Influenza: Global surveillance for epidemic and pandemic variants

Influenza viruses, unlike other viruses for which vaccines have been developed, undergo rapid and unpredictable antigenic variation in the hemagglutinin (HA), the surface glycoprotein primarily responsible for eliciting neutralizing antibodies during infection. Because of this antigenic variability and its consequences, the World Health Organization (WHO) in 1947 established an international network of collaborating laboratories to monitor the emergence and spread of new epidemic and pandemic strains of influenza. This network now includes three international WHO collaborating centers and over 100 WHO national collaborating laboratories. The primary purpose of this network is to detect, through laboratory surveillance, the emergence and spread of antigenic variants of influenza that may signal a need to update the formulation of the influenza vaccine. This laboratory surveillance network has provided the strains needed to update the vaccine as well as a repository of influenza viruses useful for studying the antigenic and genetic evolution of this virus. Knowledge gained from molecular studies on the evolution of drift variants and on the emergence of pandemic strains has made influenza a useful model for understanding the potential threat of other emerging or reemerging microbial diseases.     

多维尺度法在上海地区近年来人群甲型流感病毒抗原进化研究中的应用

目的 探讨多维尺度法在近年上海地区人群甲型流感病毒抗原进化过程中的应用,为流感的有效防控提供科学依据.方法 采用非度量多维尺度分析技术以图形化的方式形象地描述了上海地区近年来甲型流感病毒的抗原进化过程,构建了抗原图谱.结果 上海地区H3甲型流感病毒在2005年以后到现在流行株抗原进化相对比较缓慢,而甲型流感H1亚型2008年上半年流行株较之以前的疫苗株和流行株均发生了较大的抗原变异,所以在以后的流感防控工作中要警惕H1亚型的流行.结论 多维尺度分析技术得到的抗原图谱可以更直观地筛选出有流行病学意义的病毒株,为更好地推荐流感疫苗株提供科学依据.     

Antigenic distance measurements for seasonal influenza vaccine selection

Influenza vaccination is one of the major options to counteract the effects of influenza diseases. Selection of an effective vaccine strain is the key to the success of an effective vaccination program since vaccine protection can only be achieved when the selected influenza vaccine strain matches the antigenic variants causing future outbreaks. Identification of an antigenic variant is the first step to determine whether vaccine strain needs to be updated. Antigenic distance derived from immunological assays, such as hemagglutination inhibition, is commonly used to measure the antigenic closeness between circulating strains and the current influenza vaccine strain. Thus, consensus on an explicit and robust antigenic distance measurement is critical in influenza surveillance. Based on the current seasonal influenza surveillance procedure, we propose and compare three antigenic distance measurements, including Average antigenic distance (A-distance), Mutual antigenic distance (M-distance), and Largest antigenic distance (L-distance). With the assistance of influenza antigenic cartography, our simulation results demonstrated that M-distance is a robust influenza antigenic distance measurement. Experimental results on both simulation and seasonal influenza surveillance data demonstrate that M-distance can be effectively utilized in influenza vaccine strain selection.     

Genetic and antigenic characterisation of serotype A FMD viruses from East Africa to select new vaccine strains

Vaccine strain selection for emerging foot-and-mouth disease virus (FMDV) outbreaks in enzootic countries can be addressed through antigenic and genetic characterisation of recently circulating viruses. A total of 56 serotype A FMDVs isolated between 1998 and 2012, from Central, East and North African countries were characterised antigenically by virus neutralisation test using antisera to three existing and four candidate vaccine strains and, genetically by characterising the full capsid sequence data. A Bayesian analysis of the capsid sequence data revealed the viruses to be of either African or Asian topotypes with subdivision of the African topotype viruses into four genotypes (Genotypes I, II, IV and VII). The existing vaccine strains were found to be least cross-reactive (good matches observed for only 5.4–46.4% of the sampled viruses). Three bovine antisera, raised against A-EA-2007, A-EA-1981 and A-EA-1984 viruses, exhibited broad cross-neutralisation, towards more than 85% of the circulating viruses. Of the three vaccines, A-EA-2007 was the best showing more than 90% in-vitro cross-protection, as well as being the most recent amongst the vaccine strains used in this study. It therefore appears antigenically suitable as a vaccine strain to be used in the region in FMD control programmes.     

