Geometric Constraints Dominate the Antigenic Evolution of Influenza H3N2 Hemagglutinin

We have carried out a comprehensive analysis of the determinants of human influenza A H3 hemagglutinin evolution. We consider three distinct predictors of evolutionary variation at individual sites: solvent accessibility (as a proxy for protein fold stability and/or conservation), Immune Epitope Database (IEDB) epitope sites (as a proxy for host immune bias), and proximity to the receptor-binding region (as a proxy for one of the functions of hemagglutinin-to bind sialic acid). Individually, these quantities explain approximately 15% of the variation in site-wise dN/dS. In combination, solvent accessibility and proximity explain 32% of the variation in dN/dS; incorporating IEDB epitope sites into the model adds only an additional 2 percentage points. Thus, while solvent accessibility and proximity perform largely as independent predictors of evolutionary variation, they each overlap with the epitope-sites predictor. Furthermore, we find that the historical H3 epitope sites, which date back to the 1980s and 1990s, only partially overlap with the experimental sites from the IEDB, and display similar overlap in predictive power when combined with solvent accessibility and proximity. We also find that sites with dN/dS > 1, i.e., the sites most likely driving seasonal immune escape, are not correctly predicted by either historical or IEDB epitope sites, but only by proximity to the receptor-binding region. In summary, a simple geometric model of HA evolution outperforms a model based on epitope sites. These results suggest that either the available epitope sites do not accurately represent the true influenza antigenic sites or that host immune bias may be less important for influenza evolution than commonly thought.     

Immunodominance of Antigenic Site B over Site A of Hemagglutinin of Recent H3N2 Influenza Viruses

H3N2 influenza viruses have now circulated in the human population for 43 years since the pandemic of 1968, accumulating sequence changes in the hemagglutinin (HA) and neuraminidase (NA) that are believed to be predominantly due to selection for escape from antibodies. Examination of mutations that persist and accumulate led to identification of antigenically significant mutations that are contained in five antigenic sites (A-E) mapped on to the H3 HA. In early H3N2 isolates, antigenic site A appeared to be dominant while in the 1990s site B seemed more important. To obtain experimental evidence for dominance of antigenic sites on modern H3 HAs, we have measured antibodies in plasma of human subjects who received the 2006-07 trivalent subunit influenza vaccine (H3 component A/Wisconsin/67/05) or the 2008-09 formulation (H3 component A/Uruguay/716/07). Plasmas were tested against expressed HA of Wisconsin-like influenza A/Oklahoma/309/06 and site-directed mutants in antigenic site A (NNES121-124ITEG, N126T, N133D, TSSS135-138GSNA, K140I, RSNNS142-146PGSG), and antigenic site B (HL156-157KS, KFK158-160GST, NDQI189-192QEQT, A196V). "Native ELISA" analysis and escape mutant selection with two human monoclonal antibodies demonstrated that antibody E05 binds to antigenic site A and 1_C02 binds to site B. We find that most individuals, after vaccination in seasons 2006-07 and/or 2008-09, showed dominance of antigenic site B recognition over antigenic site A. A minority showed dominance of site A in 2006 but these were reduced in 2008 when the vaccine virus had a site A mutation. A better understanding of immunodominance may allow prediction of future antigenic drift and assist in vaccine strain selection.     

广东地区流感H3N2毒株血凝素基因特征、进化和变异分析

为揭示广东地区2007～2010年甲型H3N2毒株血凝素(HA)基因特征和变异,采用时空抽样方法抽样,检测广东2007～2010年甲型H3N2毒株HA基因核苷酸序列,同时检索全球HA基因序列作为对照,采用Lasergene 7.1和Mega 5.05软件对HA基因核苷酸序列进行比对和分析;并结合流行病学资料,对变异毒株进行进化速度分析;同时进行抗原分析。结果发现,广东2007～2010年H3N2毒株HA基因同义进化(Ks)和错义进化(Ka)速度分别为2.06×1E-3～2.23×1E-3核苷酸/年和1.05×1E-3～1.21×1E-3核苷酸/年,HA1较HA2的错义突变速率要高3.13倍。与疫苗株A/Perth/16/2009的HA基因比较,2009年广东毒株同源性达到98.8%～99.7%、2010年同源性达到98.0%～98.4%。在广东2007～2010年毒株中,HA1五个抗原表位均有氨基酸位点变异,尤其是2010年毒株B区(N160K)和D区(K174R/N)的变异;此外,广东2010年毒株受体结合部位(RBS)还发生K189E/N/Q和T228A置换变异;两个糖基化位点变异影响到抗原性;目前使用的H3N2疫苗株与目前流行毒株的抗原性有差异。广东地区2007～2010年的毒株中,血凝抑制抗体的抗原分析结果有差异。结果提示,目前广东乃至全球甲型H3N2毒株HA1B区和D区均有氨基酸位点变异,RBS的两个位点发生置换,糖基化位点变异影响到表位A区和B区抗原性;与WHO推荐2011年流感H3N2毒株疫苗株比较,目前流行毒株HA基因有抗原位点变异。     

