Analysis of the immunogenicity and bioactivities of a split influenza A/H7N9 vaccine mixed with MF59 adjuvant in BALB/c mice

The H7N9 influenza virus caused significant mortality and morbidity in humans during an outbreak in China in 2013. A recombinant H7N9 influenza seed with hemagglutinin (HA) and neuraminidase (NA) gene segments from A/Zhejiang/DTID-ZJU01/2013(H7N9) and six internal protein gene segments from A/Puerto Rico/8/34(H1N1; PR8) were generated using reverse genetics. We sought to determine the immunogenic, protective properties, and mechanisms of a split avian influenza A/H7N9 vaccine mixed with MF59 adjuvant in comparison to vaccines that included other adjuvant. BALB/c mice were vaccinated with two doses of different amounts and combinations of this novel A/ZJU01/PR8/2013 split vaccine with adjuvant. Mice were subsequently challenged with A/Zhejiang/DTID-ZJU01/2013(H7N9) by intranasal inoculation. We verified that MF59 enhanced the HI, MN, and IgG antibody titers to influenza antigens. Compared with alum, MF59 could more potentially induce humoral immune responses and Th2 cytokine production after virus infection, while both MF59 and alum can slightly increase NK cell activity. This split H7N9 influenza vaccine with MF59 adjuvant could effectively induce antibody production and protect mice from H7N9 virus challenge. We have selected this vaccine for manufacture and future clinical studies to protect humans from H7N9 virus infection.     

人感染H7N9禽流感的流行特征

目的：对我国发生人感染 H7N9禽流感的流行病学特点进行分析,为H7N9禽流感的预防控制提供依据。方法人感染 H7N9禽流感确诊132例的病例相关数据来源于2013年3－5月中国国家卫生和计划生育委员会网站（http：／／www ．chinapop ．gov ．cn／yjb／h7n9／list ．shtml）和我国台湾地区卫生署疾管局网站（http：／／www ． cdc ．gov ．tw／）,通过Excel 2007软件进行统计分析,总结其发病地点、时间、患者性别和年龄分布、临床症状轻重等因素。结果132例H7N9禽流感确诊病例基本为散发状态,绝大多数发生在华东地区,浙江、上海和江苏3省市较多,各占34．85％、25．00％、20．45％;病例数在前3周呈上升趋势,第3周峰值达47例,随后逐渐下降,至第6周后基本无新增确诊病例;男女性别比为2．34∶1,男性病例远多于女性;年龄分布在4～91岁,以中老年人为主,＞45岁确诊病例占75．76％;确诊病例大部分表现为重症或病死,该两者占76．52％,其中死亡38例,病死率达28．79％。结论人感染H7N9禽流感确诊病例的流行病学特征与其他禽流感例如 H5N1显然不同,具体原因有待于进一步研究确定。     

我国H7N9禽流感病毒血凝素和神经氨酸酶基因的分子进化分析

  目的  通过对安徽省安庆市人感染H7N9禽流感外环境标本检测,分析外环境中病毒污染情况,研究判断人感染H7N9的风险,为制定防控措施提供依据。  方法  于2017年1月5日 — 2月22日,采用完全随机抽样的方法,采集安庆市38家农贸市场、4例确诊人感染H7N9禽流感病例居家禽类相关标本共324份,用实时荧光定量RT-PCR方法进行甲型流感病毒通用核酸检测,对阳性标本进一步进行H7N9检测。  结果  安庆市H7N9禽流感病毒阳性率28.40 %,11个县（市、区）中有10个检出H7N9病毒阳性,阳性率90.90 % （10/11）。主城区阳性率37.68 %（26/69）高于非主城区,差异有统计学意义（χ² = 9.23,P < 0.05）;鸡、鸭、鸽子标本中,鸡标本阳性率最高31.75 %（80/252）,差异有统计学意义（χ² = 6.57,P < 0.05）;农贸市场来源的标本阳性率为29.77 %（92/309）,病例家来源的标本阳性率为0,差异有统计学意义（χ² = 6.24,P < 0.05）;不同类型标本中,咽拭子和肛拭子阳性率最高,分别为42.86 %（9/21）和40.00 %（2/5）,其次为饮水和污水;发生人感染病例前外环境标本阳性率38.71 %（12/31）高于发生后,但2者间的差异无统计学意义（χ² = 1.79,P > 0.05）。  结论  安庆市农贸市场存在H7N9禽流感病毒污染。     