Influenza A virus (H3N8) in dogs with respiratory disease, Florida

In 2004, canine influenza virus subtype H3N8 emerged in greyhounds in the United States. Subsequent serologic evidence indicated virus circulation in dog breeds other than greyhounds, but the virus had not been isolated from affected animals. In 2005, we conducted virologic investigation of 7 nongreyhound dogs that died from respiratory disease in Florida and isolated influenza subtype H3N8 virus. Antigenic and genetic analysis of A/canine/Jacksonville/2005 (H3N8) and A/canine/Miami/2005 (H3N8) found similarity to earlier isolates from greyhounds, which indicates that canine influenza viruses are not restricted to greyhounds. The hemagglutinin contained 5 conserved amino acid differences that distinguish canine from equine lineages. The antigenic homogeneity of the canine viruses suggests that measurable antigenic drift has not yet occurred. Continued surveillance and antigenic analyses should monitor possible emergence of antigenic variants of canine influenza virus.     

Characterization of antigenically and genetically similar influenza C viruses isolated in Japan during the 1999-2000 season

Between October 1999 and May 2000, a total of 28 strains of influenza C virus were isolated in four Japanese prefectures: Yamagata, Miyagi, Saitama and Hiroshima. Antigenic analysis showed that the 28 isolates were divided into three distinct antigenic groups, and viruses belonging to different antigenic groups were co-circulating in each of the four prefectures. Phylogenetic analysis of the seven protein genes demonstrated that the viruses having a similar genome composition spread in various areas of Japan during the same period. Furthermore, phylogenetic analysis showed that most of the influenza C viruses isolated in various areas of the world between the 1970s and 1980s were closely related to the contemporary Japanese viruses in all gene segments. These observations suggest that the influenza C viruses cause epidemics in some communities during the same season and that antigenically and genetically similar influenza C viruses spread throughout Japan and may be circulating worldwide.     

Relationship between haemagglutination inhibition titre and immunity to influenza in ferrets

Our understanding of the antigenic evolution of the human influenza virus is chiefly derived from experiments in which serum from influenza infected ferrets is tested against panels of virus isolates in the haemagglutination inhibition (HI) assay. The interpretation of these results has been much aided by the development of antigenic mapping techniques, which suppose that the antigenic distance between two different influenza viruses is directly proportional to their fold-difference in titre in this assay. Yet, antigenic distance is not necessarily the same as cross-protection, and high levels of protection have been observed in humans against strains to which they have low HI titres. However, no study has previously addressed the relationship between HI titre and cross-protection in ferrets: the standard animal model. This study fills this gap by analysing published data where pre-challenge HI titres are available for individual ferrets, and post-challenge outcomes have been recorded. Ultimately, this work confirms that it is the absolute, rather than relative, HI titre that determines the extent of immunity and that there is a threshold HI titre beyond which ferrets are completely protected from infection. Nevertheless, this titre is much higher in ferrets than has been suggested for humans. Further, we are consequently able to show that using distance between strains within an antigenic map to predict cross-protection between influenza viruses can be misleading.     

The selection of naturally stable candidate foot-and-mouth disease virus vaccine strains for East Africa

Foot-and-mouth disease (FMD) is a global burden on the livestock industry. The causative agent, FMD virus (FMDV), is highly infectious and exists in seven distinct serotypes. Vaccination remains the most effective control strategy in endemic regions and current FMD vaccines are made from inactivated preparations of whole virus. The inherent instability of FMDV and the emergence of new strains presents challenges to efficacious vaccine development. Currently, vaccines available in East Africa are comprised of relatively historic strains with unreported stabilities. As an initial step to produce an improved multivalent FMD vaccine we have identified naturally stable East African FMDV strains for each of the A, O, SAT1 and SAT2 serotypes and investigated their potential for protecting ruminants against strains that have recently circulated in East Africa. Interestingly, high diversity in stability between and within serotypes was observed, and in comparison to non-African A serotype viruses reported to date, the East African strains tested in this study are less stable. Candidate vaccine strains were adapted to propagation in BHK-21 cells with minimal capsid changes and used to generate vaccinate sera that effectively neutralised a panel of FMDV strains selected to improve FMD vaccines used in East Africa. This work highlights the importance of combining tools to predict and assess FMDV vaccine stability, with cell culture adaptation and serological tests in the development of FMD vaccines.     

