H9N2 avian influenza virus-like particle vaccine provides protective immunity and a strategy for the differentiation of infected from vaccinated animals

In the present study, virus-like particles (VLPs) were evaluated as a candidate poultry vaccine against avian influenza virus (AIV) subtype H9N2. Specific pathogen-free chickens received a single injection of the VLP vaccine expressing HA and M1 protein of AIV H9N2 (H9 HA VLP) at escalating doses in the presence or absence of ISA70 water-in-oil adjuvant. At 3 weeks post vaccination, we performed hemagglutination inhibition (HI) test and enzyme-linked immunosorbent assay (ELISA) to determine serological immune responses, and challenge studies using SPF chickens. A single dose of H9 HA VLP vaccine induced high levels of HI antibodies and lowered frequencies of virus isolation after the wild-type virus challenge. The addition of ISA70 adjuvant significantly increased the immunogenicity of H9 HA VLP vaccines. Furthermore, it allows differentiation of AIV-infected chickens from vaccinated chickens with an ELISA using nucleocapsid antigen, which offers a promising strategy to differentiate infected from vaccinated animals (DIVA). These results provide support for continued development of the VLP as an animal vaccine against influenza virus.     

Cross-clade protective immune responses of NS1-truncated live attenuated H5N1 avian influenza vaccines

BackgroundH5N1 highly pathogenic avian influenza (HPAI) has raised global concern for causing huge economic losses in poultry industry, and an effective vaccine against HPAI is highly desirable. Live attenuated influenza vaccine with trunctated NS1 protein as a potential strategy will be extremely useful for improving immune efficacy.MethodsA series of H5N1 avian influenza virus reassortants harboring amino-terminal 48, 70, 73, and 99 aa in NS1 proteins, along with a modified low pathogenic HA protein was generated, and named as S-HALo/NS48, S-HALo/NS70, S-HALo/NS73, and S-HALo/NS99, respectively. In addition, their biological and immunological characteristics were further analyzed.ResultsThe viruses S-HALo/NS70, S-HALo/NS73, and S-HALo/NS99, but not S-HALo/NS48, had a comparable growth property with the full-length NS1 virus, S-HALo/NSFu. Mice and chickens studies demonstrated that the viruses with truncated NS1 protein were further attenuated when compared to the virus S-HALo/NSFu. Vaccination with the virus S-HALo/NS73 in chickens induced significant cross-protection against homologous clade 2.3.4 H5 virus and heterologous clade 7.2, 2.3.2.1, and 2.3.4.4 H5 viruses.ConclusionA 70-aa amino-terminal fragment of NS1 protein may be long enough for viral replication. The recombinant virus S-HALo/NS73 is a broad-spectrum live attenuated H5N1 avian influenza vaccine candidate in chickens.     

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.     

Atypical avian influenza (H5N1)

We report the first case of avian influenza in a patient with fever and diarrhea but no respiratory symptoms. Avian influenza should be included in the differential diagnosis for patients with predominantly gastrointestinal symptoms, particularly if they have a history of exposure to poultry.     

广西禽流感H5N1亚型病毒HA基因序列分析

目的了解广西禽流感H5N1亚型病毒的基因特性.方法2011年在广西农贸市场采集污水、笼具涂抹、粪便标本,经H5亚型特异实时荧光定量PCR方法(Real-time fluorescence quantitative RT-PCR)检测,阳性样本进行病毒血凝素(hemagglutinin,HA)基因扩增后对产物直接测序,测序结果与已知参考毒株进行序列比对及系统进化分析.结果对阳性样本病毒HA基因测序获得6份HA序列,均分布在进化分支2.3.2的Ⅱ-1分支下.广西的6序列无论是氨基酸还是核苷酸的都是高度同源的,其核苷酸同源性在99.5%~100%,氨基酸同源性在99.5%~99.8%;序列测定的结果同时表明无论是受体特异性还是连接肽都是禽源的.结论2011年广西农贸市场流行的禽流感H5N1亚型病毒主要以进化分支2.3.2Ⅱ-1为主,均为禽源性的病毒.     

