甲型H1N1流感疫苗研发回顾及展望

2009年全球爆发了甲型H1N1流感大流行。为预防、控制甲流在我国的传播,减少发病率和死亡率．我国及时启动了甲流疫苗的研制和临床评价。在此过程中．疫苗生产企业、中国药品生物制品检定所、中国疾病预防控制中心（CDC）和国家药品审评中心等单位．在政府指导下统一行动、通力协作．在短短的几个月内．研制出安全有效、符合国际标准的甲流疫苗,并在全球率先开展了人群免疫接种。     

打赢阻击流感疫情“前哨战”——甲型H1N1流感疫苗研发回顾及展望：流感病毒疫苗（含甲型H1N1）生产制备和关键技术

2009年全球爆发了甲型H1N1流感人流行,为预防,控制甲流在我国的传播,减少发病率和死亡率,我国及时启动了甲型H1N1流感疫苗的研制和临床评价。这场席卷全球的突发性疫情,成为众多机构角力的竞技场——一批中国新兴生物技术公司也借此一显身手,不管是在疫苗研制,还是病毒诊断和检测方断,都取得了可喜的成绩,为打赢阻击流感疫情“前哨战”奠定了坚实的基础。     

中医成药治疗甲型H1N1 流感研究进展

近年来,中医药治疗甲型H1N1流感取得了较好的临床疗效。尽管目前临床对甲型H1N1流感的治疗仍以西药为主,但西药抗病毒药存在副作用大,易引起耐药性等缺点,限制了治疗的效果。中医治疗甲型H1N1流感则具有独特的思路,根据甲型H1N1流感不同进展阶段的不同证型,选择不同的中药进行"辨证论治",不仅能减轻西药的不良反应,而且在疾病的治疗上也有独特的功效。我们总结了近来学者们对中医成药治疗甲型H1N1流感的临床及基础研究,将甲型H1N1流感分为轻、中、重三种,并分别根据症状与中医证型相匹配,梳理了临床用于治疗甲型H1N1流感的中医成药的适应证型与作用机制。因此,在西药进行抗病毒治疗的同时,根据疾病进展不同阶段的中医的证型,选用不同的中医方药,可有效减少西药的不良反应,取得更好的疗效。     

甲型H1N1流感病毒血凝素蛋白单抗杂交瘤细胞株的制备与初步鉴定

目的:建立具有高特异、高效价的甲型H1N1流感病毒血凝素蛋白(HA)单抗的杂交瘤细胞株。方法:以纯化的昆虫杆状病毒表达的甲型H1N1流感病毒HA蛋白为免疫原免疫BALB/c小鼠,取脾细胞与Sp2/0小鼠骨髓瘤细胞融合,通过有限稀释法筛选阳性克隆,经ELISA和Western blot分析单抗的特性和特异性。结果:获得6株甲型H1N1流感HA抗原特异单克隆抗体杂交瘤细胞株,抗原肽库ELISA检测结果表明其中3株(1E12,3F12,1C11)单抗只与甲型H1N1流感HA抗原肽库反应,不与H5N1病毒HA抗原肽库反应;Western blot分析表明,单抗1B3只特异识别甲型H1N1流感HA抗原,而与其他季节性甲流病毒(H1,H3)及人禽流感H5N1病毒不反应。结论:所获杂交瘤细胞株特异性强,效价高,分泌抗体性能稳定,为分析甲型H1N1流感病毒抗原性位点、建立诊断试剂奠定了基础。     

猪源性甲型H1N1流感病毒研究概况

2009年3月在美国和墨西哥流感样患者的呼吸道标本中鉴定出新的猪源性甲型H1N1流感病毒。该病毒可人-人传播,已蔓延到112个国家和地区。为了遏制不断重组或重配的流感病毒,各国学者对甲型H1N1流感病毒的分子生物学特征、复制周期及实验室诊断做了细致的研究,以研发相应的药物或疫苗,这些成就为世界各国防控今年新鉴定的猪源性甲型H1N1流感病毒感染发挥了重要作用。现就猪源性甲型H1N1流感病毒的鉴定、基因组结构特征做一综述。     

