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.     

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.     

Immunological studies with the HA1 and HA2 polypeptides of influenza A virus haemagglutinin.

HA1 and HA2 polypeptides of influenza A virus haemagglutinin (HA) were separated in purified form using electrophoresis in SDS containing polyacrylamide gels (PAGE) or chloroform-methanol extraction. The populations of HA1 polypeptides were immunogenic but considerably less so than the intact HA molecule and induced antibody which cross-reacted with influenza A and B viruses. After absorption with heterologous influenza B virus, the cross-reacting antibodies were removed and the HA1 antisera then possessed antibodies which reacted only with the cross-reactive (CR) determinants of the HA of the homologous influenza A virus and viruses of the same subtype. Neither strain-specific (SS) nor virus-neutralizing antibodies were detected in these anti-HA1 sera. HA2 polypeptides were less immunogenic and anti-HA2 antisera after absorption with influenza B virus failed to react with influenza A virus in immuno double diffusion tests and only reacted with partially denatured HA in the more sensitive single radial diffusion tests.     

Recognition of cloned influenza virus hemagglutinin gene products by cytotoxic T lymphocytes.

The influenza A virus hemagglutinin (HA) is an integral membrane glycoprotein expressed in large quantities on infected cell surfaces and is known to serve as a target antigen for influenza virus-specific cytotoxic T lymphocytes (CTL). Despite the fact that HAs derived from different influenza A virus subtypes are serologically non-cross-reactive, the HA has been implicated by previous experiments to be a target antigen for the subset of T cells capable of lysing cells infected with any human influenza A subtype (cross-reactive CTL). To directly determine whether the HA is recognized by cross-reactive CTL, we used vaccinia virus recombinants containing DNA copies of the PR8 (A/Puerto Rico/8/34) (H1N1) or JAP (A/JAP/305) (H2N2) HA genes. When these viruses were used to stimulate HA-specific CTL and to sensitize target cells for lysis by HA-specific CTL, we found no evidence for HA recognition by cross-reactive CTL aside from a relatively small degree of cross-reactivity between H1 and H2 HAs. Results of unlabeled target inhibition studies were consistent with the conclusion that the HA is, at most, only a minor target antigen for cross-reactive CTL.     

Antigen-specific human T lymphocyte clones: viral antigen specificity of influenza virus-immune clones

Human T lymphocyte clones (TLC) specific for type A (A/Texas/1/77) influenza virus and maintained in continuous culture with T cell growth factor, were analyzed to define the cellular specificity pattern of virus recognition. A panel of TLC were stimulated with strains of serologically characterized type A influenza subtypes. Five TLC recognized all the viral subtypes; the remaining clones recognized only subtypes that shared serologically defined determinants with the immunizing subtype. In addition, the 11 TLC were analyzed for their fine antigenic specificity by using the purified viral components hemagglutinin (HA), neuraminidase (NA), matrix protein (MP), and nucleoprotein (NP). Five TLC proliferated in response to NA, four to MP, one to HA, and one to NP. None of the clones responded to the unrelated B strain influenza virus, B/Singapore. Furthermore, the fine specificity of an MP-reactive TLC was confirmed by subcloning.     

