Development of a new candidate H5N1 avian influenza virus for pre‐pandemic vaccine production

Background Highly pathogenic H5N1 avian influenza viruses currently circulating in birds have caused hundreds of human infections, and pose a significant pandemic threat. Vaccines are a major component of the public health preparedness for this likely event. The rapid evolution of H5N1 viruses has resulted in the emergence of multiple clades with distinct antigenic characteristics that require clade‐specific vaccines. A variant H5N1 virus termed clade 2.3.4 emerged in 2005 and has caused multiple fatal infections. Vaccine candidates that match the antigenic properties of variant viruses are necessary because inactivated influenza vaccines elicit strain‐specific protection. Objective To address the need for a suitable seed for manufacturing a clade 2.3.4 vaccine, we developed a new H5N1 pre‐pandemic candidate vaccine by reverse genetics and evaluated its safety and replication in vitro and in vivo. Methods A reassortant virus termed, Anhui/PR8, was produced by reverse genetics in compliance with WHO pandemic vaccine development guidelines and contains six genes from A/Puerto Rico/8/34 as well as the neuraminidase and hemagglutinin (HA) genomic segments from the A/Anhui/01/2005 virus. The multi‐basic cleavage site of HA was removed to reduce virulence. Results The reassortant Anhui/PR8 grows well in eggs and is avirulent to chicken and ferrets but retains the antigenicity of the parental A/Anhui/01/2005 virus. Conclusion These results indicate that the Anhui/PR8 reassortant lost a major virulent determinant and it is suitable for its use in vaccine manufacturing and as a reference vaccine virus against the H5N1 clade 2.3.4 viruses circulating in eastern China, Vietnam, Thailand, and Laos.     

Comparative safety, immunogenicity, and efficacy of several anti-H5N1 influenza experimental vaccines in a mouse and chicken models (Testing of killed and live H5 vaccine)

Please cite this paper as: Gambaryan et al. (2011) Comparative safety, immunogenicity, and efficacy of several anti‐H5N1 influenza experimental vaccines in a mouse and chicken models. Parallel testing of killed and live H5 vaccine. Influenza and Other Respiratory Viruses 6(3), 188–195. Objective Parallel testing of inactivated (split and whole virion) and live vaccine was conducted to compare the immunogenicity and protective efficacy against homologous and heterosubtypic challenge by H5N1 highly pathogenic avian influenza virus. Method Four experimental live vaccines based on two H5N1 influenza virus strains were tested; two of them had hemagglutinin (HA) of A/Vietnam/1203/04 strain lacking the polybasic HA cleavage site, and two others had hemagglutinins from attenuated H5N1 virus A/Chicken/Kurgan/3/05, with amino acid substitutions of Asp54/Asn and Lys222/Thr in HA1 and Val48/Ile and Lys131/Thr in HA2 while maintaining the polybasic HA cleavage site. The neuraminidase and non‐glycoprotein genes of the experimental live vaccines were from H2N2 cold‐adapted master strain A/Leningrad/134/17/57 (VN‐Len and Ku‐Len) or from the apathogenic H6N2 virus A/Gull/Moscow/3100/2006 (VN‐Gull and Ku‐Gull). Inactivated H5N1 and H1N1 and live H1N1 vaccine were used for comparison. All vaccines were applied in a single dose. Safety, immunogenicity, and protectivity against the challenge with HPAI H5N1 virus A/Chicken/Kurgan/3/05 were estimated. Results All experimental live H5 vaccines tested were apathogenic as determined by weight loss and conferred more than 90% protection against lethal challenge with A/Chicken/Kurgan/3/05 infection. Inactivated H1N1 vaccine in mice offered no protection against challenge with H5N1 virus, while live cold‐adapted H1N1 vaccine reduced the mortality near to zero level. Conclusions The high yield, safety, and protectivity of VN‐Len and Ku‐Len made them promising strains for the production of inactivated and live vaccines against H5N1 viruses.     

