HA2 subunit of influenza A H1 and H2 subtype viruses induces a protective cross-reactive cytotoxic T lymphocyte response

Influenza H1 subtype-specific CTL can be induced by secondary stimulation of a hybrid protein of the first 81 amino acids of the viral NS1 non-structural protein and the HA2 subunit of A/Puerto Rico/8/34(H1N1) hemagglutinin. In addition, a derivative of this protein with 65 amino acids deleted from the N-terminal end of HA2 can also generate H1 subtype-specific CTL in bulk cultures. CTL clones established by stimulation with the derivative protein demonstrated cross-reactive lysis of target cells infected with virus strains of the H1 and H2 subtypes. Cold target competition experiments with CTL clones as effectors demonstrated that the Ag specificity between these two hybrid proteins is identical. Adoptive transfer of the CTL clone significantly reduced virus titers in the lungs of mice infected with the virus strains of the H1 or H2 subtype but not those infected with the H3 subtype virus in vivo, which reflects the in vitro CTL clone activity. These experiments demonstrate that an epitope on the hemagglutinin that is conserved on virus strains of the H1 and H2 subtypes induces a protective CTL response. These results suggest an alternative approach for developing influenza vaccines by using conserved antigenic sites on the hemagglutinin HA2 subunit to avoid the problem of frequent antigenic mutations of the HA1 subunit antibody binding sites.     

Characterization of subtype-specific and cross-reactive helper-T-cell clones recognizing influenza virus hemagglutinin

The specificity and function of two T-cell clones derived from A/Memphis/1/71 (H3) influenza virus (Mem 71)-immune BALB/c spleen cells have been compared. One clone, X-31 clone 1, was subtype specific, proliferating in response to influenza strains of the H3 subtype only. The other, Jap clone 3, cross-reacted in proliferation assays with heterologous subtypes of influenza A, but not type B. Both clones recognized the HA1 chain of the hemagglutinin (HA) molecule and their proliferation in response to detergent-disrupted virus could be specifically inhibited by monoclonal antibodies to the HA. The T-cell clones were of the L3T4+ phenotype. Both recognized antigen in association with I-Ed, as indicated by studies with H-2 recombinant strains of mice and by blocking with monoclonal anti-I-E antibody. In vivo, both clones elicited a delayed-type hypersensitivity (DTH) reaction when inoculated into mouse footpads together with virus, X-31 clone 1 again displaying subtype specificity and Jap clone 3 being cross-reactive. The clones were also able to provide factor-mediated help in vitro to virus-primed B cells in an anti-HA antibody response. The cross-reactive T-cell clone provided help not only for B cells primed with influenza A subtype H3 and responding to H3 virus in culture, but also for H2 virus-primed B cells making anti-H2 antibody.     

Fine specificity analysis of human influenza-specific cloned cell lines

Influenza-specific human-T-cell clones, proliferating in the presence of virus-infected cells with restriction by class II molecules and displaying class II-restricted CTL activity or specific helper activity in antibody synthesis, have been analyzed for antigenic specificities. All of them were obtained by in vitro stimulation against influenza A/Texas virus. In all cases the virus specificity appeared identical in cytolytic and proliferative responses. Three of the clones were broadly cross-reactive, recognizing all or almost all type A influenza strains. The three remaining clones were subtype specific when tested with human strains and recognized the surface glycoproteins of influenza virus. One of these lines reacted with an epitope of the neuraminidase N2 while the other two recognized the hemagglutinin H3. By using a large panel of mammalian and avian influenza strains, it can be demonstrated that hemagglutinin-specific human T cells can recognize a cross-reacting determinant shared by H3 and H4 subtypes of hemagglutinin which has never been detected with antibodies.     