云南省2009～2014年甲型H1N1流感病毒HA及NA基因特征分析

了解云南省2009~2014年甲型H1N1流感病毒的流行趋势,研究HA和NA基因进化特征。对云南省近6年来上报的流感监测病例数据进行病原谱总结,挑选出23株甲型H1N1流感毒株进行HA及NA基因分析。利用MEGA 5.0软件对测序结果构建进化树分析基因同源性。2009~2014年云南省共监测到4次甲型H1N1流感流行高峰,核酸检测结果中甲型H1N1流感占检出总量的28.8%。测序结果显示,HA与NA基因均分为3个类群,检测到一株具有H275Y突变位点的毒株。甲型H1N1流感是导致本省流感流行的重要亚型之一,2009~2014年间分离的毒株主要有Goup1、Gourp7和Gourp6三个支系,绝大部分甲型H1N1流感毒株仍对神经氨酸酶抑制剂敏感。     

某高校甲型H1N1 流感确诊病例流行病学分布特征分析

目的：通过了解某高校甲型H1N1 流感病例的流行病学分布特征,为预防和控制流感在高校的蔓延提供依据。方法：以某高 校2009 年11 月6 日至2009 年11 月24 日发病并确诊的74 例甲型H1N1 流感病例为研究对象,分析并比较病例的年龄、性别、 学历层次、年级、专业、发病时间和临床症状。结果：74 例甲型H1N1 流感确诊病例均为学生,罹患率为1.63 %,其中男性占94.6 %,女性占5.4 %;病例平均年龄为20.5 岁± 2.2 岁;94.6 %的病例为本科生;本科生罹患率（4.03%）显著高于研究生（0.14%）;2006 级见习期本科生罹患率（11.05 %）显著高于其他年级学生;疫情的流行全距为19 天,发病高峰为2009 年11 月13 日至2009 年 11 月18 日;病例以发热、咳嗽、乏力、头疼等临床症状为主。结论：该高校甲型H1N1 流感确诊病例多为22岁以下的男性学生。本 研究提示加强见习学生的监测和管理、设立隔离宿舍、接种疫苗等有针对性的措施能够有效控制流感疫情在高校蔓延。     

H1N1裂解疫苗辅以C48/80佐剂滴鼻免疫小鼠能够提供抗甲流的保护作用

2009年"甲型H1N1流感"全球流行导致了数以万计人的死亡。疫苗的及时研制为预防、控制甲流的传播,减少发病率和死亡率做出了重大贡献。甲流疫苗辅以佐剂滴鼻免疫能更好地抵御甲流攻击。将A/California/7/2009(H1N1)裂解疫苗辅以化合物48/80(C48/80)佐剂滴鼻免疫雌性BALB/c小鼠,免疫一次,免疫后28 d,小鼠用致死剂量的同源病毒进行攻击。结果发现,滴鼻免疫组的抗体滴度均达到很高的水平,而且随着H1N1裂解疫苗剂量的增加,其对小鼠的保护作用越强,同时添加佐剂可以更有效提高H1N1裂解疫苗的保护效果。实验结果说明H1N1裂解疫苗辅以C48/80佐剂滴鼻免疫能够保护小鼠免受流感病毒的感染。     

抗击甲型H1N1流感大流行第一线的中国企业

自2009年3月18日墨西哥发现人感染甲型H1N1病毒疑似病例以来,一种新的猪源性H1N1型流感病毒开始在墨西哥和美国蔓延开来．并在数周内扩散到很多国家和地区．不断引起人类感染和死亡。伴随着流感疫情在全球范围内的迅速蔓延,6月初,世界卫生组织宣布把甲型H1N1流感警戒级别升至6级．甲型H1N1流感疫情已经发展成为全球性“流感大流行”。甲型H1N1流感疫情成为了全球高度关注的突发公共卫生事件。     

