Recombinant HA1 produced in E. coli forms functional oligomers and generates strain-specific SRID potency antibodies for pandemic influenza vaccines

Vaccine production and initiation of mass vaccination is a key factor in rapid response to new influenza pandemic. During the 2009-2010 H1N1 pandemic, several bottlenecks were identified, including the delayed availability of vaccine potency reagents. Currently, antisera for the single-radial immunodiffusion (SRID) potency assay are generated in sheep immunized repeatedly with HA released and purified after bromelain-treatment of influenza virus grown in eggs. This approach was a major bottleneck for pandemic H1N1 (H1N1pdm09) potency reagent development in 2009. Alternative approaches are needed to make HA immunogens for generation of SRID reagents in the shortest possible time. In this study, we found that properly folded recombinant HA1 globular domain (rHA1) from several type A viruses including H1N1pdm09 and two H5N1 viruses could be produced efficiently using a bacterial expression system and subsequent purification. The rHA1 proteins were shown to form functional oligomers of trimers, similar to virus derived HA, and elicited high titer of neutralizing antibodies in rabbits and sheep. Importantly, the immune sera formed precipitation rings with reference antigens in the SRID assay in a dose-dependent manner. The HA contents in multiple H1N1 vaccine products from different manufacturers (and in several lots) as determined with the rHA1-generated sheep sera were similar to the values obtained with a traditionally generated sheep serum from NIBSC. We conclude that bacterially expressed recombinant HA1 proteins can be produced rapidly and used to generate SRID potency reagents shortly after new influenza strains with pandemic potential are identified.     

Lessons from pandemic influenza A(H1N1): the research-based vaccine industry's perspective

As A(H1N1) influenza enters the post-pandemic phase, health authorities around the world are reviewing the response to the pandemic. To ensure this process enhances future preparations, it is essential that perspectives are included from all relevant stakeholders, including vaccine manufacturers. This paper outlines the contribution of R&D-based influenza vaccine producers to the pandemic response, and explores lessons that can be learned to improve future preparedness.The emergence of 2009 A(H1N1) influenza led to unprecedented collaboration between global health authorities, scientists and manufacturers, resulting in the most comprehensive pandemic response ever undertaken, with a number of vaccines approved for use three months after the pandemic declaration. This response was only possible because of the extensive preparations undertaken during the last decade.During this period, manufacturers greatly increased influenza vaccine production capacity, and estimates suggest a further doubling of capacity by 2014. Producers also introduced cell-culture technology, while adjuvant and whole virion technologies significantly reduced pandemic vaccine antigen content. This substantially increased pandemic vaccine production capacity, which in July 2009 WHO estimated reached 4.9 billion doses per annum. Manufacturers also worked with health authorities to establish risk management plans for robust vaccine surveillance during the pandemic. Individual producers pledged significant donations of vaccine doses and tiered-pricing approaches for developing country supply.Based on the pandemic experience, a number of improvements would strengthen future preparedness. Technical improvements to rapidly select optimal vaccine viruses, and processes to speed up vaccine standardization, could accelerate and extend vaccine availability. Establishing vaccine supply agreements beforehand would avoid the need for complex discussions during a period of intense time pressure.Enhancing international regulatory co-operation and mutual recognition of approvals could accelerate vaccine supply, while maintaining safety standards. Strengthening communications with the public and healthcare workers using new approaches and new channels could help improve vaccine uptake. Finally, increasing seasonal vaccine coverage will be particularly important to extend and sustain pandemic vaccine production capacity.     