128例人感染H7N9禽流感流行病学分析

目的 了解人感染H7N9禽流感病毒的流行病学特征.方法 对128例人感染H7N9禽流感确诊病例的相关数据进行统计,按发病地点、时间、患者年龄和性别分布、临床症状轻重等方面进行总结分析.结果 上述病例绝大多数发生在江浙沪地区.患者主要为中老年人,并且男性发病率和死亡率分别为67.19％和61.54％,明显高于女性的28.91％和23.08％.临床症状大多为重症/危重及死亡,而且不同程度临床症状的患者中,男性也明显多于女性.结论 中国大陆人感染H7N9禽流感确诊病例的流行病学特征呈现出男性的发病率与死亡率明显高于女性,患者中中老年人明显多于青少年的罕见情形.     

A highly immunogenic vaccine against A/H7N9 influenza virus

Since the first case of human infection in March 2013, continued reports of H7N9 cases highlight a potential pandemic threat. Highly immunogenic vaccines to this virus are urgently needed to protect vulnerable populations who lack protective immunity. In this study, an egg- and adjuvant-independent adenoviral vector-based, hemagglutinin H7 subtype influenza vaccine (HAd-H7HA) demonstrated enhanced cell-mediated immunity as well as serum antibody responses in a mouse model. Most importantly, this vaccine provided complete protection against homologous A/H7N9 viral challenge suggesting its potential utility as a pandemic vaccine.     

中国内地人感染H7N9禽流感疫情流行病学特征分析

目的 分析中国内地人感染H7N9禽流感疫情.方法 以2013年以来我国内地报告的确诊H7N9禽流感病例为研究对象,采用描述性流行病学方法分析病例的时间、空间和人群分布特点.结果 截至2014年6月4日中国内地共确诊人感染H7N9禽流感病例433例,死亡163人.2013年3－4月及2014年1－2月分别为2个发病高峰.病例分布在14个省(市),报告病例数前五位的省份为浙江、广东、江苏、上海、湖南,占总报告病例数的85%;病例年龄1～91岁(M=58岁);男性占70%.82%的病例在发病前有活禽相关的暴露史.共发生14起聚集性病例,其中9起发生人传人的可能性大.结论 中国内地人感染H7N9禽流感疫情有较明显的季节分布特点,同时呈现一定的区域聚集性;感染人群以老年人为主,男性多于女性;感染来源主要为禽类.     

Recombinant hemagglutinin produced from Chinese Hamster Ovary (CHO) stable cell clones and a PELC/CpG combination adjuvant for H7N9 subunit vaccine development

The novel H7N9 avian influenza A virus has caused human infections in China since 2013; some isolates from the fifth wave of infections have emerged as highly pathogenic avian influenza viruses. Recombinant hemagglutinin proteins of H7N9 viruses can be rapidly and efficiently produced with low-level biocontainment facilities. In this study, recombinant H7 antigen was obtained from engineered stable clones of Chinese Hamster Ovary (CHO) cells for subsequent large-scale production. The stable CHO cell clones were also adapted to grow in serum-free suspension cultures. To improve the immunogenicity of the recombinant H7 antigens, we evaluated the use of a novel combination adjuvant of PELC and CpG (PELC/CpG) to augment the anti-H7N9 immune responses in mice. We compared the effects with other adjuvants such as alum, AddaVax (MF59-like), and several Toll-like receptor ligands such as R848, CpG, and poly (I:C). With the PELC/CpG combination adjuvant, CHO cell-expressed rH7 antigens containing terminally sialylated complex type N-glycans were able to induce high titers of neutralizing antibodies in sera and conferred protection following live virus challenges. These data indicate that the CHO cell-expressed recombinant H7 antigens and a PELC/CpG combination adjuvant can be used for H7N9 subunit vaccine development.     

