Inferring the antigenic epitopes for highly pathogenic avian influenza H5N1 viruses

The evasion of influenza virus from host immune surveillance is mainly mediated through its surface protein hemagglutinin (HA), the main component of influenza vaccine. Thus, identification of influenza virus antigenic epitopes on HA can not only help us understand the molecular mechanisms of viral immune escape but also facilitate vaccine strain selection. Despite previous efforts, there is a lack of systematic definition of the antigenic epitopes for the highly pathogenic avian influenza (HPAI) H5N1 viruses. In this study, we infer the HA antigenic epitopes for H5N1 viruses by integrating the antigenic sites mapped from the HA of human influenza H3N2 viruses, the sites which were reported to be associated with immune escape in H5 viruses and the mutation hotspot sites identified in the evolutionary history of HPAI H5N1 viruses. We show that these inferred antigenic epitopes play significant roles in antigenic variation of HPAI H5N1 viruses. Based on inferred antigenic epitopes, we further develop a computational method to effectively predict antigenic variants for HPAI H5N1 viruses (available at http://biocloud.hnu.edu.cn/predict/html/index.html). Therefore, our work has not only inferred the antigenic epitopes for HPAI H5N1 viruses but also provided an effective computational method to assist vaccine recommendations for protection against the deadly bird flu.     

Host range restriction and pathogenicity in the context of influenza pandemic

Influenza A viruses cause pandemics at random intervals. Pandemics are caused by viruses that contain a hemagglutinin (HA) surface glycoprotein to which human populations are immunologically naive. Such an HA can be introduced into the human population through reassortment between human and avian virus strains or through the direct transfer of an avian influenza virus to humans. The factors that determine the interspecies transmission and pathogenicity of influenza viruses are still poorly understood; however, the HA protein plays an important role in overcoming the interspecies barrier and in virulence in avian influenza viruses. Recently, the RNA polymerase (PB2) protein has also been recognized as a critical factor in host range restriction, while the nonstructural (NS1) protein affects the initial host immune responses. We summarize current knowledge of viral factors that determine host range restriction and pathogenicity of influenza A viruses.     

Evolution of H5N1 avian influenza viruses in Asia

An outbreak of highly pathogenic avian influenza A (H5N1) has recently spread to poultry in 9 Asian countries. H5N1 infections have caused > or =52 human deaths in Vietnam, Thailand, and Cambodia from January 2004 to April 2005. Genomic analyses of H5N1 isolates from birds and humans showed 2 distinct clades with a nonoverlapping geographic distribution. All the viral genes were of avian influenza origin, which indicates absence of reassortment with human influenza viruses. All human H5N1 isolates tested belonged to a single clade and were resistant to the adamantane drugs but sensitive to neuraminidase inhibitors. Most H5N1 isolates from humans were antigenically homogeneous and distinct from avian viruses circulating before the end of 2003. Some 2005 isolates showed evidence of antigenic drift. An updated nonpathogenic H5N1 reference virus, lacking the polybasic cleavage site in the hemagglutinin gene, was produced by reverse genetics in anticipation of the possible need to vaccinate humans.     

Antigenic sites of H1N1 influenza virus hemagglutinin revealed by natural isolates and inhibition assays

The antigenic sites of hemagglutinin (HA) are crucial for understanding antigenic drift and vaccine strain selection for influenza viruses. In 1982, 32 epitope residues (called laboratory epitope residues) were proposed for antigenic sites of H1N1 HA based on the monoclonal antibody-selected variants. Interestingly, these laboratory epitope residues only cover 28% (23/83) mutation positions for 9 H1N1 vaccine strain comparisons (from 1977 to 2009). Here, we propose the entropy and likelihood ratio to model amino acid diversity and antigenic variant score for inferring 41 H1N1 HA epitope residues (called natural epitope residues) with statistically significant scores according to 1572 HA sequences and 197 pairs of HA sequences with hemagglutination inhibition (HI) assays of natural isolates. By combining both natural and laboratory epitope residues, we identified 62 (11 overlapped) residues clustered into five antigenic sites (i.e., A-E) which are highly correlated to the antigenic sites of H3N2 HA. Our method recognizes sites A, B and C as critical sites for escaping from neutralizing antibodies in H1N1 virus. Experimental results show that the accuracies of our models are 81.2% and 82.2% using 41 and 62 epitope residues, respectively, for predicting antigenic variants on 197 paring HA sequences. In addition, our model can detect the emergence of epidemic strains and reflect the genetic diversity and antigenic variant between the vaccine and circulating strains. Finally, our model is theoretically consistent with the evolution rates of H3N2 and H1N1 viruses and is often consistent to WHO vaccine strain selections. We believe that our models and the inferred antigenic sites of HA are useful for understanding the antigenic drift and evolution of influenza A H1N1 virus.     

