H7N9灭活疫苗联合MF59佐剂在小鼠体内诱导的长效体液免疫应答

以BALB/c小鼠为模型,探讨H7N9流感病毒灭活疫苗免疫小鼠后所诱导的长效体液免疫应答的动态变化。不同剂量的流感H7N9全病毒灭活疫苗单独或辅以MF59佐剂肌肉注射免疫小鼠一次。连续采集免疫后小鼠15个月的血清,用ELISA方法检测特异性IgG抗体水平,血凝抑制(hemagglutination inhibition,HI)试验和微量中和(microneutralization,MN)试验检测第15个月时的HI抗体和中和抗体效价。实验结果发现,小鼠血清中的特异性IgG抗体水平随时间变化持续缓慢上升,第5个月时达到顶峰,随后略有下降但一直持续平稳状态;IgG抗体滴度与疫苗剂量成正相关,且添加佐剂能提高抗体滴度。HI及MN抗体检测表明,免疫后第15个月产生的抗体能有效中和病毒,且抗体跟疫苗剂量成正比。以上研究表明,H7N9流感病毒灭活疫苗免疫小鼠一次诱导产生的特异性抗体能在较长期内保持比较平稳的抗体滴度,为小鼠提供免疫保护;增加抗原剂量和添加MF59佐剂能增加疫苗特异性抗体水平。该研究为H7N9流感疫苗产生的长期保护效应提供了一定的数据积累和参考。     

人参多糖对新甲型H1N1流感病毒灭活疫苗的免疫增强作用

在流感灭活疫苗中添加佐剂可以提高疫苗的免疫原性,节约抗原用量。一些天然中草药多糖具有潜在的佐剂效应。本文探讨了人参多糖（ginseng polysaccharide,GPS）在新甲型H1N1流感病毒裂解型灭活疫苗中的佐剂效应。将不同剂量GPS与新甲型H1N1流感病毒灭活疫苗混合,共同免疫小鼠一次,通过检测免疫后在小鼠体内诱导产生的疫苗特异性IgM、IgG、IgG1和IgG2a抗体情况来评价GPS作为流感病毒灭活疫苗佐剂的免疫增强效果,并与不添加佐剂的疫苗和加有铝佐剂的疫苗的免疫效果作比较。结果显示,GPS与铝佐剂一样能显著提高和维持疫苗特异性IgG抗体滴度,同时提高IgM抗体水平,其中800μgGPS的佐剂效果最好。因此我们认为GPS可以作为流感病毒灭活疫苗的一种候选佐剂。     

甲型H1N1流感病毒佐剂疫苗小鼠免疫效果

为了探讨甲型H1N1流感病毒氢氧化铝佐剂疫苗对小鼠的免疫作用及对小鼠繁殖性能的影响,以不同剂量、不同免疫程序免疫小鼠后定期采血;用血凝抑制(HI)方法检测血清H1N1流感病毒HI抗体滴度,观察H1N1流感病毒佐剂疫苗对小鼠受孕、产仔、哺乳的影响;比较孕鼠及非孕鼠的抗体滴度,免疫后孕鼠所产仔鼠的体重及H1N1胎传抗体水平。结果显示,以0.5μg组开始的不同剂量、不同免疫程序均可使小鼠产生90倍以上水平的H1N1流感病毒抗体;免疫后的小鼠不影响受孕、产仔及哺乳;仔鼠保护性抗体可持续1个月以上。H1N1流感病毒佐剂疫苗是一种高免疫原性的制剂,用低剂量免疫,即可产生90倍以上持续时间较长的保护性抗体。这种佐剂疫苗对小鼠的繁殖性能无明显影响,免疫产生的抗体经胎盘可垂直传递给仔鼠。     

玉竹多糖对流感病毒裂解疫苗的黏膜佐剂效应

探讨玉竹多糖(Polygonatum odoratun polysaccharides,POP)对流感病毒裂解疫苗黏膜免疫的佐剂效果。以BALB/c小鼠为模型,将H7N9流感病毒裂解疫苗单独或者添加不同剂量的POP(400、600、800、1 000和1200μg)做佐剂一次性滴鼻免疫小鼠,免疫三周后小鼠以致死剂量同源病毒攻击。实验结果表明,当在疫苗中加入1 000μg POP时,与疫苗单独免疫相比,单次滴鼻免疫就明显增强了小鼠抵抗致死量病毒感染的能力,体重丢失减少,肺部残余病毒滴度下降,存活率提高;血清中疫苗特异性IgG抗体和HI抗体以及鼻洗液中SIgA抗体都提高了;IgG亚类抗体检测显示,POP的加入显著增加了IgG1的抗体滴度,但抑制了IgG2a的产生,说明诱导的免疫应答偏向于Th2型。实验表明POP在一定剂量时能增强疫苗诱导的免疫应答,显示出黏膜佐剂效应。     