Cross-protection against H7N9 influenza strains using a live-attenuated H7N3 virus vaccine

In 2013, avian H7N9 influenza viruses were detected infecting people in China resulting in high mortality. Influenza H7 vaccines that provide cross-protection against these new viruses are needed until specific H7N9 vaccines are ready to market. In this study, an available H7N3 cold-adapted, temperature sensitive, live attenuated influenza vaccine (LAIV) elicited protective immune responses in ferrets against H7N9 viruses. The H7N3 LAIV administered alone (by intranasal or subcutaneous administration) or in a prime-boost strategy using inactivated H7N9 virus resulted in high HAI titers and protected 100% of the animals against H7N9 challenge. Naïve ferrets passively administered immune serum from H7N3 LAIV infected animals were also protected. In contrast, recombinant HA protein or inactivated viruses did not protect ferrets against challenge and elicited lower antibody titers. Thus, the H7N3 LAIV vaccine was immunogenic in healthy seronegative ferrets and protected these ferrets against the newly emerged H7N9 avian influenza virus.     

H7N9 influenza and its impact on pregnancy

This short article specifically focuses on the new emerging H7N9 influenza which has just been observed since early 2013. As a new disease, it is lack for the knowledge on the new H7N9 influenza. Here, the author will discuss on the impact of emerging H7N9 influenza on pregnancy.     

H7N9禽流感病毒感染A549细胞生物标志物研究

目的通过建立H7N9禽流感病毒和H1N1pdm09流感病毒(对照病毒)感染A549细胞模型,分析H7N9禽流感病毒感染A549细胞的特征蛋白,筛选其生物标志物,初步揭示H7N9禽流感病毒致细胞病变的蛋白分子机制。方法感染复数为0.001的H7N9和对照病毒分别感染A549细胞24、48、72 h后提取细胞总蛋白进行荧光双向差异凝胶电泳(2D-DIGE),筛选H7N9禽流感病毒感染A549细胞的可能生物标志物并进行蛋白印迹(WB)验证。结果血小板活化因子乙酰水解酶Ⅰb亚基β(PAFAH1B2)蛋白在H7N9感染A549细胞72 h后表达量急剧下降,而在空白对照组和H1N1pdm09感染A549细胞组的表达量未出现明显变化。结论 PAFAH1B2可作为H7N9感染A549细胞后期的生物标志物,其表达量的变化可能与H7N9感染患者的症状相关。     

Potency of an inactivated influenza vaccine prepared from A/duck/Mongolia/119/2008 (H7N9) against the challenge with A/Anhui/1/2013 (H7N9)

H7N9 influenza virus infection in humans was reported in China on March 31, 2013. Humans are immunologically naïve to the H7N9 subtype, for which the seasonal influenza vaccine is not effective. Thus, the development of an H7N9 influenza virus vaccine is an urgent issue. To prepare for the emergence of an influenza pandemic, we have established a library comprising more than 1300 influenza virus strains with 144 different combinations of 16 HA and 9 NA subtypes. An H7N9 virus strain isolated from a 35-year-old woman, A/Anhui/1/2013 (H7N9), was found to be antigenically similar to H7N9 influenza viruses isolated from migratory ducks. In the present study, the potency of an inactivated whole virus particle vaccine prepared from an H7N9 low pathogenic avian influenza virus, A/duck/Mongolia/119/2008 (H7N9), selected from the library, was assessed by a challenge with A/Anhui/1/2013 (H7N9). The results indicate that the test vaccine was potent enough to induce sufficient immunity to reduce the impact of disease caused by the challenge with A/Anhui/1/2013 (H7N9) in mice. The present results indicate that an inactivated whole virus particle vaccine prepared from an influenza virus strain stored in the library could be useful as a vaccine strain in case of an influenza pandemic.     