Prediction of influenza B vaccine effectiveness from sequence data

Influenza is a contagious respiratory illness that causes significant human morbidity and mortality, affecting 5–15% of the population in a typical epidemic season. Human influenza epidemics are caused by types A and B, with roughly 25% of human cases due to influenza B. Influenza B is a single-stranded RNA virus with a high mutation rate, and both prior immune history and vaccination put significant pressure on the virus to evolve. Due to the high rate of viral evolution, the influenza B vaccine component of the annual influenza vaccine is updated, roughly every other year in recent years. To predict when an update to the vaccine is needed, an estimate of expected vaccine effectiveness against a range of viral strains is required. We here introduce a method to measure antigenic distance between the influenza B vaccine and circulating viral strains. The measure correlates well with effectiveness of the influenza B component of the annual vaccine in humans between 1979 and 2014. We discuss how this measure of antigenic distance may be used in the context of annual influenza vaccine design and prediction of vaccine effectiveness.     

Recent zoonoses caused by influenza A viruses

Influenza is a highly contagious, acute illness which has afflicted humans and animals since ancient times. Influenza viruses are part of the Orthomyxoviridae family and are grouped into types A, B and C according to antigenic characteristics of the core proteins. Influenza A viruses infect a large variety of animal species, including humans, pigs, horses, sea mammals and birds, occasionally producing devastating pandemics in humans, such as in 1918, when over twenty million deaths occurred world-wide. The two surface glycoproteins of the virus, haemagglutinin (HA) and neuraminidase (NA), are the most important antigens for inducing protective immunity in the host and therefore show the greatest variation. For influenza A viruses, fifteen antigenically distinct HA subtypes and nine NA subtypes are recognised at present; a virus possesses one HA and one NA subtype, apparently in any combination. Although viruses of relatively few subtype combinations have been isolated from mammalian species, all subtypes, in most combinations, have been isolated from birds. In the 20th Century, the sudden emergence of antigenically different strains in humans, termed antigenic shift, has occurred on four occasions, as follows, in 1918 (H1N1), 1957 (H2N2), 1968 (H3N2) and 1977 (H1N1), each resulting in a pandemic. Frequent epidemics have occurred between the pandemics as a result of gradual antigenic change in the prevalent virus, termed antigenic drift. Currently, epidemics occur throughout the world in the human population due to infection with influenza A viruses of subtypes H1N1 and H3N2 or with influenza B virus. The impact of these epidemics is most effectively measured by monitoring excess mortality due to pneumonia and influenza. Phylogenetic studies suggest that aquatic birds could be the source of all influenza A viruses in other species. Human pandemic strains are thought to have emerged through one of the following three mechanisms: genetic reassortment (occurring as a result of the segmented genome of the virus) of avian and human influenza A viruses infecting the same host direct transfer of whole virus from another species the re-emergence of a virus which may have caused an epidemic many years earlier. Since 1996, the viruses H7N7, H5N1 and H9N2 have been transmitted from birds to humans but have apparently failed to spread in the human population. Such incidents are rare, but transmission between humans and other animals has also been demonstrated. This has led to the suggestion that the proposed reassortment of human and avian viruses occurs in an intermediate animal with subsequent transference to the human population. Pigs have been considered the leading contender for the role of intermediary because these animals may serve as hosts for productive infections of both avian and human viruses and, in addition, the evidence strongly suggests that pigs have been involved in interspecies transmission of influenza viruses, particularly the spread of H1N1 viruses to humans. Global surveillance of influenza is maintained by a network of laboratories sponsored by the World Health Organization. The main control measure for influenza in human populations is immunoprophylaxis, aimed at the epidemics occurring between pandemics.     