Identification of two antigenically and genetically distinct lineages of H3N8 equine influenza virus in Sweden.

Four Swedish strains of equine H3N8 influenza virus isolated from outbreaks during the last 4 years were characterized. Antigenic typing using monoclonal antibodies raised against a variety of H3N8 strains showed that the viruses are heterogeneous, the 1993 isolate being closely related to the 1991 Swedish isolate TAB/91 and the other three isolates from 1994 and 1996 being more closely related to each other. This pattern is reflected in the phylogenetic data calculated from nucleotide sequencing of the haemagglutinin genes. H3N8 equine influenza can be seen to be evolving in two distinct lineages, one European and one American. The 1993 isolate is closely related to the European lineage and is the most recent Swedish strain of this lineage to be isolated. The 1994 and 1996 isolates fit into the American lineage, which contains recent isolates from the United States and also Britain. These results indicate that American-type H3N8 viruses have become endemic in Sweden and, in light of the antigenic differences which can be observed between viruses belonging to the two lineages, we believe that equine influenza virus vaccines should be updated with an American-type virus strain.     

2003年广东省流感病毒的流行概况及特征

目的了解2003年广东省流感病毒的流行特点和抗原变异情况。方法根据广东地区流感监测资料进行分析；用交叉血凝抑制试验检测病毒的抗原性；用逆转录-聚合酶链反应扩增病毒HA1基因，纯化产物进行核苷酸序列测定。将序列和Genebank中相关序列进行比较，用MegAlign软件绘制种系发生树。结果全年分离到510株流感病毒，其中H3N2亚型流感481株，占92．4％；乙型流感29株，占7．6％。实验室证实流感爆发52起，其中50起暴发是由H3N2亚型流感病毒引起，2起暴发是由乙型流感病毒引起。H3N2亚型流感病毒抗原性和疫苗侏A／Panama／2007／99（H3N2）有所不同。类似干A／Fujian／411／02（H3N2）。在HAl区氨基酸序列上，和疫苗株A／Panama／2007／99（H3N2）同源性为92．1％，存在有25个氨基酸差异。28株乙型流感病毒属于Victoria系，抗原性类似疫苗侏B／HongKong／330／01，1株乙型病毒属于Yamagata系，抗原性不同干B／sichuan／379／99。结论2003年广东省流感活动比2002年要强；同时流行着甲型（H3N2）和2个谱系的乙型流感病毒，H3N2亚型毒侏是优势侏，抗原性发生了漂移。     

Effectiveness of seasonal influenza vaccine in Australia, 2015: An epidemiological,antigenic and phylogenetic assessment

BackgroundA record number of laboratory-confirmed influenza cases were notified in Australia in 2015, during which type A(H3) and type B Victoria and Yamagata lineages co-circulated. We estimated effectiveness of the 2015 inactivated seasonal influenza vaccine against specific virus lineages and clades.MethodsThree sentinel general practitioner networks conduct surveillance for laboratory-confirmed influenza amongst patients presenting with influenza-like illness in Australia. Data from the networks were pooled to estimate vaccine effectiveness (VE) for seasonal trivalent influenza vaccine in Australia in 2015 using the case test-negative study design.ResultsThere were 2443 eligible patients included in the study, of which 857 (35%) were influenza-positive. Thirty-three and 19% of controls and cases respectively were reported as vaccinated. Adjusted VE against all influenza was 54% (95% CI: 42, 63). Antigenic characterisation data suggested good match between vaccine and circulating strains of A(H3); however VE for A(H3) was low at 44% (95% CI: 21, 60). Phylogenetic analysis indicated most circulating viruses were from clade 3C.2a, rather than the clade included in the vaccine (3C.3a). VE point estimates were higher against B/Yamagata lineage influenza (71%; 95% CI: 57, 80) than B/Victoria (42%, 95% CI: 13, 61), and in younger people.ConclusionsOverall seasonal vaccine was protective against influenza infection in Australia in 2015. Higher VE against the B/Yamagata lineage included in the trivalent vaccine suggests that more widespread use of quadrivalent vaccine could have improved overall effectiveness of influenza vaccine. Genetic characterisation suggested lower VE against A(H3) influenza was due to clade mismatch of vaccine and circulating viruses.     

