床呼吸道标本中检出RSV阳性40例,高于普通PCR方法(31/169)。结论:建立了RSV的TaqMan探针实时定量PCR检测方法,并可用于临床鼻咽拭子样本的检测,在临床上具有较好的应用前景。     

从PRRSV BJ-4株基因组全长cDNA获得感染性病毒

对已构建的覆盖猪繁殖与呼吸综合征病毒(PRRSV)BJ-4全长cDNA的6个重组质粒进行测序,并对部分点突变进行定点回复突变,将突变片段顺次连接,获得了全长cDNA克隆pWSK-DCBA。通过体外转录获得病毒基因组RNA,将RNA与脂质体混合后直接转染MARC-145细胞,获得拯救病毒(rV68)。rV68能在MARC-145细胞上稳定传代,并可引起PRRSV特征性的细胞病变(CPE)。增殖动态分析表明,rV68在MARC-145细胞上的生长有所迟滞,达到最高滴度的培养时间比亲本病毒延迟12h,但滴毒无显著差异(P>0.05)。结果表明,构建的BJ-4全长cDNApWSK-DCBA具有感染性,为研究中国PRRSV的分子致病与免疫机制、新型疫苗等奠定了基础。     

采用半巢式RT-PCR检测兰州地区住院肺炎患儿下呼吸道分泌物中的RSV

从人呼吸道合胞病毒(hRSV)基因序列中高度保守的N基因处,设计了特异性半巢式引物,以反转录-聚合酶链式反应(RT-PCR)方法随机检测了46份兰州地区2007年春季住院肺炎患儿下呼吸道分泌物样本。结果46份样本中有19份扩增出了预期目的片段,对所有扩增产物进行基因序列测定。结果表明19份样本均有RSV感染,其检出率为41%,而且序列分析结果显示,所有RSV阳性样本均与RSVA亚型的同源性高于B亚型。     

Construction and immunogenicity of recombinant pseudorabies virus expressing the modified GP5m protein of porcine reproduction and respiratory syndrome virus

Pseudorabies virus (PRV), an alpha-herpesvirus, has been developed as a live viral vector for animal vaccines. However, the PRV recombinant virus TK⁻/gE⁻/GP5⁺ expressing GP5 of porcine reproductive and respiratory syndrome virus (PRRSV), based on the PRV genetically depleted vaccine strain TK⁻/gE⁻/LacZ⁺, scarcely stimulated the vaccinated animals to produce neutralizing antibodies against PRRSV. To develop a booster-specific immune response of such PRV recombinants, the ORF5m gene (the modified ORF5 gene having better immune responses) was substituted for the ORF5 gene and introduced into PRV TK⁻/gE⁻/LacZ⁺, resulting in a PRV recombinant named TK⁻/gE⁻/GP5m⁺, which expressed the modified GP5m protein. The recombinant virus was confirmed using PCR, Southern blotting and Western blotting. TK⁻/gE⁻/GP5m⁺ and TK⁻/gE⁻/GP5⁺ expressing the authentic GP5 protein were inoculated into Balb/c mice to evaluate their immune responses. The results indicated that the protecting neutralization antibodies (the 3/6 vaccinated mice obtained 1:16) and cell immune responses induced by TK⁻/gE⁻/GP5m⁺ against PRRSV were higher than that induced by TK⁻/gE⁻/GP5⁺. Thus, the development of the new PRV recombinant expressing the modified GP5m protein as a candidate vaccine established the basis for the study of bivalent genetic engineering vaccines against PRRSV and PRV. Translated from Journal of Biotechnology, 2005, 21(6): 858–864 [译自: 生物工程学报]