禽流感病毒H5N1血凝素基因适应性进化过程中的关键位点突变规律研究

目的:寻找导致禽流感病毒H5N1血凝素(HA)适应性进化的关键突变,建立氨基酸突变评价体系,对突变作用进行评估,印证它们与病毒适应性进化的关联性。方法:计算株频率和枝频率,寻找标记分枝,向根结点回溯寻找HA进化路径上的氨基酸突变。计算各突变位点氨基酸的频率变化、有效变换及高频次突变,基于以上几个因素建立突变评价体系。结果:建立了大规模自动化寻找突变的方法,计算得到HA进化过程中的氨基酸突变435个,通过氨基酸频率图表分析这些突变可以很好地反映病毒适应性进化过程,其中79个突变是有效变换,发生的位点为正选择位点,且多数位点落在HA抗原表位上;29个突变是高频次突变,其中多数也为有效变换,因而与病毒适应性进化密切相关。结论:大规模自动化寻找突变的方法可靠,建立的突变评价体系准确性高,找到的关键突变及位点对实验有很好的指导意义。     

Structural Analysis of the Hemagglutinin from the Recent 2013 H7N9 Influenza Virus

In March 2013, the Chinese Center for Disease Control and Prevention reported human infections with an H7N9 influenza virus, and by 20 July 2013, the numbers of laboratory-confirmed cases had climbed to 134, including 43 fatalities and 127 hospitalizations. The newly emerging H7N9 viruses constitute an obvious public health concern because of the apparent severity of this outbreak. Here we focus on the hemagglutinins (HAs) of these viruses and assess their receptor binding phenotype in relation to previous HAs studied. Glycan microarray and kinetic analyses of recombinant A(H7N9) HAs were performed to compare the receptor binding profile of wild-type receptor binding site variants at position 217, a residue analogous to one of two positions known to switch avian to human receptor preference in H2N2 and H3N2 viruses. Two recombinant A(H7N9) HAs were structurally characterized, and a mutational study of the receptor binding site was performed to analyze important residues that can affect receptor preference and affinity. Results highlight a weak human receptor preference of the H7N9 HAs, suggesting that these viruses require further adaptation in order to adapt fully to humans.     

Antigenic Mapping of the Hemagglutinin of an H9N2 Avian Influenza Virus Reveals Novel Critical Amino Acid Positions in Antigenic Sites

H9N2 influenza virus is undergoing extensive genetic and antigenic evolution, warranting detailed antigenic mapping of its hemagglutinin (HA). Through examining antibody escape mutants of an Asian avian H9N2 virus, we identified 9 critical amino acid positions in H9 antigenic sites. Five of these positions, 164, 167, 168, 196, and 207, have not been reported previously and, thus, represent novel molecular markers for monitoring the antigenic change of H9N2 virus.     