Vaccination with virus-like particles containing H5 antigens from three H5N1 clades protects chickens from H5N1 and H5N8 influenza viruses

Highly pathogenic avian influenza (HPAI) viruses, especially H5N1 strains, represent a public health threat and cause widespread morbidity and mortality in domestic poultry. Recombinant virus-like particles (VLPs) represent a promising novel vaccine approach to control avian influenza including HPAI strains. Influenza VLPs contain viral hemagglutinin (HA), which can be expressed in cell culture within highly immunogenic VLPs that morphologically and antigenically resemble influenza virions, except VLPs are non-infectious. Here we describe a recombinant VLP containing HA proteins derived from three distinct clades of H5N1 viruses as an experimental, broadly protective H5 avian influenza vaccine. A baculovirus vector was configured to co-express the H5 genes from recent H5N1 HPAI isolates A/chicken/Germany/2014 (clade 2.3.4.4), A/chicken/West Java/Subang/29/2007 (clade 2.1.3) and A/chicken/Egypt/121/2012 (clade 2.2.1). Co-expression of these genes in Sf9 cells along with influenza neuraminidase (NA) and retrovirus gag genes resulted in production of triple-clade H555 VLPs that exhibited hemagglutination activity and morphologically resembled influenza virions. Vaccination of chickens with these VLPs resulted in induction of serum antibody responses and efficient protection against experimental challenges with three different viruses including the recent U.S. H5N8 HPAI isolate. We conclude that these novel triple-clade VLPs represent a feasible strategy for simultaneously evoking protective antibodies against multiple variants of H5 influenza virus.     

Prior infection of chickens with H1N1 avian influenza virus elicits heterologous protection against highly pathogenic H5N2

Current vaccines for influenza are primarily killed whole virus vaccines that elicit antibody responses to the homologous virus but lack protection against heterologous viruses. Using chickens as a model we have explored the possibility of using a live low pathogenic avian influenza (LPAI) A/goose/AB/223/2005 H1N1 virus as a vaccine to generate protective immunity against heterologous highly pathogenic avian influenza (HPAI) A/chicken/Pensylvania/1370/1983 H5N2 virus challenge. Virus replicated in chickens infected with LPAI H1N1 but did not cause clinical disease. In addition, these chickens developed neutralizing antibodies to LPAI H1N1 virus, but not HPAI H5N2, 21 days post infection (DPI). Furthermore, peripheral blood mononuclear cells from H1N1-infected chickens at 20 DPI had antigen specific proliferation and IFN-γ secretion following antigen stimulation to H5N2 indicating a heterologous HPAI H5N2 specific cell mediated immunity (CMI) following LPAI H1N1 infection. Following challenge with HPAI H5N2 virus, all control chickens developed clinical disease, while chickens previously infected with H1N1 did not develop clinical disease and shed significantly less virus by oral and cloacal routes. These results indicated that previous infection with LPAI virus can generate heterologous CMI capable of protecting against HPAI H5N2.     

长沙市家禽市场环境中H5N1亚型禽流感病毒传播风险研究

目的 对长沙市家禽市场职业暴露人群进行禽流感病毒（AIV）H5N1亚型抗体水平和环境AIV核酸检测,并对环境中AIV H5N1亚型的血凝素（HA）基因进行测序分析。方法 抽取长沙市1个区和1个县,各选择2个城区或乡镇家禽市场进行职业暴露人群H5N1抗体和环境AIV核酸检测。利用单放射免疫扩散溶血实验（SRH）对102份家禽市场职业暴露人员血清标本进行H5N1抗体检测,real-time PCR方法检测160份家禽市场环境标本（污水、禽类粪便、羽毛和禽类笼具表面涂抹标本）AIV核酸,对4份污水H5N1亚型AIV核酸阳性标本进行HA基因RT-PCR扩增和TA克隆测序,测序结果利用Lasergene和Mega 5.0软件进行氨基酸比对和进化树构建。结果AIVH5N1抗体监测结果显示,家禽市场职业暴露人群血清H5N1抗体阳性率为25.5%（26/102）,其中乡镇和城区家禽市场职业暴露人群阳性率分别为50.0%（9/18）和25.4%（17/67）,乡镇家禽市场职业暴露人群阳性率高于城区。长沙市家禽市场环境中H5亚型AIV核酸阳性率为31.3%（50/160）,其中乡镇家禽市场阳性率为37.3%（31/83）,高于城区家禽市场24.7%（19/77）;不同标本H5亚型AIV核酸阳性率不同：污水（50.0%,24/48）、羽毛（44.5%,4/9）、禽类粪便（29.8%,14/47）和禽类笼具表面涂抹（14.3%,8/56）;差异均有统计学意义（P＜0.01）。TA克隆测序得到4个AIVH5N1亚型HA基因序列,进化树显示4个AIVH5N1亚型HA基因与中国内地和香港禽来源的AIV分离株为同一分组,属于欧亚分支;4个AIV H5N1亚型HA基因受体结合位点氨基酸序列仍然为禽源（QSG）、HA1和HA2蛋白之间连接肽为多个碱性氨基酸序列（RERRRKK或RERRGKK）,与入源AIV H5N1亚型具有相同的受体结合位点和高致病性的分子特征。结论 长沙市家禽市场环境中存在较多数量的AIVH5N1亚型,是导致职业暴露人群抗体阳性率达25.5%的原因之一;环境中存在的AIVH5N1亚型HA基因表现出来的高致病性分子特征,增加了家禽市场环境中发生AIV传播的风险。     