枸杞多糖对H5N1流感全病毒灭活疫苗的体液免疫增强作用

探讨枸杞多糖(Lycium barbarum polysaccharide,LBP)作为佐剂对H5亚型流感病毒全病毒灭活疫苗的体液免疫增强效果。将流感病毒A/Vietnam/1194/2004(H5N1)灭活疫苗与不同剂量的枸杞多糖混合后以腹腔注射的方式共同免疫小鼠,免疫后三周收集血清用于特异性抗体检测。实验中设立氢氧化铝佐剂组作对照共同评价LBP作为佐剂的免疫增强效果。结果显示,小鼠血清中针对H5灭活疫苗的特异性抗体水平在一定范围内随着LBP剂量的增加而提高。LBP在800μg剂量时血清特异性抗体水平较无佐剂组显著增强,并与氢氧化铝佐剂组大致相当。因而,LBP有可能成为一种有效的流感灭活疫苗免疫佐剂。     

阳离子脂质体DOTAP佐剂对H5N1流感病毒裂解疫苗免疫效果的影响

目的研究阳离子脂质体DOTAP佐剂对H5N1型流感病毒裂解疫苗免疫效果的影响。方法制备DOTAP阳离子脂质体流感病毒裂解疫苗(简称DOTAP流感裂解疫苗),检测其包封率。将BALB/c小鼠分为13组,分别用含0.1、1.0、10.0μg HA/只剂量以DOTAP、Al(OH)3、CPG-ODN为佐剂以及不含佐剂的流感裂解疫苗于0、21天皮下免疫,PBS作为对照组,用血凝抑制试验检测小鼠初次免疫后21、42天血清HI抗体滴度;用ELISA检测初次免疫后21、42天血清特异性IgG抗体、IgG1、IgG2a亚类抗体滴度,以及初次免疫后42天小鼠脾脏单个核细胞体外经抗原刺激后细胞因子IL-2、IL-4、IFN-γ的分泌水平。将BALB/c小鼠分为3组,分别用含不同DOTAP剂量(100、300、600μg/只)的DOTAP流感裂解疫苗于0、21天皮下免疫,检测初次免疫后21、42天小鼠血清HI抗体滴度和IgG抗体滴度。结果 DOTAP流感裂解疫苗粒径在300~400 nm,带正电荷,包封率在50%以上;DOTAP流感裂解疫苗诱导的HI抗体水平和特异性IgG抗体水平均高于流感裂解疫苗,而与铝佐剂和Cp G-ODN佐剂间差异无统计学意义;DOTAP流感裂解疫苗产生的抗体仍以IgG1亚类抗体为主,免疫后42天诱导的IgG2a亚类抗体水平高于流感裂解疫苗和铝佐剂,低于Cp G-ODN佐剂;DOTAP流感裂解疫苗免疫后既分泌高水平Th1型细胞因子IFN-γ,同时也分泌高水平Th2型细胞因子IL-4;不同DOTAP剂量的DOTAP流感裂解疫苗免疫后,其HI抗体滴度和IgG抗体滴度在低、中、高剂量组之间存在明显的量效关系。结论 DOTAP作为H5N1型流感病毒裂解疫苗的佐剂可显著提高流感裂解疫苗的免疫原性,其对体液免疫应答的增强作用不低于铝佐剂和Cp G-ODN佐剂,并具有诱导细胞免疫应答的能力。     

茯苓多糖对流感灭活疫苗的免疫增强作用

探讨茯芩多糖作为流感病毒灭活疫苗佐剂的免疫增强作用.将不同剂量(200 μg或1000 μg)的茯苓多糖分别与低剂量(0.015 μg)或高剂量(1.5 μg)流感病毒(A/PR/8)灭活疫苗共同免疫小鼠,以相应剂量灭活疫苗的单独免疫组、灭活疫苗与氢氧化铝(100 μg)共同免疫组、PBS免疫组作为对照组.一次免疫后3周收集血清,ELISA检测血清中IgG、IgG1和IgG2a的抗体水平;并用致死量(40×LD<,50>)流感病毒(A/PR/8)攻击小鼠,通过观察小鼠的体重丢失率、肺部病毒量、存活率来反映佐剂的免疫增强效果和疫苗的保护作用.结果显示,茯苓多糖能显著增加血清抗体水平,并提高小鼠抗致死量流感病毒攻击的能力,其免疫增强效果与氢氧化铝相当.茯苓多糖可作为一种新型的流感病毒灭活疫苗的免疫佐剂.     

