In silico characterization of the functional and structural modules of the hemagglutinin protein from the swine-origin influenza virus A (H1N1)-2009

The 2009 swine-origin influenza virus (S-OIV,H1N1 subtype) has developed into a new pandemic influenza as announced by the World Health Organization.In order to uncover clues about the determinants for virulence and pathogenicity of the virus,we characterized the functional modules of the surface glycoprotein hemagglutinin (HA),the most important protein in molecular epidemiology and pathogenesis of influenza viruses.We analyzed receptor binding sites,basic patch,neutralization antibody epitopes and T cell epitopes in the HA protein of the current S-OIV according to the corresponding functional and structural modules previously characterized in other H1 HA molecules or HA molecules of other subtypes.We compared their differences and similarities systematically.Based on the amino acids defined as the functional and structural modules,the HA protein of 2009 S-OIV should specifically bind to the human 2,6-receptor.The D225G/E mutation in HA,which is found in some isolates,may confer dual binding specificity to the 2,3and 2,6-receptor based on previously reported work.This HA variant contains two basic patches,one of which results in increased basicity,suggesting enhanced membrane fusion function.The 2009 S-OIV HA also has an extra glycosylation site at position 276.Four of the five antibody neutralization epitopes identified in A/RP/8/34(H1N1) were exposed,but the other was hidden by a glycosylation site.The previously identified cytotoxic T cell epitopes in various HA molecules were summarized and their corresponding sequences in 2009 S-OIV HA were defined.These results are critical for understanding the pathogenicity of the virus and host immune response against the virus.     

Crystal structure of the swine-origin A (H1N1) — 2009 influenza A virus hemagglutinin (HA) reveals similar antigenicity to that of the 1918 pandemic virus

Influenza virus is the causative agent of the seasonal and occasional pandemic flu. The current H1N1 influenza pandemic, announced by the WHO in June 2009, is highly contagious and responsible for global economic losses and fatalities. Although the H1N1 gene segments have three origins in terms of host species, the virus has been named swine-origin influenza virus (S-OIV) due to a predominant swine origin. 2009 S-OIV has been shown to highly resemble the 1918 pandemic virus in many aspects. Hemagglutinin is responsible for the host range and receptor binding of the virus and is therefore a primary indicator for the potential of infection. Primary sequence analysis of the 2009 S-OIV haemagglutinin (HA) reveals its closest relationship to that of the 1918 pandemic influenza virus, however, analysis at the structural level is necessary to critically assess the functional significance. In this report, we report the crystal structure of soluble haemagglutinin H1 (09H1) at 2.9 ?, illustrating that the 09H1 is very similar to the 1918 pandemic HA (18H1) in overall structure and the structural modules, including the five defined antiboby(Ab)-binding epitopes. Our results provide an explanation as to why sera from the survivors of the 1918 pandemics can neutralize the 2009 S-OIV, and people born around the 1918 are resistant to the current pandemic, yet younger generations are more susceptible to the 2009 pandemic.     

Glycosylation on Hemagglutinin Affects the Virulence and Pathogenicity of Pandemic H1N1/2009 Influenza A Virus in Mice

The two glycosylation sites (Asn142 and Asn177) were observed in the HA of most human seasonal influenza A/H1N1 viruses, while none in pandemic H1N1/2009 influenza A (pH1N1) viruses. We investigated the effect of the two glycosylation sites on viral virulence and pathogenicity in mice using recombinant pH1N1. The H1N1/144 and H1N1/177 mutants which gained potential glycosylation sites Asn142 and Asn177 on HA respectively were generated from A/Mexico/4486/2009(H1N1) by site-directed mutagenesis and reverse genetics, the same as the H1N1/144+177 gained both glycosylation sites Asn142 and Asn177. The biological characteristics and antigenicity of the mutants were compared with wild-type pH1N1. The virulence and pathogenicity of recombinants were also detected in mice. Our results showed that HA antigenicity and viral affinity for receptor may change with introduction of the glycosylation sites. Compared with wild-type pH1N1, the mutant H1N1/177 displayed an equivalent virus titer in chicken embryos and mice, and increased virulence and pathogenicity in mice. The H1N1/144 displayed the highest virus titer in mice lung. However, the H1N1/144+177 displayed the most serious alveolar inflammation and pathogenicity in infected mice. The introduction of the glycosylation sites Asn144 and Asn177 resulted in the enhancement on virulence and pathogenicity of pH1N1 in mice, and was also associated with the change of HA antigenicity and the viral affinity for receptor.     