Squalene-adjuvanted H7N9 virus vaccine induces robust humoral immune response against H7N9 and H7N7 viruses

Recent cases of avian influenza H7N9 have caused great concerns that virus may become transmittable between humans. It is imperative to develop an effective vaccine to fight against the pandemic potential of this H7N9 influenza virus to protect human from the disease. This study aims to investigate an optimized formulation for the development of H7N9 vaccines. Various doses of H7N9 inactivated whole or split-virus antigens (0.5, 1.5, or 3 μg based on hemagglutinin content) combined with squalene-based adjuvant (AddaVAX), aluminum hydroxide Al(OH)₃ or without adjuvant were evaluated for the efficacy of H7N9 vaccine regiments in mice. With either H7N9 whole or split-virus based vaccines, AddaVAX-adjuvanted formulations were the most immunogenic in eliciting significant humoral immune response against H7N9 virus and exhibited strong cross-reactive response in hemagglutination inhibition (HAI) and viral-neutralization assays against H7N7 virus as well. In contrast, formulations with Al(OH)₃ or without adjuvant were less immunogenic and elicited lower titers of HAI and microneutralization assays against both viruses. Dose-sparing experiments suggested that the formulation with as low as 0.004 μg of split or whole virus vaccine antigens together with 50% AddaVAX provided sufficient sero-protective HAI titers and achieved essential virus-neutralizing antibody titers against H7-subtype influenza viruses in mice. Protection experiments demonstrated that the formulation of 0.004 μg to 0.5 μg of split-virion vaccines with AddaVAX conferred full protection against viral challenge up to 100 LD50 of wild-type H7N9 virus, with 0% survival in placebo group. Taken together, our study demonstrates that squalene-based adjuvant can significantly enhance the protective efficacy of H7N9 virus vaccine and provides a useful strategy to confront the potential pandemic outbreaks of H7N9 virus.     

人口流动强度与甲型H1N1流感和人感染H7N9禽流感流行的相关性研究

目的 探讨人口流动强度与甲型H1N1流感（简称H1N1）和人感染H7N9禽流感（简称H7N9）的流行关系的模式。方法 利用2009年H1N1监测数据和2013年至2017年2月的H7N9监测数据,采用秩相关、主成分分析、Getis-Ord Gi*热点分析和对应分析探讨人口流动强度与上述两种传染病流行的相关关系。结果 人口流动强度与H1N1和H7N9的发病数均呈正相关。家禽出栏量与H7N9的发病数和人口流动强度也呈正相关。人口流动强度分别与H1N1（〖XC小五号.EPS;P〗=43.40,P<0.0001）和H7N9（〖XC小五号.EPS;P〗=51.82,P=0.0010）的热区位于对应分析图的相同区域,且均存在相同级别的热区聚集在一起,提示人口流动强度与这两种疾病均存在一定的对应关系;家禽出栏量和H7N9的热区位于对应分析图的相同区域,且热区级别大致一一对应,即存在相对严格的对应关系（〖XC小五号.EPS;P〗=36.47,P=0.0140）。人口流动强度与家禽出栏量也存在一定的对应关系,两者的热区位于对应分析图的同一区域（〖XC小五号.EPS;P〗=32.26,P=0.0410）。结论 人口流动强度与H1N1的暴发流行有相关关系,而人口流动强度与H7N9的流行关系是一种典型的“人-物流”的动物源性的传染病模式。     

Immunogenicity and safety of different dose schedules and antigen doses of an MF59-adjuvanted H7N9 vaccine in healthy adults aged 65 years and older