Predicting the Antigenic Structure of the Pandemic (H1N1) 2009 Influenza Virus Hemagglutinin

The pandemic influenza virus (2009 H1N1) was recently introduced into the human population. The hemagglutinin (HA) gene of 2009 H1N1 is derived from “classical swine H1N1” virus, which likely shares a common ancestor with the human H1N1 virus that caused the pandemic in 1918, whose descendant viruses are still circulating in the human population with highly altered antigenicity of HA. However, information on the structural basis to compare the HA antigenicity among 2009 H1N1, the 1918 pandemic, and seasonal human H1N1 viruses has been lacking. By homology modeling of the HA structure, here we show that HAs of 2009 H1N1 and the 1918 pandemic virus share a significant number of amino acid residues in known antigenic sites, suggesting the existence of common epitopes for neutralizing antibodies cross-reactive to both HAs. It was noted that the early human H1N1 viruses isolated in the 1930s–1940s still harbored some of the original epitopes that are also found in 2009 H1N1. Interestingly, while 2009 H1N1 HA lacks the multiple N-glycosylations that have been found to be associated with an antigenic change of the human H1N1 virus during the early epidemic of this virus, 2009 H1N1 HA still retains unique three-codon motifs, some of which became N-glycosylation sites via a single nucleotide mutation in the human H1N1 virus. We thus hypothesize that the 2009 H1N1 HA antigenic sites involving the conserved amino acids will soon be targeted by antibody-mediated selection pressure in humans. Indeed, amino acid substitutions predicted here are occurring in the recent 2009 H1N1 variants. The present study suggests that antibodies elicited by natural infection with the 1918 pandemic or its early descendant viruses play a role in specific immunity against 2009 H1N1, and provides an insight into future likely antigenic changes in the evolutionary process of 2009 H1N1 in the human population.     

Mutations in H5N1 Influenza Virus Hemagglutinin that Confer Binding to Human Tracheal Airway Epithelium

The emergence in 2009 of a swine-origin H1N1 influenza virus as the first pandemic of the 21st Century is a timely reminder of the international public health impact of influenza viruses, even those associated with mild disease. The widespread distribution of highly pathogenic H5N1 influenza virus in the avian population has spawned concern that it may give rise to a human influenza pandemic. The mortality rate associated with occasional human infection by H5N1 virus approximates 60%, suggesting that an H5N1 pandemic would be devastating to global health and economy. To date, the H5N1 virus has not acquired the propensity to transmit efficiently between humans. The reasons behind this are unclear, especially given the high mutation rate associated with influenza virus replication. Here we used a panel of recombinant H5 hemagglutinin (HA) variants to demonstrate the potential for H5 HA to bind human airway epithelium, the predominant target tissue for influenza virus infection and spread. While parental H5 HA exhibited limited binding to human tracheal epithelium, introduction of selected mutations converted the binding profile to that of a current human influenza strain HA. Strikingly, these amino-acid changes required multiple simultaneous mutations in the genomes of naturally occurring H5 isolates. Moreover, H5 HAs bearing intermediate sequences failed to bind airway tissues and likely represent mutations that are an evolutionary “dead end.” We conclude that, although genetic changes that adapt H5 to human airways can be demonstrated, they may not readily arise during natural virus replication. This genetic barrier limits the likelihood that current H5 viruses will originate a human pandemic.     

Newcastle Disease Virus-Vectored Vaccines Expressing the Hemagglutinin or Neuraminidase Protein of H5N1 Highly Pathogenic Avian Influenza Virus Protect against Virus Challenge in Monkeys