Immunogenicity and safety of pandemic influenza A (H1N1) 2009 vaccine: systematic review and meta-analysis

The emergence of the 2009 H1N1 pandemic has highlighted the need to have immunogenicity and safety data on the new pandemic vaccines. There is already considerable heterogeneity in the types of vaccine available and of study performed around the world. A systematic review and meta-analysis is needed to assess the immunogenicity and safety of pandemic influenza A (H1N1) 2009 vaccines. We searched Medline, EMBASE, the Cochrane Library and other online databases up to 1st October 2010 for studies in any language comparing different pandemic H1N1 vaccines, with or without placebo, in healthy populations aged at least 6 months. The primary outcome was seroprotection according to haemagglutination inhibition (HI). Safety outcomes were adverse events. Meta-analysis was performed for the primary outcome. We identified 18 articles, 1 only on safety and 17 on immunogenicity, although 1 was a duplicate. We included 16 articles in the meta-analysis, covering 17,921 subjects. Adequate seroprotection (≥70%) was almost invariably achieved in all age groups, and even after one dose and at low antigen content (except in children under 3 years receiving one dose of non-adjuvanted vaccine). Non-adjuvanted vaccine from international companies and adjuvanted vaccines containing oil in water emulsion (e.g. AS03, MF59), rather than aluminium, performed better. Two serious vaccination-associated adverse events were reported, both of which resolved fully. No death or case of Guillain-Barré syndrome was reported. The pandemic influenza (H1N1) 2009 vaccine, with or without adjuvant, appears generally to be seroprotective after just one dose and safe among healthy populations aged ≥36 months; very young children (6-35 months) may need to receive two doses of non-adjuvanted vaccine or one dose of AS03(A/B)-adjuvanted product to achieve seroprotection.     

Efficacy of a heterologous vaccine and adjuvant in ferrets challenged with influenza virus H5N1

Please cite this paper as: Vela et al. (2012) Efficacy of a heterologous vaccine and adjuvant in ferrets challenged with influenza virus H5N1. Influenza and Other Respiratory Viruses 6(5), 328–340. Background In 1997, highly pathogenic avian influenza (HPAI) viruses caused outbreaks of disease in domestic poultry markets in Hong Kong. The virus has also been detected in infected poultry in Europe and Africa. Objective The objective of this study was to determine the efficacy of a heterologous vaccine administered with and without the aluminum hydroxide adjuvant in ferrets challenged with HPAI (A/Vietnam/1203/04). Methods Animals in four of the five groups were vaccinated twice 21 days apart, with two doses of a heterologous monovalent subvirion vaccine with or without an aluminum hydroxide adjuvant and challenged with a lethal target dose of A/Vietnam/1203/04. Results All animals vaccinated with the heterologous vaccine in combination with the aluminum hydroxide adjuvant survived a lethal challenge of A/Vietnam/1203/04. Four of the eight animals vaccinated with 30 μg of the vaccine without the adjuvant survived, while two of the eight animals vaccinated with 15 μg of the vaccine without the adjuvant survived. None of the unvaccinated control animals survived challenge. Additionally, changes in virus recovered from nasal washes and post‐mortem tissues and serology suggest vaccine efficacy. Conclusions Altogether, the data suggest that the heterologous vaccine in combination with the aluminum hydroxide adjuvant offers maximum protection against challenge with A/Vietnam/1203/04 when compared to the unvaccinated control animals or animals vaccinated without any adjuvant.     

Systematic review of clinical and epidemiological features of the pandemic influenza A (H1N1) 2009