Genetic reassortment in pandemic and interpandemic influenza viruses

The human influenza pandemics of 1957 and 1968 were caused by reassortant viruses that possessed internal gene segments from avian and human strains. Whether genetic reassortment of human and avian influenza viruses occurs during interpandemic periods and how often humans are infected with such reassortants is not known. To provide this information, we used dot-blot hybridization, partial nucleotide sequencing and subsequent phylogenetic analysis to examine the 6 internal genes of 122 viruses isolated in humans between 1933 and 1992 primarily from Asia, Europe, and the Americas. The internal genes of A/New Jersey/11/76 isolated from a human fatality at Fort Dix, New Jersey in 1976 were found to be of porcine origin. Although none of the geographically and temporally diverse collection of 122 viruses was an avian-human or other reassortant, cognizance was made of the fact that there were two isolates from children from amongst 546 influenza A isolates obtained from The Netherlands from 1989–1994 which were influenza reassortants containing genes of avian origin, viruses which have infected European pigs since 1983–1985. Thus, genetic reassortment between avian and human influenza strains does occur in the emergence of pandemic and interpandemic influenza A viruses. However, in the interpandemic periods the reassortants have no survival advantage, and the circulating interpandemic influenza viruses in humans do not appear to accumulate avian influenza virus genes.     

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.     

Possible correlation between low antigenic drift of A(H1N1) influenza viruses and induction of HI antibodies

This study examined whether, during a seven-year period of low A(H1N1) influenza virus antigenic drift (1988–1989 and 1994–1995, winters), humoral antibody response of elderly volunteers to influenza vaccines could suggest a lack of antibody pressure for drift. In all the years studied A/Taiwan/1/86, the A(HIN1) vaccine component, had a low ability to induce protective hemagglutination-inhibiting (HI) antibody titres ( 1:40). However a similar low immunogenicity was found for some of the different A(H3N2) strain variants of influenza virus, co-circulating in the same period and showing a regular extent of antigenic variations.Although our data could be at least in part explained by the type of study population (elderly and repeatedly vaccinated), postepidemic serological studies did not evidence a consistently lower ability in mounting protective immune response in elderly people as compared with younger against the influenza strains studied. Therefore, our present results did not exclude a true low immunogenicity of A/Taiwan and of some A(H3N2) influenza strains, circulating in the winters examined. This suggests that, besides the necessity to evade prior immunity, additional factors could influence the frequency of influenza viruses antigenic drifts.Abbreviation HI hemagglutination inhibiting     

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.     

Directed selection of amino acid changes in the influenza hemagglutinin and neuraminidase affecting protein antigenicity

Influenza virus hemagglutinin (HA) and neuraminidase (NA) proteins elicit protective antibody responses and therefore, are used as targets for vaccination, especially the HA protein. However, these proteins are subject to antigenic drift, decreasing vaccine efficacy, and few to no studies have analyzed antigenic variability of these proteins by growing the viruses under immune pressure provided by human sera. In this work, we show that after growing different influenza virus strains under immune pressure, the selection of amino acid changes in the NA protein is much more limited than the selection in the HA protein, suggesting that the NA protein could remain more conserved under immune pressure. Interestingly, all the mutations in the HA and NA proteins affected protein antigenicity, and many of the selected amino acid changes were located at the same positions found in viruses circulating. These studies could help to inform HA and NA protein residues targeted by antibody responses after virus infection in humans and are very relevant to update the strains used for influenza virus vaccination each year and to improve the currently available vaccines.     

Vaccination with a soluble recombinant hemagglutinin trimer protects pigs against a challenge with pandemic (H1N1) 2009 influenza virus

In 2009 a new influenza A/H1N1 virus strain (“pandemic (H1N1) 2009”, H1N1v) emerged that rapidly spread around the world. The virus is suspected to have originated in swine through reassortment and to have subsequently crossed the species-barrier towards humans. Several cases of reintroduction into pigs have since been reported, which could possibly create a reservoir for human exposure or ultimately become endemic in the pig population with similar clinical disease problems as current swine influenza strains. A soluble trimer of hemagglutinin (HA), derived from the H1N1v, was used as a vaccine in pigs to investigate the extent to which this vaccine would be able to protect pigs against infection with the H1N1v influenza strain, especially with respect to reducing virus replication and excretion. In a group of unvaccinated control pigs, no clinical symptoms were observed, but (histo)pathological changes consistent with an influenza infection were found on days 1 and 3 after inoculation. Live virus was isolated from the upper and lower respiratory tract, with titres up to 10⁶ TCID₅₀ per gram of tissue. Furthermore, live virus was detected in brain samples. Control pigs were shedding live virus for up to 6 days after infection, with titres of up to 10⁵ TCID₅₀ per nasal or oropharyngeal swab. The soluble H1N1v HA trimer diminished virus replication and excretion after a double vaccination and subsequent challenge. Live virus could not be detected in any of the samples taken from the vaccinated pigs. Vaccines based on soluble HA trimers provide an attractive alternative to the current inactivated vaccines.     