H5N1 highly pathogenic avian influenza virus (HPAIV) causes periodic outbreaks in humans, resulting in severe infections with a high (60%) incidence of mortality. The circulating strains have low human-to-human transmissibility; however, widespread concerns exist that enhanced transmission due to mutations could lead to a global pandemic. We previously engineered Newcastle disease virus (NDV), an avian paramyxovirus, as a vector to express the HPAIV hemagglutinin (HA) protein, and we showed that this vaccine (NDV/HA) induced a high level of HPAIV-specific mucosal and serum antibodies in primates when administered through the respiratory tract. Here we developed additional NDV-vectored vaccines expressing either HPAIV HA in which the polybasic cleavage site was replaced with that from a low-pathogenicity strain of influenza virus [HA(RV)], in order to address concerns of enhanced vector replication or genetic exchange, or HPAIV neuraminidase (NA). The three vaccine viruses [NDV/HA, NDV/HA(RV), and NDV/NA] were administered separately to groups of African green monkeys by the intranasal/intratracheal route. An additional group of animals received NDV/HA by aerosol administration. Each of the vaccine constructs was highly restricted for replication, with only low levels of virus shedding detected in respiratory secretions. All groups developed high levels of neutralizing antibodies against homologous and heterologous strains of HPAIV and were protected against challenge with 2 × 10⁷ PFU of homologous HPAIV. Thus, needle-free, highly attenuated NDV-vectored vaccines expressing either HPAIV HA, HA(RV), or NA have been developed and demonstrated to be individually immunogenic and protective in a primate model of HPAIV infection. The finding that HA(RV) was protective indicates that it would be preferred for inclusion in a vaccine. The study also identified NA as an independent protective HPAIV antigen in primates. Furthermore, we demonstrated the feasibility of aerosol delivery of NDV-vectored vaccines.H5N1 highly pathogenic avian influenza virus (HPAIV) was first detected in human infections in 1997; previously, it had been found only in birds (11, 50). To date, this virus has been identified in 436 confirmed cases of human infection in 15 countries, 262 (60%) of which were fatal (75). The currently circulating H5N1 strains are characterized by low human-to-human transmissibility. This has been attributed, in part, to a preference for binding to α-2,3-linked sialic acids that are present in high concentrations throughout the avian respiratory tract but were thought to be found primarily in the lower human respiratory tract (57), although this explanation has been questioned (48, 49). It has also been observed that mutations in the PB2 subunit of the viral polymerase are necessary to confer the ability for the virus to be spread by aerosolized nasal droplets in ferrets (72). Whatever factors may be involved, there is widespread concern that the avian virus could mutate to enhance its transmissibility among humans, possibly resulting in a global pandemic (28, 50). For the avian H9N2 virus, which also has pandemic potential, it has been demonstrated that only five amino acid changes were sufficient for the virus to gain the ability to be spread by aerosolized nasal droplets in a ferret model (60). Thus, there is an urgent need for vaccines against HPAIV.Several vaccine strategies for HPAIV have been evaluated (reviewed in references 32 and 41), including inactivated and live attenuated vaccines. These efforts have been hampered by several factors. HPAIV strains are highly virulent for embryonated chicken eggs, the most widely used substrate for vaccine manufacture, and their rapid death following inoculation renders eggs unsuitable for efficient virus propagation. In addition, the major protective antigen, hemagglutinin (HA), administered either as a purified protein or in inactivated HPAIV virions, appears to be poorly immunogenic (69, 70). An additional factor complicating the development of HPAIV vaccines based on inactivated virus is the high cost and biohazard associated with HPAIV propagation, which must be done under enhanced biosafety level 3 (BSL-3) containment, although this problem might be addressed by the use of live attenuated reassortant influenza virus vaccines that contain the HPAIV glycoproteins on the background of an avirulent human influenza virus strain (24, 37). In addition, such reassortant strains might serve directly as live attenuated vaccines. Unfortunately, the latter approach may be limited by subtle and unpredictable incompatibility between the avian-origin glycoproteins and human-origin vaccine backgrounds acceptable for human use, which can result in overattenuation in vivo (24). There are also lingering concerns about the significant potential, with a live HPAIV vaccine, for reassortment between gene segments of the vaccine virus and circulating influenza virus strains, which might result in novel strains with unpredictable biological properties (63).We and others have been evaluating Newcastle disease virus (NDV) as a general human vaccine vector for emerging pathogens, including H5N1 HPAIV (7, 18-20, 29). NDV is an avian paramyxovirus that is antigenically unrelated to common human pathogens; hence, its use in humans should not be affected by host immunity to common pathogens. The many naturally occurring strains of NDV can be categorized into three pathotypes based on virulence in chickens: velogenic strains, causing severe disease with high mortality; mesogenic strains, causing disease of intermediate severity with low mortality; and lentogenic strains, causing mild or inapparent infections (reviewed in reference 2). Lentogenic, and sometimes mesogenic, strains of NDV are in wide use as live attenuated vaccines against velogenic NDV in poultry (2). When mesogenic or lentogenic NDV was administered to the respiratory tracts of nonhuman primates as a model for the immunization of humans, the virus was highly attenuated for replication, was shed only at low titers, appeared to remain restricted to the respiratory tract, and was highly immunogenic for the expressed foreign antigen (7). We recently demonstrated that a mesogenic strain of NDV expressing the HA protein of H5N1 HPAIV (NDV/HA) elicited high titers of neutralizing antibodies in serum following combined intranasal (i.n.) and intratracheal (i.t.) delivery in a nonhuman primate model (20). Vaccination of mice with a similar NDV-vectored vaccine protected them from HPAIV challenge (29). However, results obtained with mice do not reliably predict the efficacy of an influenza virus vaccine for human use, due to the pathophysiological and phylogenetic differences between mice and humans (71). In particular, mice may produce a potent immune response to HPAIV vaccines (64) that may not be reproduced in clinical trials (38). These considerations are especially important for a vaccine based on a live viral vector platform, since its immunogenicity, and therefore its protective efficacy, is directly linked to replication, which can differ greatly in various experimental animals versus humans (reviewed in references 6 and 9). Therefore, the protective efficacy of NDV-based vaccines against HPAIV challenge in nonhuman primate models—the closest model to humans—has remained unknown.The protease recognition sequence of the HA protein is one of the major determinants of avian influenza virus pathogenicity (62). HPAIV strains have a “polybasic” cleavage site, containing multiple basic amino acids, that is readily cleaved by ubiquitous intracellular subtilisin-like proteases, facilitating the replication and spread of the virus. In contrast, the HA cleavage site of low-pathogenicity strains contains fewer basic amino acids and depends on secretory trypsin-like proteases found in the respiratory and enteric tracts, resulting in more-localized infections (30, 62). The presence of a polybasic cleavage site in the H5 HA of any live vaccine raises some concern about the possibility of genetic exchange with circulating strains of influenza virus. It should be noted that genetic exchange involving paramyxoviruses is a rare event (14) that has been documented only once (61). However, elimination of the polybasic HA cleavage site would mitigate the effects of even this rare possibility of genetic exchange. Another concern was based on our previous finding that the HPAIV H5 HA protein is incorporated into the NDV envelope as a trimer (20), consistent with its presence in a functional form. While we previously showed that this did not enhance the pathogenicity of the NDV/HA recombinant in chickens (20), we could not rule out the possibility that it might confer an altered tropism on the NDV/HA virus in other systems. For example, a recombinant parainfluenza virus type 3 expressing the Ebola virus glycoprotein incorporated the foreign protein into its envelope, allowing cellular attachment and fusion of the vaccine virus independently of the vector''s own envelope glycoproteins (10).In addition to the HA protein, the neuraminidase (NA) protein is also present on the surfaces of influenza virus-infected cells and virions. Antibodies specific for NA are not thought to interfere with the initial viral attachment and penetration of host cells (36, 40, 54). However, NA-specific antibodies prevent the release of virus from infected cells, thereby decreasing viral spread (35), and they increase resistance to viral infection in humans (40, 47, 54). They also provide at least some protection against viruses bearing homologous or heterologous NA proteins of the same subtype in a mouse model (12, 56). NA also appears to evolve at a lower rate than HA, suggesting that NA-specific antibodies may provide broader protection than a vaccine utilizing HA alone (39). Therefore, it was important to assess the immunogenicity and protective efficacy of the HPAIV NA independently of those of HA, which has not previously been done in a human or nonhuman primate model.     