The aim of this systematic review was to summarise the clinical and epidemiological features of the pandemic influenza A (H1N1) 2009. We did a systematic search of published literature reporting clinical features of laboratory-confirmed pandemic influenza A (H1N1) 2009 from 1 April 2009 to 31 January 2010. Forty-four articles met our inclusion criteria for the review. The calculated weighted mean age of confirmed cases was 18·1 years, with the median ranging from 12 to 44 years. Cough (84·9%), fever (84·7%), headache (66·5%), runny nose (60·1%) and muscle pain (58·1%) were the most common symptoms of confirmed cases. One or more pre-existing chronic medical conditions were found in 18·4% of cases. Almost two-thirds (64%) of cases were aged between 10 and 29 years, 5·1% were aged over 50 years and only 1·1% were aged over 60 years. The confirmed case fatality ratio was 2·9% (95% CI 0·0-6·7%), an extracted average from 12 of 42 studies reporting fatal cases (937 fatal cases among 31,980 confirmed cases), which gives an overall estimated infected case fatality ratio of 0·02%. Early in the pandemic, disease occurred overwhelmingly in children and younger adults, with cough and fever as the most prevalent clinical symptoms of the confirmed cases. A high infection rate in children and young adults, with sparing of the elderly population, has implications for pandemic influenza management and control policies.     

Live attenuated H5N1 vaccine with H9N2 internal genes protects chickens from infections by both Highly Pathogenic H5N1 and H9N2 Influenza Viruses

Please cite this paper as: Nang et al. (2013) Live attenuated H5N1 vaccine with H9N2 internal genes protects chickens from infections by both Highly Pathogenic H5N1 and H9N2 Influenza Viruses. Influenza and Other Respiratory Viruses 7(2) 120–131. Background The highly pathogenic H5N1 and H9N2 influenza viruses are endemic in many countries around the world and have caused considerable economic loss to the poultry industry. Objectives We aimed to study whether a live attenuated H5N1 vaccine comprising internal genes from a cold‐adapted H9N2 influenza virus could protect chickens from infection by both H5N1 and H9N2 viruses. Methods We developed a cold‐adapted H9N2 vaccine virus expressing hemagglutinin and neuraminidase derived from the highly pathogenic H5N1 influenza virus using reverse genetics. Results and Conclusions Chickens immunized with the vaccine were protected from lethal infections with homologous and heterologous H5N1 or H9N2 influenza viruses. Specific antibody against H5N1 virus was detected up to 11 weeks after vaccination (the endpoint of this study). In vaccinated chickens, IgA and IgG antibody subtypes were induced in lung and intestinal tissue, and CD4+ and CD8+ T lymphocytes expressing interferon‐gamma were induced in the splenocytes. These data suggest that a live attenuated H5N1 vaccine with cold‐adapted H9N2 internal genes can protect chickens from infection with H5N1 and H9N2 influenza viruses by eliciting humoral and cellular immunity.     

Immunization with 1976 swine H1N1- or 2009 pandemic H1N1-inactivated vaccines protects mice from a lethal 1918 influenza infection

Please cite this paper as: Easterbrook et al. (2011) Immunization with 1976 swine H1N1‐ or 2009 pandemic H1N1‐inactivated vaccines protects mice from a lethal 1918 influenza infection. Influenza and Other Respiratory Viruses DOI: 10.1111/j.1750‐2659.2010.00191.x. Background Zoonotic infections with H1N1 influenza viruses that evolved initially from the 1918 virus (1918) and adapted to swine threatened a pandemic in 1976 (1976 swH1N1) and a novel reassortant H1N1 virus caused a pandemic in 2009–2010 (2009 pH1N1). Epidemiological and laboratory animal studies show that protection from severe 2009 pH1N1 infection is conferred by vaccination or prior infection with 1976 swH1N1 or 1918. Objectives Our aim was to demonstrate cross‐protection by immunization with 2009 pH1N1 or 1976 swH1N1 vaccines following a lethal challenge with 1918. Further, the mechanisms of cross‐protective antibody responses were evaluated. Methods Mice were immunized with 1976 swH1N1, 2009 pH1N1, 2009 seasonal trivalent, or 1918 vaccines and challenged with 1918. Cross‐reactive antibody responses were assessed and protection monitored by survival, weight loss, and pathology in mice. Results and Conclusions Vaccination with the 1976 swH1N1 or 2009 pH1N1 vaccines protected mice from a lethal challenge with 1918, and these mice lost no weight and had significantly reduced viral load and pathology in the lungs. Protection was likely due to cross‐reactive antibodies detected by microneutralization assay. Our data suggest that the general population may be protected from a future 1918‐like pandemic because of prior infection or immunization with 1976 swH1N1 or 2009 pH1N1. Also, influenza protection studies generally focus on cross‐reactive hemagglutination‐inhibiting antibodies; while hemagglutinin is the primary surface antigen, this fails to account for other influenza viral antigens. Neutralizing antibody may be a better correlate of human protection against pathogenic influenza strains and should be considered for vaccine efficacy.     