铜川市2013-2015年A型流感病毒HA基因特性研究

目的 应用生物信息学数据库和工具,了解2013-2014年铜川市A型H1N1和2013-2015年A型H3N2流感病毒血凝素(HA)基因特性及其抗原变异情况。 方法 收集铜川市哨点医院流感样病例标本,采用MDCK细胞分离流感病毒。用RT-PCR对部分A型H1N1、H3N2流感病毒进行HA基因序列扩增和纯化,序列测定,并用MEGA软件构建种系发生树分析。 结果 2013-2015年铜川市流感核酸检测标本共2 333份,流感阳性476份,阳性率20.40%。4株A型H1N1亚型流感病毒与WHO推荐A/California/07/2009(H1N1)疫苗株氨基酸序列比对,共发生11次氨基酸突变,其中3个氨基酸位点突变发生在抗原决定簇。3株A型H3N2亚型与WHO推荐的北半球2013-2014年A/Victoria/361/2011(H3N2)疫苗株比对共有9个氨基酸位点发生了突变,其中3个氨基酸位点发生在抗原决定簇;与WHO推荐的北半球2014-2015年A/Texas/50/2012(H3N2)疫苗株比对共有10个氨基酸位点发生了突变,有5个氨基酸位点发生在抗原决定簇。 结论 2013-2015年铜川市流行的A型H1N1、H3N2亚型流感病毒氨基酸发生突变导致抗原漂移。     

Influenza hemagglutinin antigenic distance measures capture trends in HAI differences and infection outcomes,but are not suitable predictive tools

Vaccination is the most effective method to combat influenza. Vaccine effectiveness is influenced by the antigenic distance between the vaccine strain and the actual circulating virus. Amino acid sequence based methods of quantifying the antigenic distance were designed to predict influenza vaccine effectiveness in humans. The use of these antigenic distance measures has been proposed as an additive method for seasonal vaccine selection. In this report, several antigenic distance measures were evaluated as predictors of hemagglutination inhibition titer differences and clinical outcomes following influenza vaccination or infection in mice or ferrets. The antigenic distance measures described the increasing trend in the change of HAI titer, lung viral titer and percent weight loss in mice and ferrets. However, the variability of outcome variables produced wide prediction intervals for any given antigenic distance value. The amino acid substitution based antigenic distance measures were no better predictors of viral load and weight loss than HAI titer differences, the current predictive measure of immunological correlate of protection for clinical signs after challenge.     

An epidemiological study of influenza viruses among Chinese farm families with household ducks and pigs.

To examine the possibility of interspecies transmission and genetic reassortment of influenza viruses on farms in Southern China, we surveyed 20 farm families living outside the city of Nanchang who raised pigs and ducks in their homes. Weekly interviews of family members and virus isolation studies of throat swabs and faecal samples, collected from September 1992 to September 1993, established the seasonal pattern of respiratory tract infections in these families and identified 11 influenza viruses (6 in humans and 5 in ducks). Most of the human isolates were type A of H3N2 subtype. Serologic studies of farm pigs indicated infection by the same human viruses circulating in family members, but there was no evidence that either swine or avian viruses had been transmitted to pigs. Eight of 156 human serum samples inhibited the neuraminidase activity of two of the duck isolates, raising the possibility of interspecies transmission of these avian viruses. Genotype analysis of duck and human isolates provided no evidence for reassortment. Our finding support the concept that intermingling of humans, pigs and ducks on Chinese farms is favourable to the generation of new, potentially hazardous strains of influenza virus.     