H3 Stalk-Based Chimeric Hemagglutinin Influenza Virus Constructs Protect Mice from H7N9 Challenge

The recent outbreak of H7N9 influenza virus infections in humans in China has raised concerns about the pandemic potential of this strain. Here, we test the efficacy of H3 stalk-based chimeric hemagglutinin universal influenza virus vaccine constructs to protect against H7N9 challenge in mice. Chimeric hemagglutinin constructs protected from viral challenge in the context of different administration routes as well as with a generic oil-in-water adjuvant similar to formulations licensed for use in humans.     

Nonlinkage of Neurovirulence Exclusively to Viral Hemagglutinin or Neuraminidase in Genetic Recombinants of A/NWS (H0N1) Influenza Virus

Genetic recombination of the neurovirulent A/NWS/cc-p (H0N1) and the non-neurovirulent A/Jap.305/57 (H2N2) influenza viruses in which hemagglutinin and neuraminidase were segregated (H0N2, H2N1) were studied for neurovirulence in mice immunosuppressed with cyclophosphamide (CPA) which permitted full expression of virulence. Both H0N2 and H2N1 recombinants replicated in the brain (in contrast to the H2N2 parent) and both produced lethal effects in CPA-treated animals. Therefore we conclude that A/NWS (H0N1) neurovirulence is not exclusively linked with either the hemagglutinin or the neuraminidase of the virus. The H0N2 and H2N1 recombinants have revealed the existence of two separate attributes of neurovirulence: (i) the capacity of virus to initiate intracerebral infection and (ii) the capacity of infection, once initiated, to produce lethal disease. These studies provide further evidence for the polygenic nature of A/NWS neurovirulence.     