Envelope Proteins Pertain with Evolution and Adaptive Mechanism of the Novel Influenza A/H1N1 in Humans

The novel swine-origin influenza A/H1N1 virus (S-OIV) first detected in April 2009 has been identified to transmit from human to human directly and is the cause of currently emerged pandemic. In this study, nucleotide and deduced amino acid sequences of hemagglutinin (HA) and neuraminidase (NA) of the S-OIV and other influenza A viruses were analyzed through bioinformatic tools for phylogenetic analysis, genetic recombination and point mutation to investigate the emergence and adaptation of the S-OIV in human. The phylogenetic analysis showed that the HA comes from triple reassortant influenza A/H1N2 and the NA from Eurasian swine influenza A/H1N1 indicating HA and NA to descend from different lineages during the genesis of the S-OIV. Recombination analysis nullified the possibility of occurrence of recombination in HA and NA denoting the role of reassortment in the outbreak. Several conservative mutations are observed in the amino acid sequences of the HA and NA and this mutated residues are identical in the S-OIV. The results reported herein suggested the notion that the recent pandemic is the result of reassortment of different genes from different lineages of two envelope proteins, HA and NA which are responsible for antigenic activity of virus. This study further suggests that the adaptive capability of the S-OIV in human is acquired by the unique mutations generated during emergence.     

Assessment of Seasonal Influenza A Virus-Specific CD4 T-Cell Responses to 2009 Pandemic H1N1 Swine-Origin Influenza A Virus

Very limited evidence has been reported to show human adaptive immune responses to the 2009 pandemic H1N1 swine-origin influenza A virus (S-OIV). We studied 17 S-OIV peptides homologous to immunodominant CD4 T epitopes from hemagglutinin (HA), neuraminidase (NA), nuclear protein (NP), M1 matrix protein (MP), and PB1 of a seasonal H1N1 strain. We concluded that 15 of these 17 S-OIV peptides would induce responses of seasonal influenza virus-specific T cells. Of these, seven S-OIV sequences were identical to seasonal influenza virus sequences, while eight had at least one amino acid that was not conserved. T cells recognizing epitopes derived from these S-OIV antigens could be detected ex vivo. Most of these T cells expressed memory markers, although none of the donors had been exposed to S-OIV. Functional analysis revealed that specific amino acid differences in the sequences of these S-OIV peptides would not affect or partially affect memory T-cell responses. These findings suggest that without protective antibody responses, individuals vaccinated against seasonal influenza A may still benefit from preexisting cross-reactive memory CD4 T cells reducing their susceptibility to S-OIV infection.The outbreak of H1N1 swine-origin influenza A virus (S-OIV) in April 2009 has raised a new threat to public health (5, 6). This novel virus (with A/California/04/09 H1N1 as a prototypic strain) not only replicated more efficiently but also caused more severe pathological lesions in the lungs of infected mice, ferrets, and nonhuman primates than a currently circulating human H1N1 virus (9). Similarly, human patients with influenza-like illness who tested negative for S-OIV had a milder clinical course than those who tested positive (13). Another major concern is the lack of immune protection against S-OIV in the human population. Initial serum analysis indicated that cross-reactive antibodies to this novel viral strain were detected in only one-third of people over 60 years of age, while humoral immune responses in the population under 60 years of age were rarely detected (3, 8). In addition, vaccination with recent seasonal influenza vaccines induced little or no cross-reactive antibody responses to S-OIV in any age group (3, 8).Only a few studies address whether preexisting seasonal influenza A virus-specific memory T cells cross-react with antigenic peptides derived from S-OIV (7). In the absence of preexisting cross-reactive neutralizing antibodies, it is likely that T-cell-mediated cellular immunity contributes to viral clearance and reduces the severity of symptoms, although virus-specific T cells cannot directly prevent the establishment of infection (10). Greenbaum and colleagues recently compared published T-cell epitopes for seasonal influenza viruses with S-OIV antigens (Ags) using a computational approach (7). Several seasonal H1N1 epitopes were found to be identical to S-OIV sequences. This implies that seasonal flu-specific memory T cells circulating in the peripheral blood of vaccinated and/or previously infected individuals are able to recognize their S-OIV homologues.The first objective of this study was to determine the extent of cross-reactivity of seasonal H1N1 influenza A virus-specific CD4 T cells with S-OIV epitopes, especially those less conserved peptide sequences. We chose 17 immunodominant DR4-restricted T-cell epitopes derived from a seasonal H1N1 strain, compared the binding of these epitopes and their S-OIV homologous peptides to DR4, tested the ability of S-OIV peptides to drive seasonal influenza virus-specific T-cell proliferation in vitro, and estimated the frequency of S-OIV cross-reactive T cells in the periphery of noninfected donors. We found that most homologous S-OIV peptides were able to activate seasonal H1N1 virus-specific CD4 T cells. The second objective was to compare the antigen dosage requirement to activate those T cells. By assessing the alternations in the functional avidities (of T cells to the cognate peptide and S-OIV homologue) due to amino acid differences in S-OIV peptides, we showed how those cross-reactive CD4 T cells differentially responded to the antigenic peptides derived from seasonal H1N1 virus or S-OIV. This study leads to the conclusion that previous exposure to seasonal H1N1 viral antigens will generate considerable levels of memory CD4 T cells cross-reactive with S-OIV.     