The mucosal and systemic immune responses elicited by a chitosan-adjuvanted intranasal influenza H5N1 vaccine

Please cite this paper as: Svindland et al. The mucosal and systemic immune responses elicited by a chitosan‐adjuvanted intranasal influenza H5N1 vaccine. Influenza and Other Respiratory Viruses DOI:10.1111/j.1750‐2659.2011.00271.x. Background Development of influenza vaccines that induce mucosal immunity has been highlighted by the World Health Organisation as a priority (Vaccine 2005;23:1529). Dose‐sparing strategies and an efficient mass‐vaccination regime will be paramount to reduce the morbidity and mortality of a future H5N1 pandemic. Objectives This study has investigated the immune response and the dose‐sparing potential of a chitosan‐adjuvanted intranasal H5N1 (RG‐14) subunit (SU) vaccine in a mouse model. Methods Groups of mice were intranasally immunised once or twice with a chitosan (5 mg/ml)‐adjuvanted SU vaccine [7·5, 15 or 30 μg haemagglutinin (HA)] or with a non‐adjuvanted SU vaccine (30 μg HA). For comparison, another group of mice were intranasally immunised with a whole H5N1 (RG‐14) virus (WV) vaccine (15 μg HA), and the control group consisted of unimmunised mice. Results The chitosan‐adjuvanted SU vaccine induced an immune response superior to that of the non‐adjuvanted SU vaccine. Compared with the non‐adjuvanted SU group, the chitosan‐adjuvanted SU vaccine elicited higher numbers of influenza‐specific antibody‐secreting cells (ASCs), higher concentrations of local and systemic antibodies and correspondingly an improved haemagglutination inhibition (HI) and single radial haemolysis (SRH) response against both the homologous vaccine strain and drifted H5 strains. We measured a mixed T‐helper 1/T‐helper 2 cytokine response in the chitosan‐adjuvanted SU groups, and these groups had an increased percentage of virus‐specific CD4⁺ T cells producing two Thelper 1 (Th1) cytokines simultaneously compared with the non‐adjuvanted SU group. Overall, the WV vaccine induced higher antibody concentrations in sera and an HI and SRH response similar to that of the chitosan‐adjuvanted SU vaccine. Furthermore, the WV vaccine formulation showed a stronger bias towards a T‐helper 1 profile than the SU vaccine and elicited the highest frequencies of CD4⁺ Th1 cells simultaneously secreting three different cytokines (INFγ⁺, IL2⁺ and INFα⁺). As expected, two immunisations gave a better immune response than one in all groups. The control group had very low or not detectable results in the performed immunoassays. Conclusion The cross‐clade serum reactivity, improved B‐ and T‐cell responses and dose‐sparing potential of chitosan show that a chitosan‐adjuvanted intranasal influenza vaccine is a promising candidate vaccine for further preclinical development.     