一株2011年出现的季节性甲型H1N1流行性感冒病毒血凝素基因特性的研究

本文通过比较2011年分离培养的1株季节性甲型H1N1流行性感冒(简称流感)病毒(A/Shanghai/1167/2011(H1N1))与历年季节性甲型H1N1流感病毒的血凝素(HA)基因,追溯该病毒的基因变异与来源,探讨该毒株的出现对流感防控工作的意义.采用反转录-聚合酶链反应(RT-PCR)方法扩增病毒的HA和神经氨酸酶(NA)片段,并进行测序;应用分子生物学软件对获得的序列进行分析,绘制基因进化树;同时,通过血凝抑制试验检测2011年下半年健康人群中该流感病毒的抗体水平.结果显示,A/Shanghai/1167/2011(H1N1)的HA基因序列与世界卫生组织(WHO)2007～2008年季节性甲型H1N1流感病毒疫苗株A/Brisbane/59/2007(H1N1)最接近,同源性达99.2%,与新型甲型H1N1流感病毒A/California/07/2009疫苗株同源性仅为72.4%.其HA基因裂解位点为PSIQSR↓GLF,尚未出现高致病性的分子特征.HA片段共编码557个氨基酸,有9个潜在的糖基化位点,序列与2009年前WHO疫苗株A/NewCaledonia/20/1999(H1N1)、A/SolomonIslands/3/2006(H1N1)和/Brisbane/59/2007(H1N1)相比,分别有15、12和4处不同,这些差异分布在Sa、Sb、Ca1、Ca2、Cb 5个抗原决定簇的氨基酸差异分别有5、5和2处.该毒株在健康人群血清的抗体阳性率为34.33%,几何平均效价(GMT)为10.38.A/Shanghai/1167/2011(H1N1)是2011年出现在上海地区的一个季节性甲型H1N1流感病毒毒株,其抗原变异与既往季节性甲型H1N1流感病毒相比不大,但在以A(H1N1)pdm09为主要流行株的年份检测到散在发生的既往季节性甲型H1N1流感病毒毒株应当引起重视,其在人群中的抗体水平较低,易引起流行,需要提高对类流感人群中此种毒株的持续监测.     

Isolation and genetic characterization of avian-like H1N1 and novel ressortant H1N2 influenza viruses from pigs in China

As pigs are susceptible to both human and avian influenza viruses, they have been proposed to be intermediate hosts or mixing vessels for the generation of pandemic influenza viruses through reassortment or adaptation to the mammalian host. In this study, we reported avian-like H1N1 and novel ressortant H1N2 influenza viruses from pigs in China. Homology and phylogenetic analyses showed that the H1N1 virus (A/swine/Zhejiang/1/07) was closely to avian-like H1N1 viruses and seemed to be derived from the European swine H1N1 viruses, which was for the first time reported in China; and the two H1N2 viruses (A/swine/Shanghai/1/07 and A/swine/Guangxi/13/06) were novel ressortant H1N2 influenza viruses containing genes from the classical swine (HA, NP, M and NS), human (NA and PB1) and avian (PB2 and PA) lineages, which indicted that the reassortment among human, avian, and swine influenza viruses had taken place in pigs in China and resulted in the generation of new viruses. The isolation of avian-like H1N1 influenza virus originated from the European swine H1N1 viruses, especially the emergence of two novel ressortant H1N2 influenza viruses provides further evidence that pigs serve as intermediate hosts or “mixing vessels”, and swine influenza virus surveillance in China should be given a high priority.     

A/H1N1流感—世界关注的焦点

2009年4月,A/H1N1流感在墨西哥和美国暴发。随后,疫情迅速蔓延到美洲、欧洲、亚洲多个国家。A/H1N1流感病毒是一种以前在人或动物身上从未观测到的新病毒。遗传进化和抗原特性分析表明该病毒和猪流感病毒密切相关,与人类的季节性流感病毒有明显区别。但是流行病学信息表明A/H1N1流感病毒只攻击人类,并在人与人之间传播,尚未发现动物向人类传播的情况。本文从A/H1N1流感病毒的生物学特性、临床特征、公共卫生意义等方面全面阐述了A/H1N1流感的最新研究进展,为正确认识和科学防控A/H1N1流感提供参考。     