Inactivated and adjuvanted whole-virion clade 2.3.4 H5N1 pre-pandemic influenza vaccine possesses broad protective efficacy against infection by heterologous clades of highly pathogenic H5N1 avian influenza virus in mice

In this study, we evaluated the immunogenicity and protective efficacy of a candidate attenuated H5N1 pre-pandemic influenza vaccine of clade 2.3.4, rgAnhui, which was reverse genetically generated from highly virulent A/Anhui/01/2005 (H5N1) wild-type virus. When a low-dose antigen (0.3 μg HA) vaccine was combined with aluminum hydroxide adjuvant, virus neutralization and anti-HA IgG antibodies induced in the sera of vaccinated mice showed similar levels as those in mice vaccinated with non-adjuvanted high-dose antigen (3 μg HA) vaccine. Serum antibodies had broad reactivity against highly pathogenic H5N1 viruses of both homologous and heterologous clades. All mice vaccinated with adjuvanted and non-adjuvanted rgAnhui vaccines at low and high antigen doses survived, without any significant weight loss, lethal challenge infection with homologous clade 2.3.4 viruses, including antigenic variant virus and heterologous clade 2.1.3. Mice vaccinated with low-dose antigen without adjuvant, however, exhibited 20% and 60% survival rates against clade 1 and clade 2.2 viruses, respectively; but, addition of adjuvant improved these rates to 80% and 100%, respectively. The data strongly suggest that aluminum hydroxide-adjuvanted rgAnhui vaccine can elicit broad cross-reactive and protective immunities against homologous and heterologous clades, and that the rgAnhui vaccine is a useful pre-pandemic H5N1 vaccine.     

Immune response of single dose vaccination against 2009 pandemic influenza A (H1N1) in the Taiwanese elderly

We conducted a multi-center, randomized and laboratory-blinded clinical trial with subgroup analyses, involving adults aged greater than 60 years old (range 61–86 years old), to investigate the immunogenicity and the potential factors affecting the immune response of a monovalent, unadjuvanted, inactivated, split-virus vaccine. A total of 107 subjects were randomized to receive 15 and 30 μg of hemagglutinin antigen in a 1:1 ratio. The immunogenicity was detected through hemagglutination inhibition (HAI) test of serum obtained before and 3 weeks after vaccination. By 3 weeks after vaccination, HAI titer ≧1:40 was observed in 75.5% and 81.1% of participants receiving 15 and 30 μg of hemagglutinin antigen, respectively. Positive seroconversion was observed in 71.7% and 81.1% of recipients of the 15 and the 30 μg, respectively. The GMTs increased by a factor of 10.7 and 17.4 in the groups of 15 and 30 μg, respectively. This study indicated that one dose of 15 μg hemagglutinin antigen without adjuvant induced protective immune response in the majority of elderly. Multivariate logistic regression analyses showed that gender, age and diabetes were statistically significant factors affecting the seroprotection rate (p = 0.04, 0.01 and 0.01, respectively) and seroconversion rate (p = 0.01, 0.01 and 0.01, respectively).     

Efficacy of live attenuated vaccines against 2009 pandemic H1N1 influenza in ferrets

The advent of the H1N1 influenza pandemic (pH1N1) in 2009 triggered the rapid production of pandemic influenza vaccines, since seasonal influenza vaccines were expected and demonstrated not to provide significant cross-protection against the newly emerged pandemic virus. To increase vaccine production capacity and further evaluate the effectiveness of different candidate pandemic influenza vaccines, the World Health Organization stimulated the evaluation of different vaccination concepts including the use of live attenuated influenza vaccines (LAIVs). Therefore, we have immunized ferrets intranasally with a single dose of pH1N1-LAIV from different manufacturers. They all induced adequate serum HI antibody titers in the ferrets and protected them against intratracheal wild-type pH1N1 virus challenge: pH1N1 virus replication in the upper respiratory tract and lungs was reduced and no disease signs or severe broncho-interstitial pneumonia were observed in any of the vaccinated ferrets. These data together with the relatively efficient production process emphasize the potential of the LAIV concept for pandemic preparedness.     