Human Cytotoxic T-Lymphocyte Repertoire to Influenza A Viruses

The murine CD8⁺ cytotoxic-T-lymphocyte (CTL) repertoire appears to be quite limited in response to influenza A viruses. The CTL responses to influenza A virus in humans were examined to determine if the CTL repertoire is also very limited. Bulk cultures revealed that a number of virus proteins were recognized in CTL assays. CTL lines were isolated from three donors for detailed study and found to be specific for epitopes on numerous influenza A viral proteins. Eight distinct CD8⁺ CTL lines were isolated from donor 1. The proteins recognized by these cell lines included the nucleoprotein (NP), matrix protein (M1), nonstructural protein 1 (NS1), polymerases (PB1 and PB2), and hemagglutinin (HA). Two CD4⁺ cell lines, one specific for neuraminidase (NA) and the other specific for M1, were also characterized. These CTL results were confirmed by precursor frequency analysis of peptide-specific gamma interferon-producing cells detected by ELISPOT. The epitopes recognized by 6 of these 10 cell lines have not been previously described; 8 of the 10 cell lines were cross-reactive to subtype H1N1, H2N2, and H3N2 viruses, 1 cell line was cross-reactive to subtypes H1N1 and H2N2, and 1 cell line was subtype H1N1 specific. A broad CTL repertoire was detected in the two other donors, and cell lines specific for the NP, NA, HA, M1, NS1, and M2 viral proteins were isolated. These findings indicate that the human memory CTL response to influenza A virus is broadly directed to epitopes on a wide variety of proteins, unlike the limited response observed following infection of mice.     

Virus specificity of human influenza virus-immune cytotoxic T cells.

The virus specificity of human in vitro cytotoxic T cell responses to influenza virus was studied with the use of peripheral blood mononuclear leukocytes from normal adult volunteers. Previous natural exposure of these donors to a variety of type A influenza viruses was documented by HI antibody titers. Cells sensitized in vitro with A/HK or A/PR8 were cytotoxic for autologous target cells infected with A/HK, A/PR8, or A/JAP 305 type A influenza viruses, but not for B/HK-infected or uninfected cells. B/HK-sensitized effector cells lysed target cells infected with B/HK but not targets infected with type A viruses. A/HK- and A/PR8-immune effector populations were shown to recognize cross-reactive antigens on A/HK- and A/PR8-infected target cells by cold target competition. Influenza-immune effector cells were cytotoxic for virus-infected autologous targets but much less so for virus-infected allogeneic targets. This self-restriction suggested that the cytotoxicity was largely T cell-mediated and was confirmed by cell separation analysis. Thus, the human secondary cytotoxic T cell response in vitro to influenza viruses is predominantly directed against cross-reactive determinants on cells infected with serologically distinct type A influenza viruses.     

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.     

A synthetic decapeptide of influenza virus hemagglutinin elicits helper T cells with the same fine recognition specificities as occur in response to whole virus

The immunogenicity of an isolated murine helper T cell determinant was studied. Mice were immunized with a synthetic peptide corresponding to amino acid residues 111-120 of the influenza PR8 hemagglutinin (HA) heavy chain, a region previously identified as a major target of the helper T cell response to the HA molecule in virus-primed BALB/c mice. Lymph node T cells from these mice were fused with BW 5147 cells to produce T hybrids for clonal analysis of their recognition specificities. Three T cell hybridoma clones, obtained from two different mice, responded to the immunizing peptide when presented by syngeneic antigen-presenting cells. All of these clones responded also to antigen provided as intact wild-type PR8 virus. The fine specificity of the peptide-induced T cell hybridomas, in response to a panel of mutant and variant influenza viruses, was indistinguishable from the fine specificities of T cells to the corresponding region of the HA1 chain of the HA molecule which had been generated by priming of mice with intact wild-type virus. These results suggest that an immunogenic determinant is contained within the 111-120 sequence that is able to elicit anti-influenza virus T cells with a similar repertoire to those elicited by immunization with whole virus.     

Genetic control and fine specificity of the immune response to a synthetic peptide of influenza virus hemagglutinin.