甲型H1N1流感合并肺炎的临床特点分析

目的通过对甲型H1N1流感合并肺炎的临床特点的分析。方法分析2009年月10月-2010年3月在我院入住的29例甲型H1N1流感合并肺炎患者的临床表现、实验室检查及胸部CT等资料。结果本组病例男性16例,女性13例。3例妊娠,13例合并有基础疾病。所有病例均有流感样前驱症状,呼吸道主要症状为发热、干咳少痰,严重者气短、呼吸困难、咯血。合并细菌感染时咯脓痰。肺部听诊无啰音或少啰音,合并哮喘时有哮鸣音,合并细菌感染时可有湿啰音。实验室检查65%白细胞不高或降低,41%心肌酶升高,58.6%存在低氧血症,35%呼吸衰竭。影像学表现多种多样：65.5%主要为单侧或双侧棉团样、团片样边界模糊高密度渗出影伴肺实变,其内见充气支气管征,病变沿支气管血管束分布。轻症及早期较局限,重症者及晚期病变融合呈双肺多发弥漫性改变。少数呈大叶及小叶性肺炎表现。预后大多良好,病死率6.9%。主要死亡原因为呼吸衰竭及大咯血。结论甲型H1N1流感合并肺炎是以甲型H1N1流感病毒肺炎为主要疾病的多种肺炎构成。甲型H1N1流感病毒肺炎临床表现具有流感病毒肺炎共性特点,其影像学表现有一定特征性。     

Characterization of a whole,inactivated influenza (H5N1) vaccine

Objectives Effective vaccines against the highly pathogenic influenza A/H5N1 virus are being developed worldwide. In Japan, two adjuvanted, inactivated, whole‐virion influenza vaccines were recently developed and licensed as mock‐up, pre‐pandemic vaccine formulations by the Ministry of Health and Labor Welfare of Japan. During the vaccine design and development process, various obstacles were overcome and, in this report, we introduce the non clinical production, immunogenicity data in human and development process that was associated with egg‐derived adjuvanted, inactivated, whole‐virion influenza A (H5N1) vaccine. Design Pilot lots of H5N1 vaccine were produced using the avirulent H5N1 reference strain A/Vietnam/1194/2004 (H5N1) NIBRG‐14 and administered following adsorption with aluminum hydroxide as an adjuvant. Quality control and formulation stability tests were performed before clinical trials were initiated (phase I‐III).
The research foundation for microbial diseases of Osaka University (BIKEN) carried out vaccine production, quality control, stability testing and the phase I clinical trial in addition to overseeing the licensing of this vaccine. Mitsubishi Chemical Safety Institute Ltd. carried out the non clinical pharmacological toxicity and safety studies and the Japanese medical association carried out the phase II/III trials. Phase I‐III trials took place in 2006. Results The production processes were well controlled by established tests and validations. Vaccine quality was confirmed by quality control, stability and pre‐clinical tests, and the vaccine was approved as a mock‐up, pre‐pandemic vaccine by the Ministry of Health and Labor Welfare of Japan. Conclusions Numerous safety and efficacy procedures were carried out prior to the approval of the described vaccine formulation. Some of these procedures were of particular importance e.g., vaccine development, validation, and quality control tests that included strict monitoring of the hemagglutinin (HA) content of the vaccine formulations.
Improving vaccine productivity, shortening the production period and improving antigen yield of the avirulent vaccine strains were also considered important vaccine development criteria.     

新甲型H1N1流感病毒与流感大流行防治研究进展

由于甲型流感病毒基因高度变异的特点,导致其对不同种属宿主亲和力、毒力、免疫原性、抗药性不断发生变化,全球新型流感大流行的风险时刻存在.因此,应加强流感特别是重症流感发病机制和有效干预措施研究,从疫苗研制、开发新型抗病毒药、加强综合治疗,特别是调节宿主免疫反应等多个方面着手,为应对可能爆发的流感大流行提供对策.     

4株野鸭源H5N1亚型禽流感病毒的全基因组鉴定

目的为了解H5N1病毒对基因进化情况,方法对4株野鸭源的H5N1禽流感病毒(A/mallard/Huadong/S/2005(S),A/mallard/Huadong/lk/2005(lk),A/mallard/Huadong/Y/2003(Y),A/mallard/Huadong/hn/2005(hn))进行全基因组测序分析,并鉴定对非免疫麻鸭的毒力。结果4株病毒全基因组序列及其氨基酸序列无明显差异,仅在HA裂解位点区,S和lk病毒的322位是Leu,329位缺失,而Y和hn在此两处分别是Gln和Lys。在遗传距离上,Y和hn比较接近,S与lk比较接近。结论根据H5亚型病毒最新分类规则,S和lk的与clade 2.3.4一致,而Y属于clade 2,hn位于clade 3。对麻鸭致病性试验表明,Y和hn是低致病性病毒,而S及lk是高致病性病毒。HA的322位的Leu和329位缺失可能是clade 2.3.4这类病毒的一个遗传进化标志。     