鼠巨细胞病毒 IE-1核酸疫苗和灭活疫苗联合免疫抗病毒感染的研究

巨细胞病毒（Cytomegalovirus,CMV）在人群中感染普遍,对婴幼儿及免疫低下人群中造成严重疾病,目前还没有针对该病毒的商品化疫苗。本研究以BALB/c小鼠为动物模型,探讨鼠巨细胞病毒（Murine cytomega-lovirus,MCMV）IE-1 DNA疫苗和MCMV灭活疫苗联合免疫抗MCMV感染的免疫保护效果。将编码IE-1基因的DNA疫苗（pIE-1）通过肌肉注射辅以电穿孔的方式对小鼠进行初免,再用全病毒灭活疫苗单独或者辅以MF59佐剂进行加强免疫,分别通过ELISA和ELISPOT方法检测到联合免疫策略在免疫组小鼠体内诱导了MC-MV特异性的抗体应答和CTL应答;免疫两周后用3＆#215;LD50致死剂量MCMV感染小鼠,疫苗对小鼠的免疫保护通过检测小鼠存活率、重要器官中的病毒滴度及体重丢失率来评价。结果显示,与单独免疫DNA疫苗或灭活疫苗相比,IE-1 DNA疫苗联合灭活疫苗组能同时在小鼠体内诱导体液免疫和细胞免疫应答,并提供小鼠完全保护;而且MF59辅以灭活疫苗免疫小鼠能增强疫苗的免疫效果。     

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。     

包裹R848的PS仿生脂质体增强H7N9流感病毒灭活疫苗的黏膜免疫效应

流感病毒是呼吸道传染病毒,增强疫苗接种诱导的黏膜免疫反应对于预防流感尤为重要。目前,流感病毒疫苗主要为病毒灭活疫苗,且通过肌肉注射接种,很难诱导产生黏膜免疫。为增强流感病毒灭活疫苗的黏膜免疫效果,本文利用薄膜分散法将与肺表面活性物质（PS）相似的磷脂以及胆固醇制备成包裹瑞喹莫德（R848）的PS脂质体（PS-R848）做黏膜佐剂,将该脂质体与H7N9流感病毒灭活疫苗混合后经鼻腔免疫BALB/c雌性小鼠,检测免疫指标及攻毒后的保护效果。与灭活疫苗添加R848组相比,血清中的免疫球蛋白G(Ig G)以及分型抗体Ig G1与Ig G2a效价、血凝抑制（HI）效价和支气管肺泡灌洗液分泌型Ig A(s Ig A)效价显著提高;诱导的细胞因子干扰素-γ(IFN-γ)、白细胞介素-2(IL-2)与IL-4的分泌量也显著增加。用同亚型流感病毒攻毒小鼠后的肺部病毒滴度较单独疫苗组降低,存活率达到100%,最大体重丢失率较单独疫苗组低且有显著差异。以上结果表明,R848仿生PS-R848作为黏膜佐剂可以有效提高H7N9流感病毒灭活疫苗的免疫效果,为开发流感病毒的黏膜疫苗做了数据积累。     

一种新发现的流感病毒—H5N1

1997年 5月 ,香港首次分离到一种新的Ａ型流感病毒———Ｈ5Ｎ1[1,2 ] ,至该年底共有 18位患者确诊被Ｈ5Ｎ1感染 ,其中 6人死亡[3 ,4 ] 。这是对人类具强毒性的流感病毒新亚型首次被确定。在呼吸系统疾病的病毒中 ,流感病毒因其有抗原性变异而不同于其它病毒 ,特别是它的表面抗原如血凝素 (ｈｅｍａｇｇｌｕｔｉｎｉｎ ,ＨＡ)和神经氨酸酶 (ｎｅｕ ｒａｍｉｎｉｄａｓｅ ,ＮＡ)。主要有两种类型变异 :抗原性漂离(因编码基因点突变的累积导致少量氨基酸改变 )和抗原性转移 (由于不同Ａ型流感病毒亚型的抗原基因的重配引起表面抗原分子广泛的…     

Anti N1 Cross-Protecting Antibodies Against H5N1 Detected in H1N1 Infected People

The A(H5N1) influenza virus pandemic may be the result of avian H5N1 adapting to humans, leading to massive human to human transmission in a context of a lack of pre-existing immunity. As A(H1N1) and A(H5N1) share the same neuraminidase subtype, anti-N1 antibodies subsequent to H1N1 infections or vaccinations may confer some protection against A(H5N1). We analysed, by microneutralization assay, the A/Vietnam/1194/04 (H5N1) anti-N1 cross-protection acquired either during A/NewCaledonia/20/99 (H1N1) infection or vaccination. In cases with documented H1N1 infection, H5N1 cross-protection could be observed only in patients born between 1930 and 1950. No such protection was detected in the sera of vaccinated individuals.     