The immune response to a synthetic peptide, H3 HA1(305-328), representing the C'-terminal 24 amino acid residues of the HA1 chain of the hemagglutinin of the H3 subtype of influenza virus is controlled by genes in the I region of the major histocompatibility complex. Mice of the H-2d haplotype are high responders and produce antibody for several months after a single injection of peptide without carrier. Mice of the H-2b, H-2k, and H-2q haplotypes are low antibody responders. Investigation of recombinant and congenic mouse strains revealed that high responsiveness requires the genes that encode the I-Ed molecule. Immunoassays, involving direct binding to analogs of this peptide and inhibition by both these analogs and synthetic epitopes, were used to analyze the specificity of the polyclonal response. In BALB/c mice, the primary antibody response is directed principally against the antigenic site 314-LKLAT-318, whereas the secondary response after a boost is predominantly directed to a distinct site, 320-MRNVPEKQT-328. The T-cell response to the peptide H3 HA1(305-328), as measured by antigen-induced proliferation of primed T cells in vitro, is also I-Ed restricted in high-responder H-2d mice and is directed against an antigenic site that does not require the four C-terminal residues unique to the H3 influenza subtype. A different epitope appears to be recognized by T cells from CBA (H-2k) mice, which proliferate to a moderate extent on exposure to the peptide but, nevertheless, do not provide help for an antibody response.     

Influenza viruses are T cell-independent B cell mitogens.

UV-inactivated influenza virus A strains of subtypes H1, H2, H3, and H6 were shown to be mitogenic for unprimed splenic lymphocytes from BALB/c mice. Representative viruses of these four subtypes all behaved as T cell-independent B cell mitogens. The magnitude of the proliferative response was determined by the subtype of the hemagglutinin molecule: H2 and H6 viruses were the most potent mitogens, and H3 viruses were moderately mitogenic, whereas H1 viruses induced only low, but significant, levels of proliferation. Mitogenesis was inhibited by antiviral sera and by monoclonal antibodies directed against hemagglutinin.     

Human Cytotoxic T Lymphocytes Directed to Seasonal Influenza A Viruses Cross-React with the Newly Emerging H7N9 Virus

In February 2013, zoonotic transmission of a novel influenza A virus of the H7N9 subtype was reported in China. Although at present no sustained human-to-human transmission has been reported, a pandemic outbreak of this H7N9 virus is feared. Since neutralizing antibodies to the hemagglutinin (HA) globular head domain of the virus are virtually absent in the human population, there is interest in identifying other correlates of protection, such as cross-reactive CD8⁺ T cells (cytotoxic T lymphocytes [CTLs]) elicited during seasonal influenza A virus infections. These virus-specific CD8⁺ T cells are known to recognize conserved internal proteins of influenza A viruses predominantly, but it is unknown to what extent they cross-react with the newly emerging H7N9 virus. Here, we assessed the cross-reactivity of seasonal H3N2 and H1N1 and pandemic H1N1 influenza A virus-specific polyclonal CD8⁺ T cells, obtained from HLA-typed study subjects, with the novel H7N9 virus. The cross-reactivity of CD8⁺ T cells to H7N9 variants of known influenza A virus epitopes and H7N9 virus-infected cells was determined by their gamma interferon (IFN-γ) response and lytic activity. It was concluded that, apart from recognition of individual H7N9 variant epitopes, CD8⁺ T cells to seasonal influenza viruses display considerable cross-reactivity with the novel H7N9 virus. The presence of these cross-reactive CD8⁺ T cells may afford some protection against infection with the new virus.     

Immunologic recognition of influenza virus-infected cells. I. Generation of a virus-strain specific and a cross-reactive subpopulation of cytotoxic T cells in the response to type A influenza viruses of different subtypes.

Parenteral immunization of mice with a given strain of type A influenza virus generates two subpopulations of cytotoxic T cells in the in vivo primary response. One subpopulation is specific for the immunizing virus; the other subpopulation cross-reacts with target cells infected with type A influenza virus of a different subtype. Both subpopulations are specific for target cells infected with type A influenza virus and optimally lyse only infected targets which are syngeneic at the H-2 gene locus. In vitro stimulation of previously primed spleen cells with cells infected with homologous virus generates both subpopulations in the secondary cytotoxic response. However, in vitro stimulation of primed cells with cells infected with heterologous type A virus of a different subtype specifically selects for the cross-reactive T-cell population. These results are discussed in terms of current models for T-cell recognition of virus-infected cells and possible mechanisms for cross-reaction between type A influenza viruses of different subtypes at the level of cytotoxic T cells.     