Reassortment Networks and the evolution of pandemic H1N1 swine-origin influenza

Prior research developed Reassortment Networks to reconstruct the evolution of segmented viruses under both reassortment and mutation. We report their application to the swine-origin pandemic H1N1 virus (S-OIV). A database of all influenza A viruses, for which complete genome sequences were available in Genbank by October 2009, was created and dynamic programming was used to compute distances between all corresponding segments. A reassortment network was created to obtain the minimum cost evolutionary paths from all viruses to the exemplar S-OIV A/California/04/2009. This analysis took 35 hours on the Cray Extreme Multithreading (XMT) supercomputer, which has special hardware to permit efficient parallelization. Six specific H1N1/H1N2 bottleneck viruses were identified that almost always lie on minimum cost paths to S-OIV. We conjecture that these viruses are crucial to S-OIV evolution and worthy of careful study from a molecular biology viewpoint. In phylogenetics, ancestors are typically medians that have no functional constraints. In our method, ancestors are not inferred, but rather chosen from previously observed viruses along a path of mutation and reassortment leading to the target virus. This specificity and functional constraint render our results actionable for further experiments in vitro and in vivo.     

长沙A(H1N1)亚型季节性流感暴发疫情的实验室诊断及其HA基因特性分析

对2009 年长沙麓山国际学校流感暴发疫情进行实验室诊断, 并探索新分离的A(H1N1)亚型流感病毒血凝素(HA)的基因特性。对流感暴发疫情的25 份鼻/咽拭子标本进行RT-PCR检测和流感病毒分离, 然后利用CEQ?8000 Genetic Analysis System对病毒分离株(A/Yuelu/314/2009)进行测序, 测序结果提交至GenBank(登录号: FJ912843)并用ClustalX和Mega4.1软件进行序列分析。结果显示, 分离出A(H1N1)亚型流感毒株18株, 检出21份A(H1N1)亚型流感病毒核酸阳性; A/Yuelu/314/2009(H1N1) HA基因序列与2008~2009 年疫苗株(A/Brisbane/59/2007)比较显示: 核苷酸和氨基酸同源性均为99%, 有6个位点的氨基酸发生了变异(V148A、S158N、G202A、I203D、A206T、W435R), 其中一个S158N氨基酸变异位于B抗原表位, HA基因序列上共有潜在糖基化位点9 个(27、28、40、71、151、176、303、497、536), 与A/Brisbane/59/2007相同且氨基酸序列保守。本实验诊断出此次流感暴发疫情的病原体为A(H1N1)型季节性流感病毒, 研究还发现A/Yuelu/314/2009(H1N1)长沙分离株与A/Brisbane/59/2007 疫苗株基因序列比较显示并未形成一个新的变种, 推测是由于分离株与疫苗株之间基因特性的改变和人群对A(H1N1)亚型流感病毒免疫力降低导致了此次长沙麓山国际学校A(H1N1)亚型流感的暴发。     