Hemagglutinin Stalk-Based Universal Vaccine Constructs Protect against Group 2 Influenza A Viruses

Current influenza virus vaccines contain H1N1 (phylogenetic group 1 hemagglutinin), H3N2 (phylogenetic group 2 hemagglutinin), and influenza B virus components. These vaccines induce good protection against closely matched strains by predominantly eliciting antibodies against the membrane distal globular head domain of their respective viral hemagglutinins. This domain, however, undergoes rapid antigenic drift, allowing the virus to escape neutralizing antibody responses. The membrane proximal stalk domain of the hemagglutinin is much more conserved compared to the head domain. In recent years, a growing collection of antibodies that neutralize a broad range of influenza virus strains and subtypes by binding to this domain has been isolated. Here, we demonstrate that a vaccination strategy based on the stalk domain of the H3 hemagglutinin (group 2) induces in mice broadly neutralizing anti-stalk antibodies that are highly cross-reactive to heterologous H3, H10, H14, H15, and H7 (derived from the novel Chinese H7N9 virus) hemagglutinins. Furthermore, we demonstrate that these antibodies confer broad protection against influenza viruses expressing various group 2 hemagglutinins, including an H7 subtype. Through passive transfer experiments, we show that the protection is mediated mainly by neutralizing antibodies against the stalk domain. Our data suggest that, in mice, a vaccine strategy based on the hemagglutinin stalk domain can protect against viruses expressing divergent group 2 hemagglutinins.     

A common neutralizing epitope conserved between the hemagglutinins of influenza A virus H1 and H2 strains.

When mice were immunized with the A/Okuda/57 (H2N2) strain of influenza virus, a unique monoclonal antibody designated C179 was obtained. Although C179 was confirmed to recognize the hemagglutinin (HA) glycoprotein by immunoprecipitation assays, it did not show hemagglutination inhibition activity to any of the strains of the three subtypes of influenza A virus. However, it neutralized all of the H1 and H2 strains but not the H3 strains. Moreover, it inhibited polykaryon formation induced by the H1 and H2 strains but not by the H3 strains. Two antigenic variants against C179 were obtained, and nucleotide sequence analysis revealed that amino acid sequences, from 318 to 322 of HA1 and from 47 to 58 of HA2, conserved among H1 and H2 strains were responsible for the recognition of C179. Since the two sites were located close to each other at the middle of the stem region of the HA molecule, C179 seemed to recognize these sites conformationally. These data indicated that binding of C179 to the stem region of HA inhibits the fusion activity of HA and thus results in virus neutralization and inhibition of cell-cell fusion. This is the first report which describes the presence of conserved antigenic sites on HA not only in a specific subtype but also in two subtypes of influenza A virus.     

The analysis of the monoclonal immune response to influenza virus. III. The relationship between stimulation of virus-primed precursor B cells by heterologous viruses and reactivity of secreted antibodies.

Individual splenic precursor B cells from BALB/c mice primed with influenza virus PR8[A/PR/8/34 (H0N1)] were stimulated in vitro in the splenic fragment culture system by homologous or various heterologous influenza viruses. The specificity of the stimulated precursor cells was determined by analysis of the antibodies secreted by the ensuing plasma cell clone in a radioimmunoassay (RIA). Viruses of the H2N2 and H3N2 subtypes were unable to stimulate hemagglutinin (HA)- or neuraminidase (NA)-committed precursor B cells but did efficiently stimulate chicken host component (ChHC)-committed precursors. Viruses of the H1N1 and H0N1 subtypes could stimulate precursors committed to any of the three viral surface components. Analysis of the fine specificity of HA-committed B cells showed that BEL(H0N1) and CAM(H1N1) stimulated almost exclusively precursors whose clonal antibody product reacted with the stimulating virus in the RIA. On the other hand, WSE and MEL (both H0N1) quite frequently were able to stimulate precursors whose clonal antibody product did not react with the stimulating virus in the RIA. These results suggest that the stimulatory interaction of viruses with the cell-bound immunoglobulin receptors is slightly less affinity dependent than the antigen-antibody interaction in the RIA.     