Heterosubtypic Antiviral Activity of Hemagglutinin-Specific Antibodies Induced by Intranasal Immunization with Inactivated Influenza Viruses in Mice

Influenza A virus subtypes are classified on the basis of the antigenicity of their envelope glycoproteins, hemagglutinin (HA; H1–H17) and neuraminidase. Since HA-specific neutralizing antibodies are predominantly specific for a single HA subtype, the contribution of antibodies to the heterosubtypic immunity is not fully understood. In this study, mice were immunized intranasally or subcutaneously with viruses having the H1, H3, H5, H7, H9, or H13 HA subtype, and cross-reactivities of induced IgG and IgA antibodies to recombinant HAs of the H1–H16 subtypes were analyzed. We found that both subcutaneous and intranasal immunizations induced antibody responses to multiple HAs of different subtypes, whereas IgA was not detected remarkably in mice immunized subcutaneously. Using serum, nasal wash, and trachea-lung wash samples of H9 virus-immunized mice, neutralizing activities of cross-reactive antibodies were then evaluated by plaque-reduction assays. As expected, no heterosubtypic neutralizing activity was detected by a standard neutralization test in which viruses were mixed with antibodies prior to inoculation into cultured cells. Interestingly, however, a remarkable reduction of plaque formation and extracellular release of the H12 virus, which was bound by the H9-induced cross-reactive antibodies, was observed when infected cells were subsequently cultured with the samples containing HA-specific cross-reactive IgA. This heterosubtypic plaque reduction was interfered when the samples were pretreated with anti-mouse IgA polyclonal serum. These results suggest that the majority of HA-specific cross-reactive IgG and IgA antibodies produced by immunization do not block cellular entry of viruses, but cross-reactive IgA may have the potential to inhibit viral egress from infected cells and thus to play a role in heterosubtypic immunity against influenza A viruses.     

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.     

Fine specificity of the in vitro antibody response to influenza virus by human blood lymphocytes

The fine specificity of anti-influenza antibody produced in vitro by human PBM stimulated with different strains of influenza virus was examined by competition binding in solid phase enzyme immunoassay. Most of the antibody produced in vitro is directed to strain-specific or cross-reactive determinants on the hemagglutinin molecule. The extent of cross-reactivity is dependent on the strain of virus used to stimulate PBM as well as the individual tested and presumably on his previous exposure to influenza viruses. PBM from some individuals produced antibody that bound to the stimulating strain of influenza virus but not to other strains of the same subtype. In other individuals, antibody was produced in vitro that cross-reacted with all viruses in the same subtype (e.g., H3N2; A/X31, A/X47, and A/Texas) but did not bind to other (H2N1 or H1N1) subtypes, and in a few individuals, extensive cross-reaction between subtypes was seen. The presence of antibody to hemagglutinin in these culture supernatants was confirmed by competition binding to highly purified hemagglutinin. This in vitro culture system allows the immunologic memory of individuals to a wide range of stimulating virus strains to be examined simultaneously in terms of specificity of the antibody response by human PBM to influenza virus after natural infection or immunization.     

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。     

A Human CD4+ T Cell Epitope in the Influenza Hemagglutinin Is Cross-Reactive to Influenza A Virus Subtypes and to Influenza B Virus