Epidemiology and Genotypic Diversity of Eurasian Avian-Like H1N1 Swine Influenza Viruses in China

Eurasian avian-like H1 N1(EA H1 N1) swine influenza virus(SIV) outside European countries was first detected in Hong Kong Special Administrative Region(Hong Kong, SAR) of China in 2001. Afterwards, EA H1 N1 SIVs have become predominant in pig population in this country. However, the epidemiology and genotypic diversity of EA H1 N1 SIVs in China are still unknown. Here, we collected the EA H1 N1 SIVs sequences from China between 2001 and 2018 and analyzed the epidemic and phylogenic features, and key molecular markers of these EA H1 N1 SIVs. Our results showed that EA H1 N1 SIVs distributed in nineteen provinces/municipalities of China. After a long-time evolution and transmission, EA H1 N1 SIVs were continuously reassorted with other co-circulated influenza viruses, including 2009 pandemic H1 N1(A(H1 N1)pdm09), and triple reassortment H1 N2(TR H1 N2) influenza viruses, generated 11 genotypes. Genotype 3 and 5, both of which were the reassortments among EA H1 N1, A(H1 N1)pdm09 and TR H1 N2 viruses with different origins of M genes, have become predominant in pig population. Furthermore, key molecular signatures were identified in EA H1 N1 SIVs. Our study has drawn a genotypic diversity image of EA H1 N1 viruses, and could help to evaluate the potential risk of EA H1 N1 for pandemic preparedness and response.     

The role of nuclear NS1 protein in highly pathogenic H5N1 influenza viruses

The non-structural protein (NS1) of influenza A viruses (IAV) performs multiple functions during viral infection. NS1 contains two nuclear localization signals (NLS): NLS1 and NLS2. The NS1 protein is located predominantly in the nucleus during the early stages of infection and subsequently exported to the cytoplasm. A nonsense mutation that results in a large deletion in the carboxy-terminal region of the NS1 protein that contains the NLS2 domain was found in some IAV subtypes, including highly pathogenic avian influenza (HPAI) H7N9 and H5N1 viruses. We introduced different mutations into the NLS domains of NS1 proteins in various strains of IAV, and demonstrated that mutation of the NLS2 region in the NS1 protein of HPAI H5N1 viruses severely affects its nuclear localization pattern. H5N1 viruses expressing NS1 protein that is unable to localize to the nucleus are less potent in antagonizing cellular antiviral responses than viruses expressing wild-type NS1. However, no significant difference was observed with respect to viral replication and pathogenesis. In contrast, the replication and antiviral defenses of H1N1 viruses are greatly attenuated when nuclear localization of the NS1 protein is blocked. Our data reveals a novel functional plasticity for NS1 proteins among different IAV subtypes.     

Diagnosis of influenza viruses with special reference to novel H1N1 2009 influenza virus

On 15 April and 17 April 2009, novel swineorigin influenza A (H1N1) virus was identifi ed in specimens obtained from two epidemiologically unlinked patients in the United States. The ongoing outbreak of novel H1N1 2009 influenza (swine influenza) has caused more than 3,99,232 laboratory confi rmed cases of pandemic influenza H1N1 and over 4735 deaths globally. This novel 2009 influenza virus designated as H1N1 A/swine/California/04/2009 virus is not zoonotic swine flu and is transmitted from person to person and has higher transmissibility then that of seasonal influenza viruses. In India the novel H1N1 virus infection has been reported from all over the country. A total of 68,919 samples from clinically suspected persons have been tested for influenza A H1N1 across the country and 13,330 (18.9%) of them have been found positive with 427 deaths. At the All India Institute of Medical Sciences, New Delhi India, we tested 1096 clinical samples for the presence of novel H1N1 influenza virus and seasonal influenza viruses. Of these 1096 samples, 194 samples (17.7%) were positive for novel H1N1 influenza virus and 197 samples (18%) were positive for seasonal influenza viruses. During outbreaks of emerging infectious diseases accurate and rapid diagnosis is critical for minimizing further spread through timely implementation of appropriate vaccines and antiviral treatment. Since the symptoms of novel H1N1 influenza infection are not specifi c, laboratory confi rmation of suspected cases is of prime importance.     