Influenza A (H5N1) Viruses from Pigs, Indonesia

Pigs have long been considered potential intermediate hosts in which avian influenza viruses can adapt to humans. To determine whether this potential exists for pigs in Indonesia, we conducted surveillance during 2005–2009. We found that 52 pigs in 4 provinces were infected during 2005–2007 but not 2008–2009. Phylogenetic analysis showed that the viruses had been introduced into the pig population in Indonesia on at least 3 occasions. One isolate had acquired the ability to recognize a human-type receptor. No infected pig had influenza-like symptoms, indicating that influenza A (H5N1) viruses can replicate undetected for prolonged periods, facilitating avian virus adaptation to mammalian hosts. Our data suggest that pigs are at risk for infection during outbreaks of influenza virus A (H5N1) and can serve as intermediate hosts in which this avian virus can adapt to mammals.     

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.     

Influenza virus-specific antibody dependent cellular cytoxicity induced by vaccination or natural infection

Influenza viruses are responsible for substantial morbidity and mortality during seasonal epidemics. Vaccination is the most effective method to prevent infection, however due to antigenic drift of the viral surface protein hemagglutinin (HA), annual influenza virus vaccination is required. In addition to seasonal viruses, certain (avian) influenza A viruses of other subtypes, like H5N1 or H7N9, cause sporadic zoonotic infections. Therefore, the availability of game-changing novel vaccines that induce “universal” immune responses to a wide variety of influenza A virus subtypes is highly desirable. The quest for universal influenza vaccines has fueled the interest in broadly-reactive antibodies specific for the stalk of hemagglutinin (HA) and biological activities of antibodies other than direct virus neutralization, like antibody-dependent cellular cytotoxicity (ADCC). In the present study, we investigated the ADCC response upon influenza virus vaccination and infection in humans using a robust ADCC assay that is based on the use of recombinant HA and a continuous NK cell line that expresses FcγRIII (CD16). This assay offers advantages over existing methods, like ease to perform and possibilities to standardize. We showed that HA-specific ADCC mediating antibodies are induced by vaccination with adjuvanted trivalent seasonal and monovalent H1N1pdm09 inactivated vaccines, and by infection with H1N1pdm09 virus. In addition, the use of chimeric influenza HA with a H1 stem but antigenically irrelevant head domain derived from an avian virus allowed detection of H1-stalk-specific ADCC mediating antibodies. This assay will facilitate the assessment of ADCC mediating serum antibodies after (universal) influenza vaccination or infection and may define ADCC activity as a correlate of (cross-) protection in the future.     

Use of monoclonal anti-haemagglutinin antibodies for the “In vitro” selection of a sequential influenza virus antigenic variant

A sequential antigenic variant of the A/Texas/77 (H3N2) influenza virus was obtained in vitro using a monoclonal antibody against the haemagglutinin (HA) of the antigenic variant V18 previously selected in vitro from the parental Texas virus. The sequential antigenic variant, designated DV1, the V18 antigenic variant and the parental A/Texas/77 viruses were used to evaluate the frequency of anti-haemagglutinin antibodies in human sera in single radial haemolysis assays. Twenty six of 100 children's sera, which contained antibodies to the parental A/Texas/77 virus, failed to react with the V18 antigenic variant. A higher proportion of sera (42%) failed to react with the DV1 antigenic variant with alterations in two different antigenic determinants with respect to the parental virus. The results are discussed in relation to the mechanism of antigenic drift and to the in vivo reaction of antigenic variants selected in vitro.Corresponding author.     

Interspecies transmission and reassortment of influenza A viruses in pigs and turkeys in the United States.

Genetic reassortment between influenza A viruses in humans and in animals and birds has been implicated in the appearance of new pandemics of human influenza. To determine whether such reassortment has occurred in the United States, the authors compared the genetic origins of gene segments of 73 swine influenza virus isolates (1976-1990), representing 11 states, and 11 turkey virus isolates (1980-1989), representing eight states. The host origin of gene segments encoding the internal proteins of H1N1 swine and turkey influenza viruses was identified by developing a dot-blot assay. All gene segments of swine influenza viruses were characteristic of influenza virus genes from that species, indicating that pigs may not be frequent participants in interspecies genetic exchange and reassortment of influenza viruses in the United States. In contrast, 73% of the turkey influenza virus isolates contained genes of swine origin. One turkey isolate was a reassortant having three genes characteristic of avian influenza virus and three of swine origin. These findings document a high degree of genetic exchange and reassortment of influenza A viruses in domestic turkeys in the United States. The molecular biologic techniques used by the authors should aid future epidemiologic studies of influenza pandemics.