Determinants of glycan receptor specificity of H2N2 influenza A virus hemagglutinin

The H2N2 subtype of influenza A virus was responsible for the Asian pandemic of 1957-58. However, unlike other subtypes that have caused pandemics such as H1N1 and H3N2, which continue to circulate among humans, H2N2 stopped circulating in the human population in 1968. Strains of H2 subtype still continue to circulate in birds and occasionally pigs and could be reintroduced into the human population through antigenic drift or shift. Such an event is a potential global health concern because of the waning population immunity to H2 hemagglutinin (HA). The first step in such a cross-species transmission and human adaptation of influenza A virus is the ability for its surface glycoprotein HA to bind to glycan receptors expressed in the human upper respiratory epithelia. Recent structural and biochemical studies have focused on understanding the glycan receptor binding specificity of the 1957-58 pandemic H2N2 HA. However, there has been considerable HA sequence divergence in the recent avian-adapted H2 strains from the pandemic H2N2 strain. Using a combination of structural modeling, quantitative glycan binding and human respiratory tissue binding methods, we systematically identify mutations in the HA from a recent avian-adapted H2N2 strain (A/Chicken/PA/2004) that make its quantitative glycan receptor binding affinity (defined using an apparent binding constant) comparable to that of a prototypic pandemic H2N2 (A/Albany/6/58) HA.     

Entry of Influenza A Virus with a α2,6-Linked Sialic Acid Binding Preference Requires Host Fibronectin

The receptor binding specificity of influenza A virus is one of the major determinants of viral tropism and host specificity. In general, avian viral hemagglutinin prefers to bind to α2,3-linked sialic acid, whereas the human viral hemagglutinin prefers to bind to α2,6-linked sialic acid. Here, we demonstrate that host fibronectin protein plays an important role in the life cycle of some influenza A viruses. Treating cells with anti-fibronectin antibodies or fibronectin-specific small interfering RNA can inhibit the virus replication of human H1N1 influenza A viruses. Strikingly, these inhibitory effects cannot be observed in cells infected with H5N1 viruses. By using reverse genetics techniques, we observed that the receptor binding specificity, but not the origin of the hemagglutinin subtype, is responsible for this differential inhibitory effect. Changing the binding preference of hemagglutinin from α2,6-linked sialic acid to α2,3-linked sialic acid can make the virus resistant to the anti-fibronectin antibody treatment and vice versa. Our further characterizations indicate that anti-fibronectin antibody acts on the early phase of viral replication cycle, but it has no effect on the initial binding of influenza A virus to cell surface. Our subsequent investigations further show that anti-fibronectin antibody can block the postattachment entry of influenza virus. Overall, these results indicate that the sialic acid binding preference of influenza viral hemagglutinin can modulate the preferences of viral entry pathways, suggesting that there are subtle differences between the virus entries of human and avian influenza viruses.     

Hemagglutinin Stalk-Reactive Antibodies Are Boosted following Sequential Infection with Seasonal and Pandemic H1N1 Influenza Virus in Mice

Previously, it has been shown that infection in humans with the pandemic swine influenza virus induces antibodies with specificity to the stalk domain of the viral hemagglutinin. Following the generation of these data, we sought to recapitulate these findings in the mouse model by sequential influenza virus infection. Mice that were inoculated with a seasonal influenza H1N1 virus followed by infection with a pandemic H1N1 strain produced higher antihemagglutinin stalk antibody titers than mice sequentially infected with drifted seasonal strains. In order to achieve antibody titers of comparable magnitude using sequential infection, mice had to be infected with 100- to 1,000-fold more of the drifted seasonal virus. The antistalk antibodies produced by these infections were influenza virus neutralizing, which illustrates the utility of the mouse model in which to study this interaction between virus and host.     

In vitro antibody response to influenza virus. II. Specificity of helper T cell recognizing hemagglutinin