Long lasting immunity in chickens induced by a single shot of influenza vaccine prepared from inactivated non-pathogenic H5N1 virus particles against challenge with a highly pathogenic avian influenza virus

An influenza vaccine was prepared from inactivated whole particles of the non-pathogenic strain A/duck/Hokkaido/Vac-1/04 (H5N1) virus using an oil adjuvant containing anhydromannitol-octadecenoate-ether (AMOE). The vaccine was injected intramuscularly into five 4-week-old chickens, and 138 weeks after vaccination, they were challenged intranasally with 100 times 50% chicken lethal dose of the highly pathogenic avian influenza (HPAI) virus A/chicken/Yamaguchi/7/04 (H5N1). All 5 chickens survived without exhibiting clinical signs of influenza, although 2 days post-challenge, 3 vaccinated chickens shed limited titres of viruses in laryngopharyngeal swabs.     

禽流感病毒研究进展及抗H7N9型病毒疫苗与抗体研究

禽流感病毒（avian influenza virus,AIV）是一种可引起急性呼吸道传染病的人畜共患病毒。自2013年我国出现了全球首例人感染H7N9型AIV病例以来,人们对该病毒产生了担忧与恐慌。AIV在全球广泛传播,人感染不同型别AIV事件也持续发生,造成了巨大的经济损失。目前尚无针对该病的特异性治疗措施与药物,...     

Probable tiger-to-tiger transmission of avian influenza H5N1

During the second outbreak of avian influenza H5N1 in Thailand, probable horizontal transmission among tigers was demonstrated in the tiger zoo. Sequencing and phylogenetic analysis of those viruses showed no differences from the first isolate obtained in January 2004. This finding has implications for influenza virus epidemiology and pathogenicity in mammals.     

Influenza virus-like particles comprised of the HA, NA, and M1 proteins of H9N2 influenza virus induce protective immune responses in BALB/c mice

Avian influenza viruses represent a growing threat for an influenza pandemic. To develop recombinant vaccine for avian influenza of the H9N2 subtype, we expressed in insect cells virus-like particles (VLPs) consisting of three structural proteins of influenza A/Hong Kong/1073/99 (H9N2) virus. Upon infection of Sf9 cells with recombinant baculoviruses, the hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins were co-expressed in the infected cells, self-assembled, and released into the culture medium as VLPs of 80–120 nm in diameter. VLPs exhibited functional characteristics of influenza virus including hemagglutination and neuraminidase activities. In BALB/c mice, VLPs elicited serum antibodies specific for influenza A/Hong Kong/1073/99 (H9N2) virus and inhibited replication of the influenza virus after challenge. Thus, VLPs represent a potential strategy for the development of human vaccines against avian influenza H9N2 viruses.     

云南省边境地区禽流感H5N1亚型病毒遗传多样性分析

目的 了解云南省边境地区禽流感H5N1亚型病毒遗传多样性.方法 2009-2011年7月在云南省边境地区采集境外家禽和野生鸟类棉拭子样品,经H5/Nl亚型特异性多重RT-PCR检测,阳性样品进行病毒HA基因扩增,克隆至pMD 18-T载体测序,并与已知参考毒株进行序列比对及系统发育分析.结果 36份阳性样品病毒HA基因测序获得15种HA序列,存在2个不同进化分支(2.3.2、2.3.4),2.3.2进一步可划分为3个进化小分支(Ⅱ-1～Ⅱ-3),2.3.4进一步可划分为2个进化小分支(Ⅰ-1和Ⅰ-2).2.3.2Ⅱ-1、Ⅱ-2毒株是新出现的H5N1亚型病毒变异株.结论 2009- 2011年7月云南省边境地区H5N1亚型病毒具有遗传多样性,病毒经历了多分支(2.3.2、2.3.4)至单一支(2.3.2)的进化过程.     