Optimal vaccination strategies for 2009 pandemic H1N1 and seasonal influenza vaccines in humans

A randomized clinical trial was conducted to assess whether the immunogenicity of seasonal and pandemic (H1N1/09) influenza vaccines is affected by the order of vaccine administration. 151 healthy adult volunteers were randomized into three groups. All groups received one dose (15 μg haemagglutinin) each of a pandemic H1N1 vaccine and a seasonal trivalent vaccine. Group 1 received the pandemic H1N1 vaccine first, followed by the seasonal vaccine 21 days later. Group 2 received vaccinations in vice versa and Group 3 received both vaccines simultaneously. Post-vaccination blood samples were collected to determine the immunogenicity by hemagglutination-inhibition (HI), microneutralization (MN), and B cell ELISPOT assays. All three vaccination strategies were well-tolerated and generated specific immune responses. However, we found a significant difference in magnitude of antibody responses to pandemic H1N1 between the three groups. Pre- or co-vaccination with the seasonal flu vaccine led to a significant reduction by 50% in HI titre to pandemic H1N1 virus after pandemic vaccination. Pre- or co-vaccination of pandemic H1N1 vaccine had no effect on seasonal flu vaccination. MN and ELISPOT assays showed a similar effect. Vaccination with pandemic H1N1 vaccine first is recommended to avoid an associated inhibitory effect by the seasonal trivalent flu vaccine. Clinical_Trials identifier: NCT01008137.     

An adjuvanted pandemic influenza H1N1 vaccine provides early and long term protection in health care workers

Mass vaccination was the most effective prophylaxis for protecting the population during the influenza H1N1 pandemic. We have evaluated the tolerability, immunogenicity and kinetics of the antibody response to a monovalent oil-in-water (AS03) adjuvanted human pandemic split influenza A/California/7/2009 H1N1 (3.75 μg haemagglutinin) vaccine in health care workers. Vaccination elicited a rapid and early protective level of haemagglutination inhibition antibody from 6 to 7 days post vaccination, and by 14 to 21 days post vaccination, up to 98% of vaccinees had protective antibody titres which persisted for at least 3 months in 84-92% of subjects. A rapid induction of protective antibody is important in reducing community spread of pandemic influenza and in helping maintain the integrity of the health care system during the pandemic.     

Evidence for differing evolutionary dynamics of A/H5N1 viruses among countries applying or not applying avian influenza vaccination in poultry

Highly pathogenic avian influenza (HPAI) H5N1 (clade 2.2) was introduced into Egypt in early 2006. Despite the control measures taken, including mass vaccination of poultry, the virus rapidly spread among commercial and backyard flocks. Since the initial outbreaks, the virus in Egypt has evolved into a third order clade (clade 2.2.1) and diverged into antigenically and genetically distinct subclades. To better understand the dynamics of HPAI H5N1 evolution in countries that differ in vaccination policy, we undertook an in-depth analysis of those virus strains circulating in Egypt between 2006 and 2010, and compared countries where vaccination was adopted (Egypt and Indonesia) to those where it was not (Nigeria, Turkey and Thailand). This study incorporated 751 sequences (Egypt n = 309, Indonesia n = 149, Nigeria n = 106, Turkey n = 87, Thailand n = 100) of the complete haemagglutinin (HA) open reading frame, the major antigenic determinant of influenza A virus. Our analysis revealed that two main Egyptian subclades (termed A and B) have co-circulated in domestic poultry since late 2007 and exhibit different profiles of positively selected codons and rates of nucleotide substitution. The mean evolutionary rate of subclade A H5N1 viruses was 4.07 × 10⁻³ nucleotide substitutions per site, per year (HPD 95%, 3.23-4.91), whereas subclade B possessed a markedly higher substitution rate (8.87 × 10⁻³; 95% HPD 7.0-10.72 × 10⁻³) and a stronger signature of positive selection. Although the direct association between H5N1 vaccination and virus evolution is difficult to establish, we found evidence for a difference in the evolutionary dynamics of H5N1 viruses among countries where vaccination was or was not adopted. In particular, both evolutionary rates and the number of positively selected sites were higher in virus populations circulating in countries applying avian influenza vaccination for H5N1, compared to viruses circulating in countries which had never used vaccination. We therefore urge a greater consideration of the potential consequences of inadequate vaccination on viral evolution.     