Long‐term vaccine‐induced heterologous protection against H5N1 influenza viruses in the ferret model

Please cite this paper as: Ducatez et al. (2012) Long‐term vaccine‐induced heterologous protection against H5N1 influenza viruses in the ferret model. Influenza and Other Respiratory Viruses 7(4), 506–512. Background Highly pathogenic H5N1 influenza viruses reemerged in humans in 2003 and have caused fatal human infections in Asia and Africa as well as ongoing outbreaks in poultry. These viruses have evolved substantially and are now so antigenically varied that a single vaccine antigen may not protect against all circulating strains. Nevertheless, studies have shown that substantial cross‐reactivity can be achieved with H5N1 vaccines. These studies have not, however, addressed the issue of duration of such cross‐reactive protection. Objectives To directly address this using the ferret model, we used two recommended World Health Organization H5N1 vaccine seed strains – A/Vietnam/1203/04 (clade 1) and A/duck/Hunan/795/02 (clade 2.1) – seven single, double, or triple mutant viruses based on A/Vietnam/1203/04, and the ancestral viruses A and D, selected from sequences at nodes of the hemagglutinin and neuraminidase gene phylogenies to represent antigenically diverse progeny H5N1 subclades as vaccine antigens. Results All inactivated whole‐virus vaccines provided full protection against morbidity and mortality in ferrets challenged with the highly pathogenic H5N1 strain A/Vietnam/1203/04 5 months and 1 year after immunization. Conclusion If an H5N1 pandemic was to arise, and with the hypothesis that one can extrapolate the results from three doses of a whole‐virion vaccine in ferrets to the available split vaccines for use in humans, the population could be efficiently immunized with currently available H5N1 vaccines, while the homologous vaccine is under production.     

关于WHO 2005年以来高致病性H5N1禽流感病毒的临床和治疗进展

自1997年中国香港特别行政区报道首例人感染H5N1禽流感病毒以来,截至2008年9月10日,这场在禽类中史无前例地持续流行、造成了人类感染并具有高病死率的疫情已经波及到15个国家和地区,总发病387例,死亡245例,其中我国疫情30例,病死率超过60%,防控形势十分严峻.本文旨在整理分析2005年以来世界卫生组织和全球对禽流感病毒临床防治研究成果及人感染H5N1禽流感病例报道的最新信息,力求提供一个防治人禽流感方面的较为全面而清晰的介绍.     

H5N1亚型和H3N2亚型禽流感病毒广西株NS基因的克隆和序列分析

目的对3株分离自广西的禽流感病毒的NS基因进行序列分析,试图从分子水平分析和了解广西地区禽流感病毒NS基因的变异特点和进化规律。方法根据GenBank上登录的已知禽流感病毒的NS基因全序列设计引物,对3株2004-2005年分离自广西的禽流感病毒株A/DK/GX/1566/04(H3N2)、A/Goose/GX/737/05(H5N1)、A/Goose/GX/2775/05(H5N1)的cDNA进行PCR扩增NS基因,并将其克隆到pMD-18T载体上,分别获得全长为855bp、834bp、837bp的NS基因全序列。结果2株H5N1亚型禽流感病毒广西株间NS基因序列的同源性为95.3%,而2株H5N1亚型禽流感病毒株与H3N2亚型禽流感病毒株之间NS基因序列的同源性为94.0%～94.1%。H5N1亚型禽流感病毒广西株的NS基因在第238-253位核苷酸处均发生了15个核苷酸缺失,与我国广东、香港、云南、湖南、湖北及东南亚等不同地区的毒株比较,NS基因的核苷酸同源性为70.2%～97.6%。在NS基因系统进化树中,3株禽流感病毒广西株都属于A亚群,但处在不同的分支上,其中A/DK/GX/1566/04(H3N2)与广东、香港2001-2003年分离株处于同一分支;A/Goose/GX/737/05(H5N1)、A/Goose/GX/2775/05(H5N1)与我国华南地区以及韩国、越南、泰国等亚洲东南部国家的2003年以后的分离株有共同起源。结论3株2004-2005年分离自广西的禽流感病毒株NS基因之间的同源性均高于94%,都属于A亚群。     