Hemagglutinin and neuraminidase matching patterns of two influenza A virus strains related to the 1918 and 2009 global pandemics

The current pandemic influenza A (H1N1) virus has revealed a complicated reassortment of various influenza A viruses. The biological study of these viruses, especially of the viral envelope proteins hemagglutinin (HA) and neuraminidase (NA), is urgently needed for the control and prevention of H1N1 viruses. We have generated H1N1-2009 and H1N1-1918 pseudotyped particles (pp) with high infectivity. Combinations of HA1918 + NA2009 and HA2009 + NA1918 also formed infectious H1N1pps, among which the HA2009 + NA1918 combination resulted in the most highly infectious pp. Our study demonstrated that some reassortments of H1N1 viruses may hold the potential to produce higher infectivity than do their ancestors.     

Effect of prior vaccination with a seasonal trivalent influenza vaccine on the antibody response to the influenza pandemic H1N1 2009 vaccine: a randomized controlled trial

Vaccination with the non-adjuvanted split-virion A/California/7/2009 influenza vaccine (pandemic H1N1 2009 vaccine) began in October 2009 in Japan. The present study was designed to assess the effect of prior vaccination with a seasonal trivalent influenza vaccine on the antibody response to the pandemic H1N1 2009 vaccine in healthy adult volunteers. One hundred and seventeen participants aged 22 to 62 were randomly assigned to two study groups. In Group 1 (the priming group), participants were first vaccinated with the seasonal trivalent influenza vaccine followed by two separate one-dose vaccinations of the pandemic H1N1 2009 vaccine, whereas in Group 2 (the non-priming group), the participants were first vaccinated with one dose of the pandemic H1N1 2009 vaccine, followed by simultaneous vaccination of the seasonal trivalent vaccine and the second dose of the pandemic H1N1 2009 vaccine. The participants in Group 2 had a seroprotection rate (SPR) of 79.7% and a seroconversion rate (SCR) of 79.7% in the hemagglutination-inhibition test after the first dose of the pandemic H1N1 2009 vaccine, indicating that the pandemic H1N1 2009 vaccine is sufficiently immunogenic. On the other hand, the participants of Group 1 had a significantly weaker antibody response, with a SPR of 60.8% and a SCR of 58.5%. These results indicate that prior vaccination with the seasonal trivalent influenza vaccine inhibits the antibody response to the pandemic H1N1 2009 vaccine. Therefore, the pandemic H1N1 2009 vaccine should be administered prior to vaccination with the seasonal trivalent influenza vaccine.     

长沙A(H1N1)亚型季节性流感暴发疫情的实验室诊断及其HA基因特性分析

对2009 年长沙麓山国际学校流感暴发疫情进行实验室诊断, 并探索新分离的A(H1N1)亚型流感病毒血凝素(HA)的基因特性。对流感暴发疫情的25 份鼻/咽拭子标本进行RT-PCR检测和流感病毒分离, 然后利用CEQ?8000 Genetic Analysis System对病毒分离株(A/Yuelu/314/2009)进行测序, 测序结果提交至GenBank(登录号: FJ912843)并用ClustalX和Mega4.1软件进行序列分析。结果显示, 分离出A(H1N1)亚型流感毒株18株, 检出21份A(H1N1)亚型流感病毒核酸阳性; A/Yuelu/314/2009(H1N1) HA基因序列与2008~2009 年疫苗株(A/Brisbane/59/2007)比较显示: 核苷酸和氨基酸同源性均为99%, 有6个位点的氨基酸发生了变异(V148A、S158N、G202A、I203D、A206T、W435R), 其中一个S158N氨基酸变异位于B抗原表位, HA基因序列上共有潜在糖基化位点9 个(27、28、40、71、151、176、303、497、536), 与A/Brisbane/59/2007相同且氨基酸序列保守。本实验诊断出此次流感暴发疫情的病原体为A(H1N1)型季节性流感病毒, 研究还发现A/Yuelu/314/2009(H1N1)长沙分离株与A/Brisbane/59/2007 疫苗株基因序列比较显示并未形成一个新的变种, 推测是由于分离株与疫苗株之间基因特性的改变和人群对A(H1N1)亚型流感病毒免疫力降低导致了此次长沙麓山国际学校A(H1N1)亚型流感的暴发。     