Molecular determinants within the surface proteins involved in the pathogenicity of H5N1 influenza viruses in chickens

Although it is established that the cleavage site and glycosylation patterns in the hemagglutinin (HA) play important roles in determining the pathogenicity of H5 avian influenza viruses, some viruses exist that are not highly pathogenic despite possessing the known characteristics of high pathogenicity (i.e., their HA contains multiple basic amino acids at the cleavage site and has glycosylation patterns similar to that of the highly pathogenic H5 viruses). Currently little is known about the H5N1 viruses that fall into this intermediate category of pathogenicity. We have identified strains of H5N1 avian influenza viruses that have markers typical of high pathogenicity but distinctly differ in their ability to cause disease and death in chickens. By analyzing viruses constructed by reverse-genetic methods and containing recombinant HAs, we established that amino acids 97, 108, 126, 138, 212, and 217 of HA, in addition to those within the cleavage site, affect pathogenicity. Further investigation revealed that an additional glycosylation site within the neuraminidase (NA) protein globular head contributed to the high virulence of the H5N1 virus. Our findings are in agreement with previous observations that suggest that the activities of the HA and NA proteins are functionally linked.     

Characterization of an artificial swine-origin influenza virus with the same gene combination as H1N1/2009 virus: a genesis clue of pandemic strain

Pandemic H1N1/2009 influenza virus, derived from a reassortment of avian, human, and swine influenza viruses, possesses a unique gene segment combination that had not been detected previously in animal and human populations. Whether such a gene combination could result in the pathogenicity and transmission as H1N1/2009 virus remains unclear. In the present study, we used reverse genetics to construct a reassortant virus (rH1N1) with the same gene combination as H1N1/2009 virus (NA and M genes from a Eurasian avian-like H1N1 swine virus and another six genes from a North American triple-reassortant H1N2 swine virus). Characterization of rH1N1 in mice showed that this virus had higher replicability and pathogenicity than those of the seasonal human H1N1 and Eurasian avian-like swine H1N1 viruses, but was similar to the H1N1/2009 and triple-reassortant H1N2 viruses. Experiments performed on guinea pigs showed that rH1N1 was not transmissible, whereas pandemic H1N1/2009 displayed efficient transmissibility. To further determine which gene segment played a key role in transmissibility, we constructed a series of reassortants derived from rH1N1 and H1N1/2009 viruses. Direct contact transmission studies demonstrated that the HA and NS genes contributed to the transmission of H1N1/2009 virus. Second, the HA gene of H1N1/2009 virus, when combined with the H1N1/2009 NA gene, conferred efficient contact transmission among guinea pigs. The present results reveal that not only gene segment reassortment but also amino acid mutation were needed for the generation of the pandemic influenza virus.     

甲型H1N1流感病毒血凝素蛋白单抗杂交瘤细胞株的制备与初步鉴定

目的:建立具有高特异、高效价的甲型H1N1流感病毒血凝素蛋白(HA)单抗的杂交瘤细胞株。方法:以纯化的昆虫杆状病毒表达的甲型H1N1流感病毒HA蛋白为免疫原免疫BALB/c小鼠,取脾细胞与Sp2/0小鼠骨髓瘤细胞融合,通过有限稀释法筛选阳性克隆,经ELISA和Western blot分析单抗的特性和特异性。结果:获得6株甲型H1N1流感HA抗原特异单克隆抗体杂交瘤细胞株,抗原肽库ELISA检测结果表明其中3株(1E12,3F12,1C11)单抗只与甲型H1N1流感HA抗原肽库反应,不与H5N1病毒HA抗原肽库反应;Western blot分析表明,单抗1B3只特异识别甲型H1N1流感HA抗原,而与其他季节性甲流病毒(H1,H3)及人禽流感H5N1病毒不反应。结论:所获杂交瘤细胞株特异性强,效价高,分泌抗体性能稳定,为分析甲型H1N1流感病毒抗原性位点、建立诊断试剂奠定了基础。     