BackgroundThe number of human influenza A (H7N9) infections has escalated since 2013 with high resultant mortality. We conducted a phase II, randomized, partially-blinded trial to evaluate the safety and immunogenicity of an MF59-adjuvanted inactivated, split virion, H7N9 influenza vaccine (H7N9 IIV) administered at various dose levels and schedules in older adults.Methods479 adults ≥ 65 years of age in stable health were randomized to one of six groups to receive either 3.75, 7.5 or 15 µg of influenza A/Shanghai/02/2013 (H7N9) IIV adjuvanted with MF59 given as a 3-dose series either on days 1, 28 and 168 or on days 1, 57 and 168. Immunogenicity was assessed using both hemagglutination inhibition (HAI) and microneutralization (MN) assays prior to and 28 days following each dose. Safety was assessed through 1 year following the last dose.ResultsSubjects in all groups had only modest immune responses, with the HAI GMT < 20 after the second vaccine dose and <29 after the third vaccine dose. HAI titers ≥ 40 were seen in <37% of subjects after the second dose and <49% after the third dose. There were no significant differences seen between the two dose schedules. MN titers followed similar patterns, although the titers were approximately two-fold higher than the HAI titers. Logistic regression modeling demonstrated no statistically significant associations between the immune responses and age, sex or body mass index whereas recent prior receipt of seasonal influenza vaccine significantly reduced the HAI response [OR 0.13 (95% CI 0.05, 0.33); p < 0.001]. Overall, the vaccine was well tolerated. Two mild potentially immune mediated adverse events occurred, lichen planus and guttate psoriasis.ConclusionsMF59-adjuvanted H7N9 IIV was only modestly immunogenic in the older adult population following three doses. There were no significant differences in antibody responses noted among the various antigen doses or the two dose schedules.     

浙江省人感染H7N9禽流感病毒的基因组序列分析

目的分析浙江省2013年4月初一例人感染甲型H7N9禽流感患者的病原学和基因组序列特征.方法提取患者标本的病毒RNA并用荧光定量RT-PCR方法检测,一步法RT-PCR扩增H7N9禽流感病毒基因组的8个片段,测序并拼接出基因组序列.下载目前已经公布的H7N9禽流感病毒和其他H7、N9亚型的病毒的HA和NA序列,采用Mega 5.1软件对其进行序列比对并构建进化树.根据测序的结果分析该例H7N9禽流感病毒的序列变异情况.结果该患者标本甲型流感、H7亚型和N9亚型均为阳性,通过扩增基因组各片段并进行测序.对血凝素和神经氨酸酶基因进行比对和进化树构建,结果显示该H7N9病毒HA基因与A/duck/Zhejiang/12/2011(H7N3)的亲缘关系最近,NA基因与A/wild bird/Korea/A14/2011 (H7N9)的亲缘关系最近.编码内部蛋白的6个基因均与中国大陆近两年的H9N2毒株最为相似.该标本的病毒HA蛋白发生了Q226L突变,而与已经公布的人感染H7N9禽流感毒株均不一致的是PB2未发生E627K突变.结论从该份临床标本完成了检测和基因组各片段的扩增与测序.该病毒与已报道的人感染H7N9禽流感病毒同源性高,存在Q226L等重要位点的突变,但PB2未发生E627K突变.     

基于离散型地理信息的H7N9流感病毒动态时空传播模式

目的 基于离散型的地理信息构建了H7N9病毒时空动态传播模式图,探索H7N9病毒的空间传播路径。方法 选取全球共享禽流感数据倡议组织（GISAID）中中国以人类为宿主的H7N9流感病毒血凝素（HA）和神经酰胺酶（NA）基因序列,利用BioEdit 7.0软件进行多序列比对。在贝叶斯理论框架下,运用BEAST 1.8.2软件构建H7N9时空传播模型,采用symmetric substitution model和贝叶斯随机搜索变量选择（BBSVS）方法,对H7N9的历史传播轨迹进行推断和检验。以Google Earth软件展现H7N9病毒的时空传播动态图。结果 感染人类的H7N9病毒起源于上海或杭州,最早可追溯到2012年10月。并在2013年3、4月份开始传向邻近省份,同年8、9月份传播加剧,3个月内传向10余处地区。结论 基于基因核苷酸序列和空间地理信息,追踪了H7N9病毒历史传播轨迹,为早期流感疫情的防控及病毒的溯源提供线索。     

Improving immunogenicity of influenza virus H7N9 recombinant hemagglutinin for vaccine development