新甲型H1N1(2009)病毒的早期分子特征

摘要：【目的】本世纪首次流感大流行的病原属于甲型H1N1流感病毒,在遗传特性和抗原等方面都有别于人群中流行多年的季节性H1N1流感病毒。为了深入了解病毒的遗传特性,跟踪病毒的演化趋势,及时发现具有流行病学意义的变异株,本研究对早期分离的甲型H1N1(2009)病毒的分子特性进行了详细分析。【方法】通过GenBank的流感资源中心下载相关毒株的基因组信息, 序列分析采用DNAStar软件包的EditSeq和MegAlign比较与病毒致病性和宿主特异性相关的氨基酸变化情况。以A/California/07/2009（H1N1）作为新甲型H1N1(2009)的代表株进行详细的分子特征分析。【结果】A/California/07/2009不具备高致病性流感病毒的分子特征;病毒编码的11个蛋白大部分保留有猪流感病毒的分子特征,同时也具有一些禽和人流感病毒的特征;PB1-F2在11aa,57aa和87aa后发生断裂,具有古典猪H1N1和人H1N1双重特点,这是甲型H1N1（2009）病毒一个特有的分子特征。【结论】首次详细分析了新甲型H1N1（2009）病毒的分子特征。随着病毒在人群中的进一步适应和持续存在,这些分子特征将发生变化,应该特别关注这些变化对病毒的传播力和致病性的影响。     

Influenza A (H1N1) 2009 Antibodies in Residents of New South Wales,Australia, after the First Pandemic Wave in the 2009 Southern Hemisphere Winter

Background

The first wave of pandemic influenza A(H1N1)2009 (pH1N1) reached New South Wales (NSW), Australia in May 2009, and led to high rates of influenza-related hospital admission of infants and young to middle-aged adults, but no increase in influenza-related or all-cause mortality.

Methodology/Principal Findings

To assess the population rate of pH1N1 infection in NSW residents, pH1N1-specific haemagglutination inhibition (HI) antibody prevalence was measured in specimens collected opportunistically before (2007–2008; 474 specimens) and after (August–September 2009; 1247 specimens) the 2009 winter, and before the introduction of the pH1N1 monovalent vaccine. Age- and geographically-weighted population changes in seroprevalence were calculated. HI antibodies against four recent seasonal influenza A viruses were measured to assess cross-reactions. Pre- and post-pandemic pH1N1 seroprevalences were 12.8%, and 28.4%, respectively, with an estimated overall infection rate of 15.6%. pH1N1 antibody prevalence increased significantly - 20.6% overall - in people born since 1944 (26.9% in those born between 1975 and 1997) but not in those born in or before 1944. People born before 1925 had a significantly higher pH1N1 seroprevalence than any other age-group, and against any seasonal influenza A virus. Sydney residents had a significantly greater change in prevalence of antibodies against pH1N1 than other NSW residents (19.3% vs 9.6%).

Conclusions/Significance

Based on increases in the pH1N1 antibody prevalence before and after the first pandemic wave, 16% of NSW residents were infected by pH1N1 in 2009; the highest infection rates (27%) were among adolescents and young adults. Past exposure to the antigenically similar influenza A/H1N1(1918) is the likely basis for a very high prevalence (49%) of prepandemic cross-reacting pH1N1 antibody and sparing from pH1N1 infection among people over 85 years. Unless pre-season vaccine uptake is high, there are likely to be at least moderate rates including some life-threatening cases of pH1N1 infection among young people during subsequent winters.