江苏首例甲型H1N1(2009)流感病毒分离及其血凝素分子遗传特征分析

2009年6月12日,江苏确诊首例甲型H1N1(2009)病例。通过细胞和鸡胚分离系统,我们分离到一株具有较高血凝活性的病毒,命名为A/Jiangsu/1/2009。为了跟踪病毒的变异情况,我们开展了病毒的全基因组测序工作,在此基础上对其血凝素基因(Haemagglutinin,HA)的遗传特性进行了详细研究。分离株HA蛋白不具有多碱基HA裂解位点,具有低致病性流感病毒特点。与参考株A/California/04/2009相比,分离株A/Jiangsu/1/2009HA蛋白的有4个氨基酸发生了突变,但都不在已知的抗原位点上。分离株有5个潜在糖基化位点,这与近年来古典猪H1N1和北美三源重配猪H1病毒完全一致,保留了古典猪H1的特点。与禽流感H1病毒相比,分离株HA蛋白受体结合位点上的E190D和G225D发生突变,这可能成为新甲型H1N1(2009)在人际间传播的一个重要分子基础。此外,其它受体结合位点上相关氨基酸同时具有人和猪流感病毒的特点。本研究首次对早期流行的甲型H1N1(2009)流感病毒的HA蛋白的分子遗传特征进行了详细研究,对进一步监测病原变异具有重要指导意义。     

Comparative analysis of hemagglutinin of 2009 H1N1 influenza A pandemic indicates its evolution to 1918 H1N1 pandemic

To gain insight into the possible origin of the hemagglutinin of 2009 outbreak, we performed its comparative analysis with hemagglutinin of influenza viral strains from 2005 to 2008 and the past pandemics of 1977, 1968, 1957 and 1918. This insilico analysis showed a maximum sequence similarity between 2009 and 1918 pandemics. Primary structure analysis, antigenic and glycosylation site analyses revealed that this protein has evolved from 1918 pandemic. Phylogenetic analysis of HA amino acid sequence of 2009 influenza A(H1N1) viruses indicated that this virus possesses a distinctive evolutionary trait with 1918 influenza A virus. Although the disordered sequences are different among all the isolates, the disordered positions and sequences between 2009 and 1918 isolates show a greater similarity. Thus these analyses contribute to the evidence of the evolution of 2009 pandemic from 1918 influenza pandemic. This is the first computational evolutionary analysis of HA protein of 2009 H1N1 pandemic.     

Increased pathogenicity of a reassortant 2009 pandemic H1N1 influenza virus containing an H5N1 hemagglutinin

A novel H1N1 influenza virus emerged in 2009 (pH1N1) to become the first influenza pandemic of the 21st century. This virus is now cocirculating with highly pathogenic H5N1 avian influenza viruses in many parts of the world, raising concerns that a reassortment event may lead to highly pathogenic influenza strains with the capacity to infect humans more readily and cause severe disease. To investigate the virulence of pH1N1-H5N1 reassortant viruses, we created pH1N1 (A/California/04/2009) viruses expressing individual genes from an avian H5N1 influenza strain (A/Hong Kong/483/1997). Using several in vitro models of virus replication, we observed increased replication for a reassortant CA/09 virus expressing the hemagglutinin (HA) gene of HK/483 (CA/09-483HA) relative to that of either parental CA/09 virus or reassortant CA/09 expressing other HK/483 genes. This increased replication correlated with enhanced pathogenicity in infected mice similar to that of the parental HK/483 strain. The serial passage of the CA/09 parental virus and the CA/09-483HA virus through primary human lung epithelial cells resulted in increased pathogenicity, suggesting that these viruses easily adapt to humans and become more virulent. In contrast, serial passage attenuated the parental HK/483 virus in vitro and resulted in slightly reduced morbidity in vivo, suggesting that sustained replication in humans attenuates H5N1 avian influenza viruses. Taken together, these data suggest that reassortment between cocirculating human pH1N1 and avian H5N1 influenza strains will result in a virus with the potential for increased pathogenicity in mammals.     