Quantitative Characterization of Glycan-Receptor Binding of H9N2 Influenza A Virus Hemagglutinin

Avian influenza subtypes such as H5, H7 and H9 are yet to adapt to the human host so as to establish airborne transmission between humans. However, lab-generated reassorted viruses possessing hemagglutinin (HA) and neuraminidase (NA) genes from an avian H9 isolate and other genes from a human-adapted (H3 or H1) subtype acquired two amino acid changes in HA and a single amino acid change in NA that confer respiratory droplet transmission in ferrets. We previously demonstrated for human-adapted H1, H2 and H3 subtypes that quantitative binding affinity of their HA to α2→6 sialylated glycan receptors correlates with respiratory droplet transmissibility of the virus in ferrets. Such a relationship remains to be established for H9 HA. In this study, we performed a quantitative biochemical characterization of glycan receptor binding properties of wild-type and mutant forms of representative H9 HAs that were previously used in context of reassorted viruses in ferret transmission studies. We demonstrate here that distinct molecular interactions in the glycan receptor-binding site of different H9 HAs affect the glycan-binding specificity and affinity. Further we show that α2→6 glycan receptor-binding affinity of a mutant H9 HA carrying Thr-189→Ala amino acid change correlates with the respiratory droplet transmission in ferrets conferred by this change. Our findings contribute to a framework for monitoring the evolution of H9 HA by understanding effects of molecular changes in HA on glycan receptor-binding properties.     

Antigenic variability in Neuraminidase protein of Influenza A/H3N2 vaccine strains (1968 - 2009)

Antigenic drift and shift involving the surface proteins of Influenza virus gave rise to new strains that caused epidemics affecting millions of people worldwide over the last hundred years. Variations in the membrane proteins like Hemagglutinin (HA) and Neuraminidase (NA) necessitates new vaccine strains to be updated frequently and poses challenge to effective vaccine design. Though the HA protein, the primary target of the human immune system, has been well studied, reports on the antigenic variability in the other membrane protein NA are sparse. In this paper we investigate the molecular basis of antigenic drift in the NA protein of the Influenza A/H3N2 vaccine strains between 1968 and 2009 and proceed to establish correlation between antigenic drift and antigen-antibody interactions. Sequence alignments and phylogenetic analyses were carried out and the antigenic variability was evaluated in terms of antigenic distance. To study the effects of antigenic drift on the protein structures, 3D structure of NA from various strains were predicted. Also, rigid body docking protocol has been used to study the interactions between these NA proteins and antibody Mem5, a 1998 antibody.     

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.     

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.     

流感病毒H3N2血凝素基因和神经氨酸酶基因在大肠杆菌中的表达

克隆、表达和鉴定流感病毒H3N2 HA,NA基因序列,为制备抗体和基因工程疫苗打下基础。在成功克隆流感病毒H3N2全长HA、NA基因并测序的基础上,将部分基因序列克隆到表达载体pET32a(+)上,全基因序列克隆到表达载体pGEX4T-1上,构建了重组表达质粒pET32a(+)/HA(截短),pET32a(+)/NA(截短),pGEX4T-1/HA,转化大肠杆菌BL21/Rosetta,IPTG诱导表达,利用Ni2+亲和层析柱和GSTrap 4B亲和层析柱对重组蛋白进行纯化,并用Western blotting和ELISA方法检测其抗原性。结果显示,重组蛋白在大肠杆菌中可以高效表达,SDS-PAGE显示其相对分子质量与预计大小一致。ELISA和Western blotting试验证实,重组蛋白具有良好的抗原性。成功克隆和表达了流感病毒H3N2 HA、NA基因序列,可为流感病毒H3N2诊断试剂和疫苗的开发等进一步的研究提供参考。     

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.