A bivalent influenza VLP vaccine confers complete inhibition of virus replication in lungs

The conventional egg-grown influenza vaccines are trivalent. To test the feasibility of using multivalent influenza virus-like particles (VLPs) as an alternative influenza vaccine, we developed cell-derived influenza VLPs containing the hemagglutinin (HA) of the H1 subtype virus A/PR/8/34 or the H3 subtype virus A/Aichi/2/68 (X31). Mice immunized intramuscularly with bivalent influenza VLPs containing H1 and H3 HAs induced neutralizing activities against the homologous and closely related H1N1 strains A/PR/8/34 and A/WSN/33 as well as the H3N2 strains A/Aichi/2/68 (X31) and A/Hong Kong/68, but not the A/Philippines/2/82 strain isolated 14 years later. HA sequence and structure analysis indicated that antigenic distance could be a major factor in predicting cross-protection by VLP vaccines. The bivalent influenza VLP vaccine demonstrated advantages in broadening the protective immunity after lethal challenge infections when compared to a monovalent influenza VLP vaccine. High levels of the inflammatory cytokine IL-6 were observed in na?ve or unprotected immunized mice but not in protected mice upon lethal challenge. These results indicate that multivalent influenza VLP vaccines can be an effective antigen for developing safe and alternative vaccine to control the spread of influenza viruses.     

Molecular characterization of serotype Asia-1 foot-and-mouth disease viruses in Pakistan and Afghanistan; emergence of a new genetic Group and evidence for a novel recombinant virus

Foot-and-mouth disease (FMD) is endemic in Pakistan and Afghanistan. The FMD virus serotypes O, A and Asia-1 are responsible for the outbreaks in these countries. Diverse strains of FMDV, even within the same serotype, co-circulate. Characterization of the viruses in circulation can facilitate appropriate vaccine selection and tracing of outbreaks. The present study characterized foot-and-mouth disease serotype Asia-1 viruses circulating in Pakistan and Afghanistan during the period 1998-2009. Phylogenetic analysis of FMDV type Asia-1 revealed that three different genetic Groups of serotype Asia-1 have circulated in Pakistan during this time. These are Group-II, -VI and, recently, a novel Group (designated here as Group-VII). This new Group has not been detected in neighbouring Afghanistan during the study period but viruses from Groups I and -II are in circulation there. Using near complete genome sequences, from FMD viruses of serotypes Asia-1 and A that are currently circulating in Pakistan, we have identified an interserotypic recombinant virus, which has the VP2-VP3-VP1-2A coding sequences derived from a Group-VII Asia-1 virus and the remainder of the genome from a serotype A virus of the A-Iran05(AFG-07) sub-lineage. The Asia-1 FMDVs currently circulating in Pakistan and Afghanistan are not efficiently neutralized by antisera raised against the Asia-1/Shamir vaccine strain. Thus, new Asia-1 vaccine strains may be required to block the spread of the current Asia-1 viruses.     

Antigenic analysis of SAT 2 serotype foot-and-mouth disease virus isolates from Zimbabwe using monoclonal antibodies.

This paper compares strains of foot-and-mouth disease (FMD) serotype SAT (South African Territories) 2 viruses isolated from Zimbabwe and other African countries using monoclonal antibodies (MAb). A sandwich-ELISA was used to examine the relative binding of anti-SAT 2 MAb to the various viruses. The MAb-binding profiles of viruses isolated from field samples were compared using hierarchical cluster analysis. Viruses were obtained from game animals, mainly African buffalo (Syncerus caffer) which is the natural host and reservoir for SAT serotypes in Africa, and from cattle showing clinical signs of FMD, as well as from animals suspected of carrying the virus subclinically. Some isolates have been adapted for use as vaccine strains. The results showed that most of the Zimbabwe isolates collected between 1989 and 1992 were an antigenically closely-related group. Although differences were observed between Zimbabwe isolates collected between 1989 and 1992 and those collected in 1987, there was no correlation with the different MAb binding patterns within the 1987 group and the epidemiological information received from the field. Similar profiles were observed for many SAT 2 viruses, including viruses isolated over a 50-year period and from geographically distant areas. This indicates an inherent stability in antigenic profiles of SAT 2 viruses. The MAb panel was capable of assessing antigenic variation, since very different profiles were obtained for some isolates. The work also allowed comparison and characterization of anti-type SAT 2 MAb from different laboratories. The findings are discussed with reference to selection of vaccine strains.     