The hemagglutinin protein (HA) of the influenza virus family is a major antigen for protective immunity. Thus, it is a relevant target for developing vaccines. Here, we describe a human CD4(+) T cell epitope in the influenza virus HA that lies in the fusion peptide of the HA. This epitope is well conserved in all 16 subtypes of the HA protein of influenza A virus and the HA protein of influenza B virus. By stimulating peripheral blood mononuclear cells (PBMCs) from a healthy adult donor with peptides covering the entire HA protein based on the sequence of A/Japan/305/1957 (H2N2), we generated a T cell line specific to this epitope. This CD4(+) T cell line recognizes target cells infected with influenza A virus seasonal H1N1 and H3N2 strains, a reassortant H2N1 strain, the 2009 pandemic H1N1 strain, and influenza B virus in cytotoxicity assays and intracellular-cytokine-staining assays. It also lysed target cells infected with avian H5N1 virus. We screened healthy adult PBMCs for T cell responses specific to this epitope and found individuals who had ex vivo gamma interferon (IFN-γ) responses to the peptide epitope in enzyme-linked immunospot (ELISPOT) assays. Almost all donors who responded to the epitope had the HLA-DRB1*09 allele, a relatively common HLA allele. Although natural infection or standard vaccination may not induce strong T and B cell responses to this highly conserved epitope in the fusion peptide, it may be possible to develop a vaccination strategy to induce these CD4(+) T cells, which are cross-reactive to both influenza A and B viruses.     

Importance of subtype in the immune response to the pre-S(2) region of the hepatitis B surface antigen. I. T cell fine specificity.

Previous studies of murine T cell recognition of the pre-S(2) region of the hepatitis B surface Ag (HBsAg) identified high (H-2b,d,q), intermediate (H-2s,k), and low to nonresponder (H-2f) haplotypes. However, these studies utilized the y subtype of HBsAg. The purpose of this study was to examine the influence of viral subtype on T cell recognition of the pre-S(2) region and to identify specific T cell recognition sites in a panel of H-2 congenic strains. Immunization with pre-S(2) containing HBsAg particles of the d and y subtypes indicated that T cell recognition of the pre-S(2) region is predominantly subtype-specific in murine strains of eight different H-2 haplotypes. Furthermore, the B10.M strain (H-2f) classified as a T cell nonresponder to the y subtype of the pre-S(2) region responds efficiently to the d subtype, indicating that pre-S(2) responder status can be subtype-dependent as well as subtype-specific. Studies using a truncated pre-S(2) polypeptide and synthetic peptides illustrated that the C-terminal sequence (p148-174) of the pre-S(2) region is the dominant focus of T cell recognition in multiple murine strains. Specifically, 17 distinct T cell recognition sites were defined within the C-terminal half of the pre-S(2) region. The fine specificity of T cell recognition of the pre-S(2) region was dependent on the H-2 haplotype of the responding strain. T cell recognition of all 17 sites was subtype specific, which is consistent with the fact that the C-terminal sequence is highly polymorphic between the d and y subtypes of the pre-S(2) region. Lastly, it was shown that the ability of synthetic peptides to elicit T cells cross-reactive with the native pre-S(2) region was variable and depended on the nature of the immunizing peptide. The pre-S(2)-containing HBsAg vaccines currently in clinical trials are composed of ra single subtype, either d or y. The results of this study suggest that both subtypes should be incorporated to increase the frequency of T cell responders to the pre-S(2) region, and to insure Th cell memory relevant to infection with hepatitis B virus of either the d or y subtypes.     

H1亚型猪流感病毒HA基因DNA疫苗的构建及其对Balb/c小鼠的免疫效果评价

以A/Swine/Guangdong/LM/2004(H1N1)猪流感病毒HA基因为模板,通过RT-PCR技术扩增出HA基因,并将其克隆到pCI-neo真核表达载体中,成功构建重组表达质粒pCI-HA,瞬时转染vero E6和293T细胞,通过免疫过氧化物酶单层细胞试验(Immunoperoxidase monolayer assay ,IPMA)、间接免疫荧光试验(indirect immunofluorescence assay, iIFA)和蛋白免疫印迹（Western blot,WB）实验证明,HA基因能够在哺乳动物细胞中有效表达并具有良好的生物学活性。将重组质粒三次免疫8w雌性Balb/c小鼠后,ELISA试验和中和试验结果表明该重组质粒能够诱导小鼠产生较高的抗体滴度,并具有良好的中和活性。因此为H1亚型猪流感DNA疫苗的研究奠定了理论基础。