Vaccine-mediated protection of pigs against infection with pandemic H1N1 2009 swine influenza A virus requires a close antigenic match between the vaccine antigen and challenge virus

Swine influenza A virus (SwIV) infection has considerable economic and animal welfare consequences and, because of the zoonotic potential, can also have public health implications. The 2009 pandemic H1N1 ‘swine-origin’ infection is now endemic in both pigs and humans. In Europe, avian-like H1_avN1, human-like H1_huN2, human-like swine H3N2 and, since 2009, pandemic H1N1 (pH1N1) lineage viruses and reassortants, constitute the dominant subtypes. In this study, we used a swine pH1N1 challenge virus to investigate the efficacy of whole inactivated virus vaccines homologous or heterologous to the challenge virus as well as a commercial vaccine. We found that vaccine-mediated protection was most effective when vaccine antigen and challenge virus were homologous and correlated with the specific production of neutralising antibodies and a cellular response to the challenge virus. We conclude that a conventional whole inactivated SwIV vaccine must be antigenically matched to the challenge strain to be an effective control measure.     

Comparative efficacy of North American and antigenically matched reverse genetics derived H5N9 DIVA marker vaccines against highly pathogenic Asian H5N1 avian influenza viruses in chickens

Highly pathogenic (HP) H5N1 avian influenza has become endemic in several countries in Asia and Africa, and vaccination is being widely used as a control tool. However, there is a need for efficacious vaccines preferably utilizing a DIVA (differentiate infected from vaccinated animals) marker strategy to allow for improved surveillance of influenza in vaccinated poultry. Using a reverse genetics approach, we generated Asian rgH5N9 vaccine strain deriving the hemagglutinin gene from A/chicken/Indonesia/7/2003 (H5N1) with modification of the cleavage site to be low pathogenic (LP) and N9 neuraminidase gene from the North American LP A/turkey/Wisconsin/1968 (H5N9) virus. The recombinant rgH5N9, A/turkey/Wisconsin/1968 (H5N9) A/chicken/Hidalgo/232/1994 (H5N2), and wild type HP A/chicken/Indonesia/7/2003 (H5N1) viruses were used to prepare inactivated oil-emulsified whole virus vaccines. Two weeks after vaccination, chickens were challenged with either Asian HP H5N1 viruses, A/chicken/Indonesia/7/2003 (W.H.O. clade 2.1) or A/chicken/Supranburi Thailand/2/2004 (W.H.O. clade 1.0). The H5 HA1 of the North American vaccine strains exhibited 12% amino acid differences including amino acid changes in the major antigenic sites as compared to the Asian HP H5N1 challenge viruses, serologically exhibited substantial antigenic difference, but still provided 100% protection from mortality. However, challenge virus shedding was significantly higher in chickens immunized with antigenically distinct American lineage vaccines as compared to the antigenically matched Asian rgH5N9 and the wild type Asian H5N1 vaccine. The antibody response to the heterologous subtype neuraminidase proteins were discriminated in vaccinated and infected chickens using a rapid fluorescent 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid sodium salt as substrate for neuraminidase inhibition assay. This study demonstrates the value of using a vaccine containing antigenically matched H5 hemagglutinin for control of HP H5N1 avian influenza in poultry and the potential utility of a heterologous neuraminidase as a DIVA marker.     

Safety,immunogenicity and efficacy of poxvirus-based vector vaccines expressing the haemagglutinin gene of a highly pathogenic H5N1 avian influenza virus in pigs

This study investigates the safety, immunogenicity and efficacy of different pox-vector vaccines expressing the haemagglutinin of a highly pathogenic (HP) H5N1 avian influenza virus (AIV) (A/chicken/Indonesia/7/03) in pigs. Pigs were vaccinated twice, with a 4-week interval, with a fowlpox (TROVAC^®), a canarypox (ALVAC^®), or a vaccinia (NYVAC) vector vaccine combined with an oil-in-water adjuvant, with the unadjuvanted NYVAC, or left unvaccinated. Six weeks after the second vaccination, all pigs were challenged intra-tracheally with low pathogenic (LP) H5N2 AIV A/chicken/Belgium/150/99. Sera were examined in haemagglutination inhibition (HI) tests against the H5N1 AIV from which the vaccine haemagglutinin derived, the challenge virus and the human A/Vietnam/1194/04 HPAIV. After challenge pigs were compared for H5N2 virus replication in the trachea and 4 lung lobes at 24 or 72 h post-challenge. Vaccination was well tolerated by all animals. Antibody titres peaked 2 weeks after the second vaccination and were 2- to 4-fold higher against the vaccine virus than heterologous H5 viruses. The NYVAC and ALVAC adjuvanted vaccines consistently induced higher antibody titres than TROVAC or NYVAC without adjuvant. Following challenge, the H5N2 challenge virus was isolated from all unvaccinated pigs, while 19 out of 21 vaccinates showed complete virological protection. Pox-vector vaccines were safe, immunogenic and efficacious against challenge with a heterologous H5 AIV, offering an alternative to classical inactivated vaccines. It remains to be seen whether they would protect against a swine-adapted H5 virus, which may replicate 100–1000 times better than our challenge virus.     