Influenza A: From highly pathogenic H5N1 to pandemic 2009 H1N1. Epidemiology and clinical features

The last decade has seen the emergence of two new influenza A subtypes and they have become a cause of concern for the global community. These are the highly pathogenic H5N1 influenza A virus (H5N1) and the Pandemic 2009 influenza H1N1 virus. Since 2003 the H5N1 virus has caused widespread disease and death in poultry, mainly in south East Asia and Africa. In humans the number of cases infected with this virus is few but the mortality has been about 60%. Most patients have presented with severe pneumonia and acute respiratory distress syndrome. The second influenza virus, the pandemic H1N1 2009, emerged in Mexico in March this year. This virus acquired the ability for sustained human to human spread and within a few months spread throughout the world and infected over 4 lakh individuals. The symptoms of infection with this virus are similar to seasonal influenza but it currently affecting younger individuals more often. Fortunately the mortality has been low. Both these new influenza viruses are currently circulating and have different clinical and epidemiological characteristics.     

Evolution and control of H5N1

The evolutionary dynamics of the H5N1 virus present a challenge for conventional control measures. Efforts must consider the regional aspects of endemic H5N1.The H5N1 virus has spread across Asia, Europe and Africa, and has infected birds in several endemic areas, including China, Indonesia, Vietnam and Egypt. H5N1 outbreaks pose a massive threat for the poultry industry and, ultimately, for human health [1]. However, the rapid spread of the virus also offers the opportunity to study and learn from its dynamics in the wild. The insights gained should inform new public health policies and preventive actions against a possible pandemic.Progress in influenza research has been impressive. In particular, the application of reverse genetics has led to the identification of mutations and reassortment changes that determine virus virulence. Perhaps the most significant results come from the two now infamous studies, published in Nature and Science, about the generation of recombinant H5N1 viruses that are transmissible in ferrets [2,3]. These advances show that we are steadily elucidating influenza virus at the molecular level. By contrast, our understanding of the dynamics of highly pathogenic influenza virus in the environment remains limited [4,5].Highly pathogenic avian influenza (HPAI) is an important poultry disease. The major reservoir of the virus is wild waterfowl, and infected birds are usually asymptomatic as a result of long-term evolutionary adaptation [1,6]. After transmission from wild waterfowl to poultry, however, avian influenza viruses occasionally become highly pathogenic and can cause mortalities of up to 100% within 48 h of infection. The standard method for controlling an HPAI outbreak is the testing and culling of all infected poultry, and the setting up of a concentric control area around the infected flock.The HPAI H5N1 virus, circulating in Eurasia and Africa, emerged in China around 1997 [1] but it only infected terrestrial birds at the time. Continuous transmission in poultry eventually allowed the virus to evolve, resulting in large outbreaks in China in 2005 with high mortality in wild waterfowl. The virus spread rapidly, probably though migratory birds, to Central Asia, Europe, the Middle East and Africa. Such ‘east to west'' movements of H5N1 viruses over comparably long distances have not since been recorded. Moreover, migrating wildfowl have begun to spread the virus intermittently between Asia and Siberia [7]. This H5N1 lineage is the longest-circulating HPAI virus that has been reported, and it has reached epizootic levels in both domestic and wild bird populations.…the challenge is to understand the evolution of H5N1 to better predict new strains that could become a serious threat for human healthOne of the striking characteristics of the H5N1 lineage, in contrast with other HPAI, is its infectivity toward mammals. H5N1 can be directly transmitted from birds to humans and cause severe disease, although it has a significantly lower transmissibility than seasonal influenza viruses [1]. So far, 608 cases of human H5N1 infections have been reported with 59% mortality [5]. Most human infections have resulted from close contact with H5N1-infected poultry or poultry products, and no sustained human–human transmission has as yet been documented. Nonetheless, a potential H5N1 pandemic remains a great concern for public health.The viruses that caused the five influenza pandemics since 1900 arose by two mechanisms: reassortment among avian, human and swine influenza viruses, and accumulation of mutations in an avian influenza virus [1,8]. Triple reassortment between avian H5N1, swine H3N1 and H1N1 viruses, and double reassortment between avian H5N1 and H9N2 viruses has already been reported in Asia, which raises concerns about new reassortment viruses that could infect humans [9,10]. Meanwhile, research has identified some 80 genetic mutations that could increase infectivity of avian influenza viruses in mammals, and thus potentially facilitate avian influenza evolution to generate a pandemic strain [8,11]. H5N1 strains with some of these mutations have often been found in bird populations [5] and in human H5N1 strains [12]. Indeed, specific mutations that could confer switching in receptor-binding specificity were reported in H5N1-infected patients in Thailand [13]. The two controversial studies published in Nature and Science also showed how a handful of mutations might enable the H5N1 virus to be transmitted between humans [2,3]. Pathogenic variants of the H5N1 virus with a higher pandemic potential could naturally evolve; the challenge is to understand the evolution of H5N1 to better predict new strains that could become a serious threat for human health.