Intraperitoneal immunization of mice with liver influenza virus was shown to induce helper T (TH) cells with specificity for the hemagglutinin (HA). The interaction of virus-primed TH cells with purified HA was studied independently of B cell reactivity to the same antigen by using the generation of nonspecific help as an index of activation of HA-specific TH cells. TH cells from mice primed with any of the H3 viruses A/Aichi/68 X A/Bel/42 (H3N1), A/Memphis/102/72 X A/Bel/42 (H3N1) or A/Port Chalmers/73 (H3N2) were strongly cross-reactive towards HA of other strains within the H3 subtype. In addition, several examples of cross-reactivity towards HA of a different subtype were observed, usually of a lower magnitude. TH cells from mice primed to any of the H3 viruses above or to A/Bel/42 (H1N1) virus cross-reacted with the HA of A/Japan/305/57 (H2); furthermore, priming with A/Bel/42 or with A/Jap/305/57 X A/Bel/42 (h2N1) virus yielded TH cells that cross-reacted with certain of the H3 HA preparations. The cross-reactivity observed between subtypes was not due to the common chicken host carbohydrate component of HA, since no response to the purified type A HA preparations was obtained with T cells from mice primed with egg-grown influenza B/Hong-Kong/8/73 virus. The results indicate that HA of different subtypes may share cross-reactive antigenic determinants recognized by TH cells. Within a subtype, HA are highly cross-reactive with respect to tH cell recognition.     

禽流感病毒A型和H5亚型RT-PCR检测试剂盒研究

目的　检测和鉴定A型、H5亚型禽流感病毒 (AIV) ,研发一种高效实用的检测手段。方法　根据Ming ShiuhLee报道的文献设计、合成引物 ,采用反转录和PCR一步法对A型、H5亚型禽流感病毒cDNA进行扩增和电泳鉴定 ,组装成禽流感病毒RT PCR试剂盒 ,对H1～ 15亚型AIV参考株、38份AIV国内分离株进行检测试验。结果　建立了A型、H5亚型禽流感病毒RT PCR检测方法 ,并在此基础上组装试剂盒 ,用A型试剂盒检测时 ,全部AIV毒株均为阳性 ,能检测 1 10 2 4血凝单位禽流感病毒 ;用H5亚型试剂盒检测时 ,仅有H5亚型AIV参考株和 19株H5亚型AIV分离株呈阳性 ,其余H1～H4、H6～H15参考株和H7、H9分离株以及 1株H5分株均为阴性 ,能检测1 6 4血凝单位禽流感病毒。 2种试剂盒对实验感染鸡病料检出率均为 10 0 %。结论　研制的AIVA型、H5亚型RT PCR试剂盒具有特异性强、敏感性高、稳定性和重复性好的特点。     

Induction of immune response to influenza virus with anti-idiotypic antibodies.

Anti-idiotypic (anti-Id) antibodies were raised in rabbits against five monoclonal antibodies (MAbs) specific for different antigenic sites on the hemagglutinin (HA) of influenza virus Mem71H-BelN (H3N1) [A/Memphis/1/71 (H3N2) x A/Bel/42 (H1N1)]. Each of the anti-Id sera was directed predominantly towards a unique (private) idiotype of the immunizing MAb, none of the five idiotypes being detectable in pooled BALB/c antisera against Mem71H-BelN virus or on most other anti-HA MAbs tested. Partial idiotypic sharing was observed, however, between certain MAbs, from different mice, having the same or similar epitope specificity for HA. When used as immunogens in BALB/c mice, two of the five anti-Id preparations induced antibodies that reacted with Mem71H-BelN virus and displayed neutralizing activity. Mice of other inbred strains responded similarly, indicating that the response was not genetically restricted by the Igh locus. From their pattern of reactivity with mutants of Mem71H-BelN virus with known single amino acid substitutions in the HA molecule, the antiviral antibodies elicited by anti-Id antibodies were shown to be directed to the same antigenic site on A/Memphis/1/71 HA as the original immunizing MAb (site A or site E, respectively). However, several of these antisera were shown to contain additional distinct subpopulations of antibodies specific for heterologous influenza A virus strains, either of the H3 subtype or of a different HA subtype (H1 or H2). Since the induction of antibodies to HA of different subtypes is not a feature of the antibody response to influenza virus itself, their induction by anti-Id antibodies merits further investigation.     

Antigenic variation of influenza A virus nucleoprotein detected with monoclonal antibodies.