A systematic review and meta-analysis of cross-reactivity of antibodies induced by oil-in-water emulsion adjuvanted influenza H5N1 virus monovalent vaccines

BackgroundCross-clade immunogenic stockpiled H5N1 vaccines may decrease the morbidity and transmission of infection during the initial phase of influenza pandemic. Meta-analysis of cross-reactive antibodies induced by oil-in-water emulsion adjuvanted (OWEA) influenza H5N1 virus monovalent vaccines with circulating heterologous H5N1 virus strains, isolated from human infections was performed.MethodsLiterature search of MEDLINE, EMBASE, Web of Knowledge, The Cochrane Library, ClinicalTrials.gov, and International Standard Randomised Controlled Trial Number registry was conducted up through December 1, 2015. Methodologically qualified studies were included for (1) use of two doses of licensed OWEA (AS03 or MF59) egg-derived, inactivated influenza H5N1 virus monovalent vaccine, (2) participant age between 18 and 64 years, and (3) evaluation of immunogenicity outcome for one or more subclade. Meta-analysis assessed the cross-reactivity of antibodies elicited by clade 1 adjuvanted vaccine strain against clade 2.1 virus strain (A/Vietnam/1194/2004 vs. A/Indonesia/05/2005); and separately against clade 2.2 virus strain (A/Vietnam/1194/2004 vs. A/turkey/Turkey/1/05); and clade 2.1 adjuvanted vaccine strain against clade 1 virus strain (A/Indonesia/05/2005 vs. A/Vietnam/1194/2004). Quantitative publication bias and influence analysis was conducted to evaluate potential impact of unpublished or new studies on the robustness of meta-analysis.ResultsOf 960 articles, 53 qualified for quality assessment and 15 studies met the inclusion criteria. All assessed clade pairs elicited cross-reactive antibodies (clade 1 against clade 2.1 and 2.2; clade 2.1 against clade 1, 2.2, and 2.3). Heterologous strains of same sub-clade are likely to elicit higher cross-reactive antibodies.ConclusionsOWEA influenza H5N1 virus monovalent vaccines exhibit broad cross-clade immunogenicity, a desired feature for vaccine stockpiling not yet demonstrated by unadjuvanted vaccines. In case of an impending H5N1 virus pandemic, stockpiled OWEA influenza H5N1 virus monovalent vaccines may allow population priming that could slow down the course of pandemic and could offer additional time needed for development of an effective strain specific vaccine supply.     

北京市首例人禽流感H5N1病例的感染来源调查

目的 调查北京市首例人禽流感(H5N1)病例的感染来源.方法 通过对患者家属和其他关键人物访谈,检测患者标本以及流行病学关联的环境标本,对患者可能的感染来源进行追踪调查.结果 该患者在其发病前5天曾经接触过宰杀的鸭子(购自河北省燕郊某市场活禽摊位),采集该摊位及邻近活禽摊位的环境标本10份,经PCR方法检测有6份标本为H5N1亚型病毒核酸阳性,并通过鸡胚分离出5株H5N1亚型病毒.将从环境标本中分离的H5N1亚型病毒与患者体内分离的H5N1亚型病毒(禽源、属于Clade 2.3.4)进行全基因组序列比对,氨基酸同源性高达99.8%～100%.结论 流行病学和病原学双重证据表明,北京市首例人禽流感H5N1病例的感染来源为接触携带H5N1亚型禽流感病毒的鸭子.     