…continuous replication of H5N1 virus in Egypt has provided a valuable opportunity to study the impact of genetic evolution on phenotypic variation without reassortmentThe evolutionary dynamics of the Egyptian H5N1 strains provide clues to understanding the pandemic potential of H5N1. The virus was introduced only once in Egypt, in early 2006, and spread among a variety of bird species, including chickens, ducks, turkeys, geese and quail [14]. The virus rapidly evolved to form a phylogenetically distinct clade that has since diverged into multiple sublineages [15]. Thus, continuous replication of H5N1 virus in Egypt has provided a valuable opportunity to study the impact of genetic evolution on phenotypic variation without reassortment.After diversification in local bird populations, some new H5 sublineages have emerged in Egypt with a higher affinity for human-type receptors. Indeed, since their emergence in 2008, almost all human H5N1 strains in Egypt have been phylogenetically grouped into these new sublineages, which can be transmitted to humans with a higher efficacy than other avian influenza viruses. This might explain why, since 2009, Egypt has had the highest number of human cases of H5N1 infection, with more than 50% of the cases worldwide [5]. Fortunately, these Egyptian H5N1 sublineages still do not have binding affinity for receptors in the upper respiratory tract and, therefore, do not sustain transmission in humans. However, it increases the risk of H5N1 variants that are better adapted to humans after viral replication in infected patients.…Egypt is regarded as the country with the highest H5N1 pandemic potential worldwideThe Egyptian H5N1 sublineages are also diversifying antigenically in the field, as some are no longer crossreactive to other co-circulating sublineages [15]. Moreover, faint traces of species-specific evolutionary changes have been detected [16], implying a change in their host species. It shows that the H5N1 virus has undergone significant diversification in Egypt during the past seven years. Of greater concern, however, are Egyptian H5N1 strains that carry mammalian influenza virus type PB2 and have lost the N-linked 158 glycosylation site in the top region of haemagglutinin [15,17], both of which can potentially facilitate viral transmission to humans. The genetic diversification of H5N1 virus in Egypt represents an increasing pandemic potential, and Egypt is regarded as the country with the highest H5N1 pandemic potential worldwide [18].A similar situation exists in other geographical areas. Multiple clades and sublineages of H5N1 are co-circulating in Asia, occasionally enabling reassortment events within and beyond the viral subtypes in the field [19,20]. Several H5N1 strains with enhanced binding affinity to human-type receptors have been reported in Indonesia [12]. Similarly, avian and swine H5N1 strains with an altered receptor-binding preference have been isolated sporadically in Indonesia and Laos [21,22]. As in other areas, distinct groups of H5N1 viruses are circulating amongst themselves and with other avian influenza viruses, generating diverse viral phenotypes in nature. The evolutionary dynamics of H5N1 might even accelerate in the wild. H5N1 viruses diverge genetically in ducks [23]; they can transfer the virus over long distances by migration. Thus, the H5N1 virus has established a complex life cycle in nature with accelerated evolutionary dynamics. The pandemic threat of H5N1 remains a serious concern and might be increasing.Control measures based on isolating and culling are still the gold standard for controlling the early phase of an H5N1 outbreak, and worked against the H5N1 outbreaks in Hong Kong in 1997 and in Thailand in 2004 [4]. However, this measure failed in several countries and made H5N1 endemic. Cross-border circulation of H5N1 further complicates implementation of a classical control strategy based on culling in the infected area.In response, public health officials in several countries, including Egypt and Indonesia, advocate poultry vaccination as a preventive or adjunct control measure [1]. Although vaccination does not completely prevent infections, its proper use can help to control avian influenza outbreaks by reducing virus transmission from infected animals. However, it can also increase vaccine-driven evolution among avian influenza viruses. The endemic status of H5N1, which can cause devastating local epidemics, puts pressure on health officers to use a vaccine or a vaccination strategy that might eventually increase selective pressure and thereby accelerate H5N1 evolution. Given the high mutability and diversity of circulating viruses, it seems best to avoid using a vaccine based on a strain from a different geographical area because there would only be a partial antigen match; such a heterologous vaccine would only be effective in the short term compared with a homologous vaccine. During past control of H5N1 epidemics using imported vaccines, escape mutants have emerged within about a year of the start of vaccination, which made the epidemic even worse [14]. When a vaccination strategy is implemented in an endemic area, the vaccine seed strain should be selected from the same geographical area to try to get the longest possible protection. Vaccine seed virus selection must be periodically revised to produce well-matched and efficacious vaccines.Close communication and workshops hold the greatest potential for controlling the H5N1 virusIn most cases, H5 vaccine for an endemic area comes from a foreign supplier. It would be necessary to enable foreign manufacturers to produce customized H5 vaccines based on epidemic strains from different areas. The best approach might be a plasmid-based reverse genetics system to construct vaccine seed viruses [1]. In egg-based production, which is the basis of flu vaccine production, the seed virus needs to be adapted for high growth. This time-consuming step carries the risk of antigenic changes during vaccine production. Yet, advances in influenza reverse genetics have led to the development of cell culture systems to produce recombinant viruses, which would enable rapid genetic mutagenesis and reassortment. Once reverse genetics generates a virus genome that is well adapted to growth in cell culture, the haemagglutinin and neuraminidase genes can be easily interchanged with those of other influenza viruses. In addition, virus growth in cell culture can shorten production time, which increases the probability of selecting a seed virus antigenically appropriate for the upcoming flu season, and enables a rapid increase in production if necessary [24].A control strategy imposed without consideration of regional customs will not be successfulGiven the zoonotic risks of influenza viruses to both humans and animals, the establishment of a vaccine production