Development of Eurasian H7N7/PR8 high growth reassortant virus for clinical evaluation as an inactivated pandemic influenza vaccine

Avian-to-human transmission of the high pathogenicity (HP) H7N7 subtype avian influenza viruses in the Netherlands during 2003 caused zoonotic infections in 89 people, including a case of acute fatal respiratory distress syndrome. Public health emergency preparedness against H7N7 avian influenza viruses with pandemic potential includes the development of vaccine candidate viruses. In order to develop a high growth reassortant vaccine candidate virus, low pathogenicity (LP) A/mallard/Netherlands/12/2000 (H7N3) and A/mallard/Netherlands/2/2000 (H10N7) strains were selected as donors of the H7 haemagglutinin and N7 neuraminidase genes, respectively. The donor viruses exhibited high amino acid sequence homology with the surface glycoproteins of A/Netherlands/219/03 H7N7 virus (NL219), an isolate recovered from the fatal human case. Adhering to the seasonal influenza vaccine licensure regulations, we generated a H7N7/PR8 reassortant containing desired surface glycoprotein genes from the mallard viruses and internal genes of A/Puerto Rico/8/34 human vaccine strain (H1N1). Antigenic analysis revealed that the vaccine candidate virus confers broad antigenic cross-reactivity against contemporary Eurasian and the North American H7 subtype human isolates. Mice immunized with formalin inactivated (FI) H7N7/PR8 whole virus vaccine with or without aluminum hydroxide adjuvant conferred clinical protection from mortality and reduced pulmonary replication of the NL219 challenge virus. The FI H7N7/PR8 whole virus vaccine also afforded cross-protection in mice at the pulmonary level against antigenically distinct North American LP A/Canada/444/04 (H7N3) human isolate. The vaccine candidate virus satisfied the agricultural safety requirements for chickens, proved safe in mice, and has entered in phase-I human clinical trial in the United States.     

Influenza Vaccine Research funded by the European Commission FP7-Health-2013-Innovation-1 project

Due to influenza viruses continuously displaying antigenic variation, current seasonal influenza vaccines must be updated annually to include the latest predicted strains. Despite all the efforts put into vaccine strain selection, vaccine production, testing, and administration, the protective efficacy of seasonal influenza vaccines is greatly reduced when predicted vaccine strains antigenically mismatch with the actual circulating strains. Moreover, preparing for a pandemic outbreak is a challenge, because it is unpredictable which strain will cause the next pandemic. The European Commission has funded five consortia on influenza vaccine development under the Seventh Framework Programme for Research and Technological Development (FP7) in 2013. The call of the EU aimed at developing broadly protective influenza vaccines. Here we review the scientific strategies used by the different consortia with respect to antigen selection, vaccine delivery system, and formulation. The issues related to the development of novel influenza vaccines are discussed.     

季节性流感监测中病毒分子变异分析的意义

目的探讨季节性流感监测的同时,针对人群中循环的优势毒株开展基因变异分析的意义。方法以2007年深圳市流感监测系统累积的数据为基础,按周分析不同监测医院流感样病例占门诊和急诊就诊者的比例,并对病毒型别进行常规分离鉴定,对优势毒株扩增HA1区基因片段,并进行基因变异分析。结果深圳市流感样病例2007年流感样病例（ILI）比例为6．67％,以低年龄为高发人群;按市、区医院和社区健康服务中心等分别分析,ILI的比例按周分布一致,呈现明显的夏季高峰,最活跃季节在5～7月;ILI暴发疫情比散发病例提早2周出现。流感病毒分离率为11．63％,优势株为H3N2亚型（占59．5％）,B型次之（占37．0％）。H3N2亚型流感病毒HA1基因进化分析分成两支,WHO当年疫苗株和我国代表株仅与1～4月毒株接近,而与5月之后分离株产生了遗传距离,虽然还不能确定新的变异株出现,但提示A／sz／9／2007和A／sz122／2007两个分离株值得高度关注。B型流感病毒HAl基因进化分析发现,2007年以Yamagata系最活跃。结论季节性流感监测中开展病毒分子变异分析,可以进一步查明人群中所发生的优势毒株的特性,便于流行病学综合分析。     