Safety and immunogenicity of prepandemic H5N1 influenza vaccines: A systematic review of the literature

A systematic review was performed to assess the safety and immunogenicity of the prepandemic H5N1 influenza vaccines licensed so far. A bibliographic search according to the COSI protocol was carried out and 8 of 235 potentially relevant publications were selected. Quality assessment was defined with both CASP and Jadad checklists. Taken together, the results from the present systematic review suggest that the inactivated split-virion formulation that includes a low antigen dose (3.8 μg) and an oil-in-water emulsion-based adjuvant, represents the best option in the case of a pandemic, due to its antigen-sparing capacity and its favorable safety profile.     

一例孕妇感染禽流感病毒(H5N1)死亡的调查

目的探讨2005年11月安徽省铜陵市人民医院报告的一例不明原因肺炎孕妇死亡的病因.方法访谈病例发病前后的相关知情人,查阅临床病志,对病例和病死禽的密切接触者进行医学观察.采集病例的气管吸取物,应用逆转录-聚合酶链反应（RT-PCR）和实时荧光定量PCR法,检测A/H5N1亚型特异的核酸片段,接种SPF鸡胚进行病毒分离.结果病例发病前4天有明确的病死禽直接暴露史,病例和病死禽的122名密切接触者经10天医学观察均健康.病毒性肺炎伴血白细 胞和淋巴细胞计数的显著下降,迅速进展的呼吸衰竭和多器官衰竭是其主要临床特征.病例的气管吸取物A/H5N1的HA和NA基因阳性,分离到的A/Anhui/1/2005（H5N1）的8个基因节段均为禽源的,血凝素抗原的受体链接肽仍为多个碱性氨基酸.结论该病例为首次报告感染禽流感病毒（H5N1）发病、死亡的孕妇.孕妇妊娠期间的免疫耐受状态可能是导致其感染,引起相应临床表现和进程的重要原因.     

Evaluation of conserved and variable influenza antigens for immunization against different isolates of H5N1 viruses

The combination of rapid evolution and high mortality in human cases of infections has raised concerns that the H5N1 avian influenza virus may become a new, possibly severe, pandemic virus. Vaccination is likely to be the most efficient strategy to mitigate the impact of the next influenza pandemic. The present study evaluates B and T cell immune responses generated by the H5N1 viral antigens, hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), or the M2 ion channel in parallel, expressed from a DNA vaccine vehicle. Protection studies of immunized mice challenged with 100 LD50 of homologous or heterologous H5N1 viruses indicate that HA afforded better protection than the NA, NP or M2 DNA vaccines. The antibody response was also higher in HA-vaccinated mice as determined by hemagglutination inhibition (HI) and neutralizing antibodies (NAB) assays. Interestingly, the T cell response was higher against HA than against NA, NP or M2 and was detectable at low doses of the DNA–HA vaccine capable of inducing complete protection, despite the absence of a detectable B cell response. This study emphasizes the need to evaluate the relationship between both arms of the adaptive immune responses in regards to protective efficacy against influenza virus.     

Improved neutralizing antibody response in the second season after a single dose of pandemic (H1N1) 2009 influenza vaccine in HIV-1-positive adults

Neutralizing antibody titers were determined before and after a single dose of pandemic (H1N1) 2009 influenza vaccine in HIV-1-positive Japanese adults in the first season of the pandemic and in those in the second season who had already received the vaccine in the first season. The antibody response rate at 2-month post-vaccination increased significantly from 49.0% (50/102, 95%CI: 39.0-59.1%) in the 2009/2010 season to 66.7% (42/63, 95%CI: 53.7-78.1%) in the 2010/2011 season. Geometric mean antibody titers (fold dilution) at baseline, at 2 months, and at 4 months also increased significantly from 4.4 (95%CI: 3.3-5.7), 19.0 (95%CI: 13.4-26.8) and 13.7 (95%CI: 9.3-20.2), respectively, in the 2009/2010 season to 8.3 (95%CI: 5.8-11.7), 47.0 (95%CI: 32.2-68.6) and 38.2 (95%CI: 23.8-61.4), respectively, in the 2010/2011 season. Although the vaccine response was low in the first season, it was improved in the second season.     