system applicable to both human and animal infections is an urgent issue. The capacity of vaccine production needs to be flexible for seasonal, pre-pandemic and pandemic vaccines. Advances in genetic engineering facilitate in vitro control of human- and avian-type receptor expression on cultured cells, which should allow both human and avian influenza viruses to grow in the same system. As vaccine production capacity based on cell culture develops, commercial production of H5N1 vaccines tailored to each geographical area should become possible. In addition, emergency vaccination guidelines, such as pre-pandemic vaccine stockpiling, expanding and accelerating vaccine production and setting vaccination priorities, should be formulated in a business–government partnership, to ensure pandemic preparation. There is no guarantee that the H5N1 virus will be the next pandemic influenza strain. However, exploring options for versatile vaccine manufacturing is a key to controlling zoonotic influenza viruses, including H5N1.The complexity of H5N1 ecology also makes control of endemic H5N1 by vaccination a complex task. The problem is that antigenically different groups of viruses, which are not crossreactive, are often co-circulating in endemic areas. Circulation of viruses in each sublineage is not restricted in terms of geography or host species, which complicates efforts to use a vaccine produced against antigens from a single virus strain [15]. Of greater concern, H5N1 virus infects a variety of bird species [1], which means the vaccination targets have expanded. Bird species differ in their optimal vaccination protocol—for example, the single vaccination used routinely in chickens does not induce an adequate immune response in turkeys, which require multi-dose vaccination at an older age [25]. Furthermore, rearing many bird species and their hybrid breeds in uncontrolled confinement is common in H5N1 endemic countries, especially in rural areas. Therefore, the immunogenicity of existing vaccines is probably inadequate to protect all target species with a single vaccination scheme. Endemic H5N1 already forces public health officials to redefine vaccine development policy to improve both vaccine immunogenicity and vaccination regime.Unfortunately, it is unlikely that science will ever produce a clear answer as to when, where and how the next pandemic influenza virus will emergeToday, there are numerous techniques that could overcome these problems by increasing immunogenic potency and crossreactivity. Innovative vaccine formats—multivalent, universal, nasal and synthetic vaccines—possibly coupled with the use of adjuvants, could improve the global vaccine supply [24]. These new technologies should be applied as soon as possible. Nevertheless, no single technique can probably resolve the underlying complexity of H5N1 dynamics. Over-reliance on vaccination might therefore only worsen the situation. Vaccination can help control endemic H5N1 only when administered as part of an integrated control programme that includes surveillance, culling, restricting host movement and enhanced quarantine and biosecurity.The complex evolutionary dynamics of the H5N1 virus are challenging host species barriers and the ecology brings H5N1 into close proximity to humans [1]. The close link between the virus and humans is a multifaceted phenomenon that can affect health in myriad ways. Thus, we need to redefine control strategies to address the nature of H5N1 dynamics. Surveillance is the basis of infection control in the field. Wild birds and their predators should be included as surveillance targets, thereby expanding the H5N1 host species range. Another drawback is the fact that epidemiological studies focus mainly on virus genotyping. Although genetic data is informative, the diversity of H5N1 viruses makes characterization based only on genetic traits difficult. Characterization of viral phenotypes—antigenicity, receptor-binding preference, pathogenicity and transmissibility—is equally important for investigating the evolutionary dynamics of H5N1 viruses in nature. We would need techniques to determine easily viral phenotype, in particular new rapid diagnostic systems that can be used for timely epidemiological investigations and rapid infection control measures [1]. For example, portable kits that can determine virus receptor specificity would allow field testing of whether a particular avian influenza virus strain has adapted to human-type receptors, thereby adding a new dimension for characterizing and assessing H5N1 outbreaks.Our perception of H5N1 control should change from short-term hunting to long-term controlThe large-scale slaughter of all known and suspected infected birds in H5N1 endemic countries is hugely expensive in terms of execution costs and compensation for lost poultry. Financial assistance from international organizations might be needed to promote the thorough implementation of such a policy. However, H5N1 endemic countries are not all poor nations and some have already built a certain level of technology infrastructure. Thus, transfer of epidemiological skills and concepts to local health officers and scientists is a priority. Overseas collaborations between technologically developed countries and their institutions, and H5N1 endemic countries and their institutions, should be established at a functional level. Close communication and workshops hold the greatest potential for controlling the H5N1 virus. Such projects supported by governments and funding agencies would encourage establishment of bilateral and multilateral relationships between developed countries and the developing countries, which are the epicentres of H5N1 outbreaks. Sharing information about risk and risk management is one of the key methods for reducing the threat of future H5N1 epidemics.Globalization has had major benefits for international travel and trade, and sharing of information. The improvements in information technology have dramatically increased the speed and ease of data flow [26]. Intelligence networks facilitate instantaneous sharing of information and enable global warnings about potential hazards as well as problem-solving. Moreover, collaborative research centres, which have been established on reciprocal bases between scientifically advanced countries and institutes and overseas partner countries and institutes in Asia, Africa and Latin