人用H5N1禽流感疫苗检定评价

目的选用H5N1禽流感毒种（R1194和R1203）,按设计的生产工艺,制成的疫苗进行全面检定,选取其中较好毒株生产的疫苗,用于以后人用禽流感疫苗临床前研究的动物免疫。方法利用从国外引进2株H5N1禽流感病毒,设计生产3种疫苗（全病毒、裂解-1、裂解-2）的纯化工艺,优化纯化条件,全面检定制成的疫苗,比较2毒种生产疫苗的质量。结果2毒株在接种滴度104EID50/ml（半数鸡胚感染剂量/ml）,培养温度34.5℃,培养时间3 d的条件下,病毒增殖滴度较高;检定结果证明,制成的所有疫苗中反映质量的内毒素、卵清蛋白、总蛋白、总蛋白与血凝素之比这4项指标,明显优于国家标准;3种工艺疫苗的电镜形态完全不同;经十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）,全病毒疫苗和裂解-2疫苗的蛋白条带形态相似,但裂解-1疫苗在32 KD左右的条带缺失;毒种R1203株疫苗的产量明显高于R1194株。结论设计的3种生产疫苗的纯化工艺,均可生产出高质量的疫苗;毒种R1203株的疫苗产量高于毒种R1194株;可用R1203毒株,按3种不同工艺生产的疫苗接种动物,观察免疫效果。     

Dose response effects of avian influenza (H7N7) vaccination of chickens: Serology,clinical protection and reduction of virus excretion

Knowledge of the relation between the antigen content of inactivated avian influenza (AI) vaccines, the serological response after vaccination and protection of vaccinated animals is important for the choice of optimal vaccines and vaccination regimes as well as for the assessment of criteria for the licensing of new AI-vaccines. We studied this relation in a dose response study using inactivated H7N7 avian influenza vaccines with varying antigen content. The serological response depended on the antigen content of the vaccines. Anti-AI antibodies were detected most frequently with ELISA, followed by the virus neutralisation test and the haemagglutination inhibition (HI) assay. Chickens with measurable HI-antibody titers, using homologous H7N7 antigen, were all protected against clinical disease after challenge with highly pathogenic A/chicken/Netherlands/621557/03 H7N7 virus. However, in these chickens high levels of virus could still be present on days 2–4 after challenge. The reduction of virus titers after challenge, depended on the antigen content of the vaccines as well as on the serum antibody titers. While 10 haemagglutinating units (HAU), equivalent to 0.8 μg haemagglutinin (HA) protein, per vaccine dose was sufficient for prevention of clinical disease, 128 HAU (9 μg HA) per dose was required for reduction of virus titers in all chickens to 10³ egg-infectious dose 50% (EID₅₀) or less. In order to reduce virus titers below 10³ EID₅₀ per swab a HI-antibody titer of 64 was required. After use of the vaccine with the highest antigen content, challenge still induced a booster of antibody titers which is indicative of replication of challenge virus.     

Generation of a reassortant avian influenza virus H5N2 vaccine strain capable of protecting chickens against infection with Egyptian H5N1 and H9N2 viruses

BackgroundAvian influenza H5N1 viruses have been enzootic in Egyptian poultry since 2006. Avian influenza H9N2 viruses which have been circulating in Egyptian poultry since 2011 showed high replication rates in embryonated chicken eggs and mammalian cells.MethodsTo investigate which gene segment was responsible for increasing replication, we constructed reassortant influenza viruses using the low pathogenic H1N1 PR8 virus as backbone and included individual genes from A/chicken/Egypt/S4456B/2011(H9N2) virus. Then, we invested this finding to improve a PR8-derived H5N1 influenza vaccine strain by incorporation of the NA segment of H9N2 virus instead of the NA of H5N1. The growth properties of this virus and several other forms of reassortant H5 viruses were compared. Finally, we tested the efficacy of this reassortant vaccine strain in chickens.ResultsWe observed an increase in replication for a reassortant virus expressing the neuraminidase gene (N2) of H9N2 virus relative to that of either parental viruses or reassortant PR8 viruses expressing other genes. Then, we generated an H5N2 vaccine strain based on the H5 from an Egyptian H5N1 virus and the N2 from an Egyptian H9N2 virus on a PR8 backbone. This strain had better replication rates than an H5N2 reassortant strain on an H9N2 backbone and an H5N1 reassortant on a PR8 backbone. This virus was then used to develop a killed, oil-emulsion vaccine and tested for efficacy against H5N1 and H9N2 viruses in chickens. Results showed that this vaccine was immunogenic and reduced mortality and shedding.DiscussionOur findings suggest that an inactivated PR8-derived H5N2 influenza vaccine is efficacious in poultry against H5N1 and H9N2 viruses and the vaccine seed replicates at a high rate thus improving vaccine production.     