Estimating the protection afforded by foot-and-mouth disease vaccines in the laboratory

Foot-and-mouth disease (FMD) vaccines must be carefully selected and their application closely monitored to optimise their effectiveness. This review covers serological techniques for FMD vaccine quality control, including potency testing, vaccine matching and post-vaccination monitoring. It also discusses alternative laboratory procedures, such as antigen quantification and nucleotide sequencing, and briefly compares the approaches for FMD with those for measuring protection against influenza virus, where humoral immunity is also important. Serology is widely used to predict the protection afforded by vaccines and has great practical utility but also limitations. Animals differ in their responses to vaccines and in the protective mechanisms that they develop. Antibodies have a variety of properties and tests differ in what they measure. Antibody-virus interactions may vary between virus serotypes and strains and protection may be affected by the vaccination regime and the nature and timing of field virus challenge. Finally, tests employing biological reagents are difficult to standardise, whilst cross-protection data needed for test calibration and validation are scarce. All of this is difficult to reconcile with the desire for simple and universal criteria and thresholds for evaluating vaccines and vaccination responses and means that oversimplification of test procedures and their interpretation can lead to poor predictions. A holistic approach is therefore recommended, considering multiple sources of field, experimental and laboratory data. New antibody avidity and isotype tests seem promising alternatives to evaluate cross-protective, post-vaccination serological responses, taking account of vaccine potency as well as match. After choosing appropriate serological tests or test combinations and cut-offs, results should be interpreted cautiously and in context. Since opportunities for experimental challenge studies of cross-protection are limited and the approaches incompletely reflect real life, more field studies are needed to quantify cross-protection and its correlation to in vitro measurements.     

Analysis of antigenic variation in equine 2 influenza A viruses

Influenza outbreaks involving viruses of the H3N8 subtype (equine 2) often occur in vaccinated horses. For this reason, a series of influenza viruses of the H3N8 subtype were examined to determine if antigenic variation could be detected in isolates during the period 1963-81. Antigenic analyses with post-infection ferret sera and monoclonal antibodies showed that the haemagglutinins of recent isolates were antigenically distinguishable from the prototype A/eq/Miami/1/63 and that antigenically distinguishable groups of equine 2 viruses co-circulate in the horse population. Based on these studies, it is recommended that a recent equine strain, A/equine/Fontainebleu/1/79 or A/equine/Kentucky/1/81, serve as an additional prototype strain for this subtype.     

Genetic and antigenic relationship of foot–and–mouth disease virus serotype O isolates with the vaccine strain O1/BFS

Foot–and–mouth disease serotype O viruses (FMDV/O) are responsible for the most outbreaks in FMD endemic countries. O1/BFS is one of the recommended FMD/O vaccine strains by World Reference Laboratory for FMD. In the current study, FMDV/O1 BFS vaccine strain and serotype O field isolates (45) were analyzed phylogenetically and antigenically to gain more insight into the genetic and antigenic characteristics of the vaccine strain and field isolates.O1/BFS showed similarity with 89% of the field isolates using a virus neutralization test (VNT). The P1 region encoding the FMDV capsid was sequenced and analysed for 46 strains of FMDV/O. Phylogenetic analysis showed these viruses originated from five continents and covered eight of 11 reported topotypes. Five isolates that demonstrated low antigenic similarities with O1/BFS were analyzed for their antigenic variation at the known neutralizing antigenic sites. Three of the five isolates demonstrated unique amino acid substitutions at various antigenic sites. No unique amino acid substitutions were observed for the other two unmatched isolates. Positively selected residues were identified on the surface of the FMD virus capsid supporting that it is important to continuously monitor field isolates for their antigenic and phenotypic changes.In conclusion, the vaccine strain O1/BFS is likely to confer protection against 89% of the 45 FMDV/O isolates based on VNT. Thus O1/BFS vaccine strain is still suitable for use in global FMD serotype O outbreak control. Combining data from phylogenetic, molecular and antigenic analysis can provide improvements in the process of vaccine selection.