America, are important players in information networking—for instance the Institute Pasteur Network, the Mahidol Oxford Tropical Medicine Research Unit and Japan Initiative for Global Research Network on Infectious Diseases. Linking such laboratory-based networks should be the next step. This would have a profound synergistic effect by maximizing research capacity, human resources and geographic coverage to build a robust global-scale network for infection control.However, regional socio-cultural issues can be a significant concern for virus control wherever accepted values and scientific understanding might differ. Multiple local and regional factors—customs, religion, politics and economics—can affect H5N1 control in an area. Successful implementation of an H5N1 control strategy depends largely on mutual understanding and consideration of local idiosyncrasies.Some examples from Egypt show how regional identity can be closely linked with local public health initiatives. Egypt is an Islamic nation and bird meat is an important source of animal protein, and the only source in some rural areas [14]. A large proportion of Egyptian households in rural areas raise poultry. Although broiler and layer chickens are raised under modern hygienic controls on commercial farms, backyard birds are raised in open uncontrolled farms, leaving them free to interact with other birds (Fig 1A). The poultry meat trade depends mainly on live bird markets in traditional bazaars (Fig 1B), because of a preference for freshly slaughtered poultry. Pigeon towers are built on farms, backyards and roofs throughout villages to raise pigeons for eating. Generally, birds in Egypt are raised in proximity to humans (Fig 1C), which presents an increasing risk of human H5N1 infection in Egypt and establishment of endemic H5N1 in birds nationwide.Open in a separate windowFigure 1Socio-cultural traditions in rearing birds for food in Egypt. (A) Free rearing of backyard birds. (B) Live birds at a downtown market. (C) An example of the intertwined relationship between birds and humans.Such regional identity is inseparable from socio-cultural contexts, making fundamental change virtually impossible. Although there are many scenarios in which a local public health system could be improved by food safety standards and veterinary inspection or short-term closing of live bird markets for virus clearance, H5N1 control measures have to be implemented whilst respecting the intrinsic socio-cultural traditions in the region. A control strategy imposed without consideration of regional customs will not be successful. It is the local health officers and scientists who are best suited to address the enormous complexity and breadth of issues required for H5N1 control. They also experience H5N1 outbreaks in their area on a regular basis and have a great incentive to be involved in infection control. Therefore, it is important to include local expertise in planning and implementing a control strategy.Science in an area such as infectious disease research can no longer be viewed as independent of societal needs…Science is frequently looked at as if it can produce a ‘silver bullet'' to solve every problem. Early success in vaccine and antibiotic development also created a false sense of optimism that scientific methods could eliminate the risk of infection. However, the reality has turned out to be different—some infectious diseases remain uncontrollable and far from eradication. Given the mutable and diversifying nature of avian influenza viruses, there is a significant possibility that different avian influenza subtypes and strains do not follow a single evolutionary pathway. Unfortunately, it is unlikely that science will ever produce a clear answer as to when, where and how the next pandemic influenza virus will emerge. Our perception of H5N1 control should change from short-term hunting to long-term control. The ecology of H5N1 virus brings it into close proximity to humans. The most important strategy is to minimize contact between terrestrial poultry and wild waterfowl to segregate H5N1 in poultry, because H5N1 spread would be uncontrollable if it established a stable equilibrium in waterfowl. For example, H5N1 viruses in Siberia have not been consistently isolated each year from carcasses and faeces of wildfowl migrating from Asia [7]. This implies that H5N1 circulation in the wild still largely depends on occasional introduction from poultry. It is possible that trials to limit H5N1 infection in poultry would lead to a reduction in viral spread and a dwindling evolutionary path in nature. Infection control policy must abandon fixed strategies in favour of flexible ones to keep pace with the evolutionary dynamics of pathogens such as H5N1 (Fig 2).Open in a separate windowFigure 2Changing dynamics of H5N1 virus in the field. Endemic H5N1 virus diversifies in nature, making traditional control measures extremely difficult.Today''s infection control strategy is becoming largely dependent on the reliability and accuracy of information networking. However, the vast flood of scientific information can hide erroneous information and easily mislead the public [26]. Of greater concern, globalization has prompted the centralization of capital and resources, which can lead to an overemphasis on certain research topics. As a consequence, research projects are often short term, without consideration of effects that might have a long-term social impact [27]. This has led to a debate about whether to limit publication of certain types of research or keep scientific information completely accessible. There is probably no easy answer to this. Our global society needs a more mature approach to support research projects that are accurate reflections of societal needs in public health. At the same time, the increasing links between science and society put more pressure on science to play a greater role in society. This is a serious dilemma—how to use science to solve societal problems whilst maintaining its autonomy [27]. Science in an area such as infectious disease research can no longer be viewed as independent of societal needs; we need to establish a balance between the pursuit of independent basic research and its application for solving clinical problems and crises.? Open in a separate windowYohei WatanabeOpen in a separate windowKazuyoshi IkutaOpen in a separate windowMadiha S Ibrahim     