The S190R mutation in the hemagglutinin protein of pandemic H1N1 2009 influenza virus increased its pathogenicity in mice

Human influenza viruses preferentially bind to sialic acid-α2,6-galactose (SAα2,6Gal) receptors, which are predominant in human upper respiratory epithelia, whereas avian influenza viruses preferentially bind to SAα2,3Gal receptors. However, variants with amino acid substitutions around the receptor-binding sites of the hemagglutinin (HA) protein can be selected after several passages of human influenza viruses from patients’ respiratory samples in the allantoic cavities of embryonated chicken eggs. In this study, we detected an egg-adapted HA S190R mutation in the pandemic H1N1 virus 2009 (pdmH1N1), and evaluated the effects of this mutation on receptor binding affinity and pathogenicity in mice. Our results revealed that residue 190 is located within the pocket structure of the receptor binding site. The single mutation to arginine at position 190 slightly increased the binding affinity of the virus to the avian receptor and decreased its binding to the long human α2,6-linked sialic acid receptor. Our study demonstrated that the S190R mutation resulted in earlier death and higher weight loss in mice compared with the wild-type virus. Higher viral titers at 1 dpi (days post infection) and diffuse damage at 4 dpi were observed in the lung tissues of mice infected with the mutant virus.     

N-Linked Glycosylation of the Hemagglutinin Protein Influences Virulence and Antigenicity of the 1918 Pandemic and Seasonal H1N1 Influenza A Viruses

The hemagglutinin (HA) protein is a major virulence determinant for the 1918 pandemic influenza virus; however, it encodes no known virulence-associated determinants. In comparison to seasonal influenza viruses of lesser virulence, the 1918 H1N1 virus has fewer glycosylation sequons on the HA globular head region. Using site-directed mutagenesis, we found that a 1918 HA recombinant virus, of high virulence, could be significantly attenuated in mice by adding two additional glycosylation sites (asparagine [Asn] 71 and Asn 286) on the side of the HA head. The 1918 HA recombinant virus was further attenuated by introducing two additional glycosylation sites on the top of the HA head at Asn 142 and Asn 172. In a reciprocal experimental approach, deletion of HA glycosylation sites (Asn 142 and Asn 177, but not Asn 71 and Asn 104) from a seasonal influenza H1N1 virus, A/Solomon Islands/2006 (SI/06), led to increased virulence in mice. The addition of glycosylation sites to 1918 HA and removal of glycosylation sites from SI/06 HA imposed constraints on the theoretical structure surrounding the glycan receptor binding sites, which in turn led to distinct glycan receptor binding properties. The modification of glycosylation sites for the 1918 and SI/06 viruses also caused changes in viral antigenicity based on cross-reactive hemagglutinin inhibition antibody titers with antisera from mice infected with wild-type or glycan mutant viruses. These results demonstrate that glycosylation patterns of the 1918 and seasonal H1N1 viruses directly contribute to differences in virulence and are partially responsible for their distinct antigenicity.     

Glycosylation focuses sequence variation in the influenza A virus H1 hemagglutinin globular domain

Antigenic drift in the influenza A virus hemagglutinin (HA) is responsible for seasonal reformulation of influenza vaccines. Here, we address an important and largely overlooked issue in antigenic drift: how does the number and location of glycosylation sites affect HA evolution in man? We analyzed the glycosylation status of all full-length H1 subtype HA sequences available in the NCBI influenza database. We devised the “flow index” (FI), a simple algorithm that calculates the tendency for viruses to gain or lose consensus glycosylation sites. The FI predicts the predominance of glycosylation states among existing strains. Our analyses show that while the number of glycosylation sites in the HA globular domain does not influence the overall magnitude of variation in defined antigenic regions, variation focuses on those regions unshielded by glycosylation. This supports the conclusion that glycosylation generally shields HA from antibody-mediated neutralization, and implies that fitness costs in accommodating oligosaccharides limit virus escape via HA hyperglycosylation.     

Mutations in Hemagglutinin and Polymerase Alter the Virulence of Pandemic A(H1N1) Influenza Virus

To study the pathogenicity factors of the pandemic A(H1N1) influenza virus, a number of mutant variants of the A/Hamburg/5/2009 (H1N1)pdm09 strain were obtained through passage in chicken embryos, mouse lungs, and MDCK cell culture. After 17 lung-to-lung passages of the A/Hamburg/5/2009 in mice, the minimum lethal dose of the derived variant decreased by five orders of magnitude compared to that of the parental virus. This variant differed from the original virus by nine amino acid residues in the following viral proteins: hemagglutinin (HA), neuraminidase (NA), and components of the polymerase complex. Additional passaging of the intermediate variants and cloning made it possible to obtain pairs of strains that differed by a single amino acid substitution. Comparative analysis of replicative activity, receptor specificity, and virulence of these variants revealed two mechanisms responsible for increased pathogenicity of the virus for mice. Thus, (1) substitutions in HA (Asp225Gly or Gln226Arg) and compensatory mutation decreasing the charge of HA (Lys123Asn, Lys157Asn, Gly158Glu, Asn159Asp, or Lys212Met) altered viral receptor-binding specificity and restored the functional balance between HA and NA; (2) Phe35Leu substitution in the PA protein increased viral polymerase activity.     