人用H5N1禽流感疫苗免疫动物效果观察

目的将用禽流感毒种R1203株按3种生产工艺生产的疫苗进行动物接种,观察疫苗的免疫效果。方法用不同剂量的3种工艺生产的禽流感疫苗,分别免疫大鼠和家兔,肌肉接种2针（隔14 d）,首针后14和28 d静脉采血,测定接种1针和2针后,动物血清中血凝抑制抗体和中和抗体的产生情况。结果大鼠的抗体反应较家兔敏感;大鼠和家兔在疫苗接种1针后14 d,都能产生血抑抗体,滴度可达1∶40,但中和抗体滴度都很低,几乎测不到;接种2针后,血抑抗体或中和抗体反应都显著高于第1针;裂解-2苗接种大鼠和家兔后的抗体反应普遍高于另2种疫苗,且家兔量-效反应明显。结论裂解-2疫苗优于其他2种;中和抗体能正确反应疫苗的质量;H5N1禽流感疫苗的效力试验可用于免疫家兔测定中和抗体和临床前研究的动物试验,用15μg血凝素（HA）接种家兔2针,血清中和抗体滴度达1：40或以上。     

An inactivated H5N2 vaccine reduces transmission of highly pathogenic H5N1 avian influenza virus among native chickens

Vaccination against H5N1 highly pathogenic avian influenza in endemically affected areas is a potentially attractive option for local prevention and control. In Indonesia the majority of local outbreaks have occurred in back yard flocks with native chickens, and it is therefore of interest to determine whether these birds can be protected against infection by vaccination. To this end two transmission experiments were carried out with H5N1 virus (A/chicken/Legok/2003) in vaccinated and unvaccinated native chickens. The vaccine contained an inactivated heterologous H5N2 strain (A/turkey/England/N28/73 H5N2). Birds were vaccinated at 4 and 7 weeks of age and challenged at 10 weeks of age. During 10 days post-challenge tracheal and cloacal swabs were taken for virus isolation, and serum blood was collected regularly to measure haemaglutinin inhibiting (HI) antibody responses. The results show that transmission of H5N1 virus was rapid and efficient in unvaccinated birds, that infection and transmission were completely prevented in vaccinated birds, and that vaccinated birds that were exposed to unvaccinated inoculated birds were still protected from infection. These findings indicate that vaccination with a heterologous H5N2 vaccine is able to prevent virus transmission in flocks of native chickens.     

Enhanced cross-lineage protection induced by recombinant H9N2 avian influenza virus inactivated vaccine

Background

Antigenic drift of H9N2 low pathogenic avian influenza viruses (AIV) may result in vaccination failure in the poultry industry and thus a cross-protective vaccine against H9N2 AIV is highly desirable.

H5N1禽流感病毒血凝素和神经氨酸酶基因在杆状病毒中的共表达

目的构建共表达H5N1禽流感病毒血凝素(HA)和神经氨酸酶(NA)蛋白的杆状病毒表达系统。方法利用PCR方法分别扩增A/Hubei/1/2010(H5N1)病毒的HA和NA基因,克隆至经改造带有对虾白斑综合病毒(WSSV)早期启动子iel的mpFastBac Dual载体,构建mpFast Bac-HA-NA表达质粒,转化DH10Bac感受态细胞,用获得的Bacmid-HA-NA穿梭质粒转染sf9细胞,获得重组杆状病毒Bac-HA-NA;提取重组杆状病毒Bac-HA-NA的DNA并使用PCR方法检测;利用Western blot检测HA和NA表达,并分别检测重组杆状病毒Bac-HA-NA红细胞凝集能力和神经氨酸酶活性。结果经PCR鉴定,构建的质粒Bacmid-HA-NA正确;Western blot检测表明Bacmid-HANA能在sf9细胞中有效表达HA和NA蛋白,生物活性检测表明Bac-HA-NA能引起红细胞凝集并具有神经氨酸酶活性。结论获得了能同时表达禽流感病毒HA和NA的重组杆状病毒Bac-HA-NA,为进一步研究流感疫苗奠定了基础。