H5N1 avian influenza in China

H5N1 highly pathogenic avian influenza virus was first detected in a goose in Guangdong Province of China in 1996. Multiple genotypes of H5N1 viruses have been identified from apparently healthy waterfowl since 1999. In the years 2004–2008, over 100 outbreaks in domestic poultry occurred in 23 provinces and caused severe economic damage to the poultry industry in China. Beginning from 2004, a culling plus vaccination strategy has been implemented for the control of epidemics. Since then, over 35420000 poultry have been depopulated, and over 55 billion doses of the different vaccines have been used to control the outbreaks. Although it is logistically impossible to vaccinate every single bird in China due to the large poultry population and the complicated rearing styles, there is no doubt that the increased vaccination coverage has resulted in decreased disease epidemic and environmental virus loading. The experience in China suggests that vaccination has played an important role in the protection of poultry from H5N1 virus infection, the reduction of virus load in the environment, and the prevention of H5N1 virus transmission from poultry to humans. Supported by the Key Animal Infectious Disease Control Program of the Ministry of Agriculture, the Chinese National S&T Plan(Grant No. 2004BA519A-57), National Key Basic Research and Development Program of China (Grant Nos: 2005CB523005, 2005CB523200).     

New Orf Virus (Parapoxvirus) Recombinant Expressing H5 Hemagglutinin Protects Mice against H5N1 and H1N1 Influenza A Virus

Previously we demonstrated the versatile utility of the Parapoxvirus Orf virus (ORFV) as a vector platform for the development of potent recombinant vaccines. In this study we present the generation of new ORFV recombinants expressing the hemagglutinin (HA) or nucleoprotein (NP) of the highly pathogenic avian influenza virus (HPAIV) H5N1. Correct foreign gene expression was examined in vitro by immunofluorescence, Western blotting and flow cytometry. The protective potential of both recombinants was evaluated in the mouse challenge model. Despite adequate expression of NP, the recombinant D1701-V-NPh5 completely failed to protect mice from lethal challenge. However, the H5 HA-expressing recombinant D1701-V-HAh5n mediated solid protection in a dose-dependent manner. Two intramuscular (i.m.) injections of the HA-expressing recombinant protected all animals from lethal HPAIV infection without loss of body weight. Notably, the immunized mice resisted cross-clade H5N1 and heterologous H1N1 (strain PR8) influenza virus challenge. In vivo antibody-mediated depletion of CD4-positive and/or CD8-posititve T-cell subpopulations during immunization and/or challenge infection implicated the relevance of CD4-positive T-cells for induction of protective immunity by D1701-V-HAh5n, whereas the absence of CD8-positive T-cells did not significantly influence protection. In summary, this study validates the potential of the ORFV vectored vaccines also to combat HPAIV.     

Computational studies of H5N1 influenza virus resistance to oseltamivir

Influenza A (H5N1) virus is one of the world's greatest pandemic threats. Neuraminidase (NA) inhibitors, oseltamivir and zanamivir, prevent the spread of influenza, but drug‐resistant viruses have reduced their effectiveness. Resistance depends on the binding properties of NA‐drug complexes. Key residue mutations within the active site of NA glycoproteins diminish binding, thereby resulting in drug resistance. We performed molecular simulations and calculations to characterize the mechanisms of H5N1 influenza virus resistance to oseltamivir and predict potential drug‐resistant mutations. We examined two resistant NA mutations, H274Y and N294S, and one non‐drug‐resistant mutation, E119G. Six‐nanosecond unrestrained molecular dynamic simulations with explicit solvent were performed using NA‐oseltamivir complexes containing either NA wild‐type H5N1 virus or a variant. MM_PBSA techniques were then used to rank the binding free energies of these complexes. Detailed analyses indicated that conformational change of E276 in the Pocket 1 region of NA is a key source of drug resistance in the H274Y mutant but not in the N294S mutant.