Monoclonal antibodies were used to study antigenic variation in the nucleoprotein of influenza A viruses. We found that the nucleoprotein molecule of the WSN/33 strain possesses at least five different determinants. Viruses of other influenza A virus subtypes showed antigenic variation in these nucleoprotein determinants, although changes in only one determinant were detected in H0N1 and animal strains. The nucleoprotein of human strains isolated from 1933 through 1979 could be divided into six groups, based on their reactivities with monoclonal antibodies; these groups did not correlate with any particular hemagglutinin or neuraminidase subtype. Our results indicate that antigenic variation in the nucleoproteins of influenza A viruses proceeds independently of changes in the viral surface antigens and suggest that point mutations and genetic reassortment may account for nucleoprotein variability.     

辽宁省2016‒2017年H3N2亚型流感病毒 HA1基因特征分析

摘要：目的 了解2016?2017年辽宁省H3N2亚型流感病毒基因变异情况及流行株与疫苗株的匹配情况。方法 采用逆转录聚合酶链反应（RT-PCR）对分离得到的H3N2亚型流感毒株的HA1基因进行扩增,扩增片段经测序与近年来WHO推荐的北半球疫苗株进行比对和基因特征分析。结果 进化分析表明,2016?2017年H3N2亚型流感病毒与近三年的疫苗株均不在同一分支上;基因特性分析中,所有病毒均在A、B抗原决定簇上发生了两处以上的变异;19株病毒的受体结合位点131位氨基酸发生了新的变异;20株病毒中有1株突变产生了新的半胱氨酸,提示可能有新的二硫键产生;糖基化位点并未检测到新的突变。结论 2016?2017年辽宁省H3N2亚型流感病毒的抗原性及基因特性均发生了一定的变化,但变异程度不大,应密切关注疫苗株对流感病毒的免疫效果及流感毒株的变异情况。     

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.     

<Emphasis Type="Italic">In silico</Emphasis> 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 2,3- and 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.     

Formation of mixed hemagglutinin trimers during double infection with influenza virus strains of the same subtype

Formation of hemagglutinin spikes in the course of the mixed infection of cell culture by two influenza virus strains belonging to the same antigenic subtype or to different subtypes was studied by means of immunoprecipitation of [14C]-labelled hemagglutinins from cell lysates. The immunoprecipitates were further analysed by polyacrylamide gel electrophoresis. Lysates of separately infected cells mixed before lysis were used as control samples. The analysis of immunoprecipitates revealed the formation of chimeric hemagglutinin spikes in the cells infected by the strains possessing hemagglutinins of the same subtype but not in the cells infected by the strains of different subtypes (H1 and H3). The results are discussed in connection with the homology of amino-acid sequences of influenza virus hemagglutinins.     

Subtyping of influenza virus A hemagglutinin with a hybridization microarray

An oligonucleotide microarray for influenza A hemagglutinin subtyping was presented. The number of probes for the determination of each subtype of hemagglutinin (H1-H13, H15, H16, pandemic flu H1N1) varied from 13 to 28. When testing the microarray using 40 type-A influenza virus isolates, the hemagglutinin subtypes were unambiguously determined for 36 specimens.     

Changes in the phenotypic properties of highly pathogenic influenza A virus of H5N1 subtype induced by N186I and N186T point mutations in hemagglutinin

The change in the phenotypic properties resulting from amino acid substitutions in the hemagglutinin (HA) molecule is an important link in the evolutionary process of influenza viruses. It is believed to be one of the mechanisms of the emergence of highly pathogenic strains of influenza A viruses, including subtype H5N1. Using the site-directed mutagenesis, we introduced mutations in the HA gene of the H5N1 subtype of influenza A virus. The obtained virus variants were analyzed and compared using the following parameters: optimal pH of conformational transition (according to the results of the hemolysis test), specificity of receptor binding (using a set of synthetic analogues of cell surface sialooligosaccharides), thermoresistance (heat-dependent reduction of hemagglutinin activity), virulence in mice, and the kinetics of replication in chicken embryos, and reproductive activity at different temperatures (RCT-based). N186I and N186T mutations in the HA protein increased the virulence of the original virus in mice. These mutations accelerated virus replication in the early stages of infection in chicken embryos and increased the level of replication at late stages. In addition, compared to the original virus, the mutant variants replicated more efficiently at lower temperatures. The obtained data clearly prove the effect of amino acid substitutions at the 186 position of HA on phenotypic properties of the H5N1 subtype of influenza A.