Human infections of novel avian influenza A virus (H7N9) emerged in early 2013 and caused about 40% case-fatality through 2017. Therefore, development of influenza H7N9 vaccines is critical for pandemic preparedness. Currently, there are three means of production of commercial influenza vaccines: egg-based, mammalian cell-based, and insect cell-based platforms. The insect cell-based platform has the advantage of high speed in producing recombinant protein. In this study, we evaluate the stability and immunogenicity of two different influenza H7 HA expression constructs generated using the baculovirus system, including membrane-based full-length HA (mH7) and secreted ectodomain-based H7 (sH7). The mH7 construct could form an oligomer-rosette structure and had a high hemagglutinin (HA) titer 8192. In contrast to mH7, the sH7 construct could not form an oligomer-rosette structure and did not have HA titer before cross-linking with anti-His antibody. Thermal stability tests showed that the sH7 and mH7 constructs were unstable at 43?°C and 52?°C, respectively. In a mice immunization study, the mH7 construct but not the sH7 construct could induce robust HI and neutralizing antibody titers. In conclusion, further development of the mH7 vaccine candidate is desirable.     

江西省人感染禽流感病例流行病学特征分析

目的 分析江西省不同亚型人感染禽流感病例的流行特征,为江西省禽流感防控策略的制定和优化提供科学依据。方法 以江西省2013年1月至2016年4月报告的人感染禽流感确诊病例为研究对象,采用流行病学个案调查方法,分析病例的流行病学特征。结果 江西省共报告人禽流感确诊病例18例,其中H7N9病例14例,H10N8病例3例,H5N6病例1例;死亡4例,病死率22.22%。88.89%（16/18）的病例发生在1 - 2、4和12月,61.11%（11/18）的病例出现在鄱阳湖周边地区;病例年龄中位数为62岁（23～80岁）,男性占55.56%（10/18）;17例病例均有明确的禽类暴露史,病例可疑暴露场所环境相关标本中H7N9、H10N8禽流感病毒阳性标本检出率分别为12.24%和5.19%。结论 江西省人感染禽流感病例在冬春季高发,且存在一定的地域集聚性,多数病例发病前有禽类或活禽市场暴露史。建议实施“加强活禽市场管理,控制发病风险”控制措施,并主动开展禽流感病毒病原学监测和血清学调查,系统收集轻症病例和隐性感染者信息,全面分析和掌握我省人禽流感流行特征,为科学防控人禽流感疫情提供依据。     

H7N3 live attenuated influenza vaccine has a potential to protect against new H7N9 avian influenza virus

After recent emergence of new avian influenza A(H7N9) viruses in humans many people and Governments are asking about H7 influenza vaccine which could provide cross-protection against new viruses, until H7N9 vaccine is prepared from a relevant strain. Here we scientifically justify that available H7N3 live attenuated influenza vaccine (LAIV) can be protective against H7N9 viruses due to the presence of conserved immune epitopes in its hemagglutinin. We used Immune Epitope Database analysis resource to predict B-cell and CTL epitopes distributed across H7N3 HA molecule and assessed their identity with new H7N9 viruses at near 70% and 60% of the epitopes, respectively. In addition, we tested serum samples of volunteers participated in phase I clinical trial of H7N3 LAIV for the presence of anti-H7N9 hemagglutination-inhibition and neutralizing antibodies and found seroconversions in 44.8% of vaccinated persons, which suggests the potential of H7N3 LAIV to protect against new H7N9 avian influenza viruses.     

Influenza H7N9 LAH-HBc virus-like particle vaccine with adjuvant protects mice against homologous and heterologous influenza viruses

The long alpha-helix (LAH) region located in influenza virus hemagglutinin (HA) shows conservation among different influenza A strains, which could be used as a candidate target of influenza vaccines. Moreover, the hepatitis B virus core protein (HBc) is a carrier for heterologous epitopes in eliciting effective immune responses. We inserted the LAH region of H7N9 influenza virus into the HBc and prepared the LAH-HBc protein, which were capable of self-assembly into virus-like particles (VLP), by using E. coli expression system. Intranasal immunization of the LAH-HBc VLP in combination with chitosan adjuvant or CTB¹ adjuvant in mice could induce both humoral and cellular immune responses effectively and provide complete protection against lethal challenge of homologous H7N9 virus or heterologous H3N2 virus, as well as partial protection against lethal challenge of heterologous H1N1 virus. These results provide a proof of concept for LAH-HBc VLP vaccine that would be fast and easy to be produced and might be an ideal candidate as a rapid-response tool against a future influenza pandemic.