H5N1亚型禽流感病毒NS1基因在大肠杆菌和昆虫细胞中的表达

目的构建大肠杆菌表达载体pET-NS1和昆虫杆状病毒转移载体pFast-NS1,将H5N1亚型禽流感病毒NS1基因分别在大肠杆菌和昆虫细胞中进行表达,表达产物用Western blot进行检测分析。方法酶切含有NS1基因的质粒pUC-NS1,分别克隆进大肠杆菌表达载体pET-28a(+)和杆状病毒转移载体pFastBac HT A中,分别获得表达载体pET-NS1和pFast-NS1。将pET-NS1转化大肠杆菌BL21,以异丙基硫代半乳糖苷(IPTG)进行诱导表达;将pFast-NS1转化DH10Bac感受态细胞,提取重组Bac-NS1DNA,以M13为通用引物作PCR鉴定,阳性Bac-NS1用脂质体转染sf9细胞,72h后收集感染细胞。大肠杆菌表达产物和细胞表达产物分别裂解后作SDS-PAGE和Western blot分析。结果成功构建了大肠杆菌表达载体pET-NS1和昆虫杆状病毒转移载体pFast-NS1,大肠杆菌和昆虫细胞中表达的融合蛋白Western blot都能检测到特异性条带。结论NS1基因在大肠杆菌和昆虫细胞中得到成功表达,为获得大量NS1蛋白进行功能研究及抗体制备奠定了基础。     

Phase II,randomized, open,controlled study of AS03‐adjuvanted H5N1 pre‐pandemic influenza vaccine in children aged 3 to 9 years: follow‐up of safety and immunogenicity persistence at 24 months post‐vaccination

Background

An AS03-adjuvanted H5N1 influenza vaccine elicited broad and persistent immune responses with an acceptable safety profile up to 6 months following the first vaccination in children aged 3–9 years.

Methods

In this follow-up of the Phase II study, we report immunogenicity persistence and safety at 24 months post-vaccination in children aged 3–9 years. The randomized, open-label study assessed two doses of H5N1 A/Vietnam/1194/2004 influenza vaccine (1·9 μg or 3·75 μg hemagglutinin antigen) formulated with AS03_A or AS03_B (11·89 mg or 5·93 mg tocopherol, respectively). Control groups received seasonal trivalent influenza vaccine. Safety was assessed prospectively and included potential immune-mediated diseases (pIMDs). Immunogenicity was assessed by hemagglutination-inhibition assay 12 and 24 months after vaccination; cross-reactivity and cell-mediated responses were also assessed. ({"type":"clinical-trial","attrs":{"text":"NCT00502593","term_id":"NCT00502593"}}NCT00502593).

Results

The safety population included 405 children. Over 24 months, five events fulfilled the criteria for pIMDs, of which four occurred in H5N1 vaccine recipients, including uveitis (n = 1) and autoimmune hepatitis (n = 1), which were considered to be vaccine-related. Overall, safety profiles of the vaccines were clinically acceptable. Humoral immune responses at 12 and 24 months were reduced versus those observed after the second dose of vaccine, although still within the range of those observed after the first dose. Persistence of cell-mediated immunity was strong, and CD4⁺ T cells with a T_H1 profile were observed.

Conclusions

Two doses of an AS03-adjuvanted H5N1 influenza vaccine in children showed low but persistent humoral immune responses and a strong persistence of cell-mediated immunity, with clinically acceptable safety profiles up to 24 months following first vaccination.