中国大陆首例人感染禽流感病毒(H5N1)的调查与确认

目的确认2005年10月湖南省湘潭县发生的一起家庭聚集性（姐弟二人）不明原因肺炎病例的病因.方法访谈病例发病前后的相关知情人,重现病家暴露环境及发病时间序列,查阅临床病志,对病例和病死禽的密切接触者进行医学观察.采集病例咽拭子,应用逆转录-聚合酶链反应（RT-PCR）和实时荧光定量PCR法,检测A/H5N1亚型特异的核酸片段,接种SPF鸡胚进行病毒分离.应用马血球血凝抑制试验和微量中和试验检测患者急性期和恢复期血清抗A/H5特异性抗体滴度.结果姐（病例1）弟（病例2）二人分别在其家鸡开始死亡2天和4天后出现发热和肺炎症状,病例1死于急性呼吸窘迫综合征和多器官功能衰竭,病例2痊愈.病例1病后第8天的血清A/H5抗体阴性,病例2急性期和恢复期血清抗体滴度呈4倍以上升高.192名密切接触者中,仅1名（接诊过病例1的医生）出现上呼吸道症状,但血清标本经微量中和试验阴性.结论病例2为中国大陆第一例感染禽流感病毒（H5N1）的确诊病例,病例1为临床诊断病例.二者发病最可能是感染了相同家庭环境中病死禽传播的H5N1病毒,调查中未发现两病例之间有互相传播的证据.     

Challenges of global surveillance during an influenza pandemic

Surveillance is an essential foundation for monitoring and evaluating any disease process, and is especially critical when new disease agents appear. The H1N1 influenza pandemic of 2009 tested the capacities of countries to detect, assess, notify and report events as required by the 2005 International Health Regulations (IHR). As detailed in the IHR, the World Health Organization drew on official reports from Member States as well as unofficial sources (e.g., media alerts) to quickly report and disseminate information about the appearance of the novel influenza virus. The pre-existing Global Influenza Surveillance Network for virological surveillance also provided crucial information for rapid development of a vaccine and for detection of changes in the virus. However, the pandemic also highlighted a number of shortcomings in global epidemiological surveillance for respiratory disease. These included the lack of standards for reporting illness, risk factor and mortality data, and a mechanism for systematic reporting of epidemiological data. Such measures would have facilitated direct comparison of data between countries and improved timely understanding of the characteristics and impact of the pandemic. This paper describes the surveillance strategies in place before the pandemic and the methods that were used at global level to monitor the pandemic. Enhancements of global surveillance are proposed to improve preparedness and response for similar events in the future.     

Evaluation of multivalent H2 influenza pandemic vaccines in mice

Subtype H2 Influenza A viruses were the cause of a severe pandemic in the winter of 1957. However, this subtype no longer circulates in humans and is no longer included in seasonal vaccines. As a result, individuals under 50 years of age are immunologically naïve. H2 viruses persist in aquatic birds, which were a contributing source for the 1957 pandemic, and have also been isolated from swine. Reintroduction of the H2 via zoonotic transmission has been identified as a pandemic risk, so pre-pandemic planning should include preparation and testing of vaccine candidates against this subtype. We evaluated the immunogenicity of two inactivated, whole virus influenza vaccines (IVV) in mice: a monovalent IVV containing human pandemic virus A/Singapore/1/1957 (H2N2), and a multivalent IVV containing human A/Singapore/1/1957, avian A/Duck/HongKong/319/1978 (H2N2), and swine A/Swine/Missouri/2124514/2006 (H2N3) viruses. While both vaccines induced protective immunity compared to naïve animals, the multivalent formulation was advantageous over the monovalent in terms of level and breadth of serological responses, neutralization of infectious virus, and reduction of clinical disease and respiratory tissue replication in mice. Therefore, multivalent pandemic H2 vaccines containing diverse viruses from animal reservoirs, are a potential option to improve the immune responses in a pre-pandemic scenario where antigenic identity cannot be predicted.