高滴度感染力H5N1亚型禽流感病毒假病毒颗粒的制备

利用一个瞬时共转染系统,将H5N1亚型禽流感病毒的血凝素（Hemagglutinin,HA）蛋白与神经氨酸酶（Neuraminidase,NA）蛋白整合到鼠白血病病毒假病毒颗粒表面,包装成表达HA与NA的假病毒颗粒,通过透射电子显微镜形态学观察、感染滴度分析、血凝试验和中和试验研究其生物学特性。研究获得了高滴度感染力的H5N1假病毒颗粒（H5N1 Pseudotyped particle,H5N1Pp）,H5N1Pp的感染力滴度为1E8 Pp/mL,形态、血凝活性及中和特性均与野生H5N1病毒相似,结果为H5N1病毒受体、HA与NA的功能、中和抗体、抗病毒药物开发研究的开展建立了平台。     

Genetic Requirement for Hemagglutinin Glycosylation and Its Implications for Influenza A H1N1 Virus Evolution

Influenza A virus has evolved and thrived in human populations. Since the 1918 influenza A pandemic, human H1N1 viruses had acquired additional N-linked glycosylation (NLG) sites within the globular head region of hemagglutinin (HA) until the NLG-free HA head pattern of the 1918 H1N1 virus was renewed with the swine-derived 2009 pandemic H1N1 virus. Moreover, the HA of the 2009 H1N1 virus appeared to be antigenically related to that of the 1918 H1N1 virus. Hence, it is possible that descendants of the 2009 H1N1 virus might recapitulate the acquisition of HA head glycosylation sites through their evolutionary drift as a means to evade preexisting immunity. We evaluate here the evolution signature of glycosylations found in the globular head region of H1 HA in order to determine their impact in the virulence and transmission of H1N1 viruses. We identified a polymorphism at HA residue 147 associated with the acquisition of glycosylation at residues 144 and 172. By in vitro and in vivo analyses using mutant viruses, we also found that the polymorphism at HA residue 147 compensated for the loss of replication, virulence, and transmissibility associated with the presence of the N-linked glycans. Our findings suggest that the polymorphism in H1 HA at position 147 modulates viral fitness by buffering the constraints caused by N-linked glycans and provide insights into the evolution dynamics of influenza viruses with implications in vaccine immunogenicity.     

An Influenza A/H1N1/2009 Hemagglutinin Vaccine Produced in Escherichia coli

Background

The A/H1N1/2009 influenza pandemic made evident the need for faster and higher-yield methods for the production of influenza vaccines. Platforms based on virus culture in mammalian or insect cells are currently under investigation. Alternatively, expression of fragments of the hemagglutinin (HA) protein in prokaryotic systems can potentially be the most efficacious strategy for the manufacture of large quantities of influenza vaccine in a short period of time. Despite experimental evidence on the immunogenic potential of HA protein constructs expressed in bacteria, it is still generally accepted that glycosylation should be a requirement for vaccine efficacy.

Methodology/Principal Findings

We expressed the globular HA receptor binding domain, referred to here as HA_63–286-RBD, of the influenza A/H1N1/2009 virus in Escherichia coli using a simple, robust and scalable process. The recombinant protein was refolded and purified from the insoluble fraction of the cellular lysate as a single species. Recombinant HA_63–286-RBD appears to be properly folded, as shown by analytical ultracentrifugation and bio-recognition assays. It binds specifically to serum antibodies from influenza A/H1N1/2009 patients and was found to be immunogenic, to be capable of triggering the production of neutralizing antibodies, and to have protective activity in the ferret model.

Conclusions/Significance

Projections based on our production/purification data indicate that this strategy could yield up to half a billion doses of vaccine per month in a medium-scale pharmaceutical production facility equipped for bacterial culture. Also, our findings demonstrate that glycosylation is not a mandatory requirement for influenza vaccine efficacy.