Recombinant multi-epitope vaccine induce predefined epitope-specific antibodies against HIV-1

Monoclonal antibody 2F5 recognizing ELDKWA-epitope on HIV-1 gp41 has significant neutralization potency against 90% of the investigated viruses of African, Asia, American and European strains, but antibodies responses to ELDKWA-epitope in HIV-1 infected individuals were very low. Based on the epitope-vaccine strategy suggested by us, a recombinant glutathione S-transferase (GST) fusion protein (GST-MELDKWAGELDKWAGELDKWAVDIGPGRAFYGPGRAFYGPGRAFY) as vaccine antigen containing three repeats of neutralizing epitope ELDKWA on gp41 and GPGRAFY on gp120 was designed and expressed in Escherichia coli. After vaccination course, the recombinant multi-epitope vaccine could induce high levels of predefined multi-epitope-specific antibodies in mice. These antibodies in sera could bind to both neutralizing epitopes on gp41 peptide, V3 loop peptide and recombinant soluble gp41 (aa539-684) in ELISA assay (antisera dilution: 1:1,600-25,600), while normal sera did not. Moreover, these antibodies in sera could recognize the CHO-WT cells which expressed HIV-1 envelope glycoprotein on the cell surfaces, indicating that the predefined epitope-specific antibodies could recognize natural envelope protein of HIV-1 though these antibodies were induced by recombinant multi-epitope-vaccine. These experimental results suggested a possible way to develop recombinant multi-epitope vaccine inducing multi-antiviral activities against HIV-1.     

A candidate vaccine against influenza virus intensively improved the immunogenicity of a neutralizing epitope

BACKGROUND: The hemagglutinin (HA) of influenza viruses is one of the major targets of the humoral response. The role of serum antibody to HA in the protection against infection has been demonstrated by long-standing observation. In previous studies, we suggested that an epitope vaccine might be a new strategy against the virus. METHODS: HA sequences of 491 H3 subtype strains from the influenza sequence database were compared and analyzed. To acquire information on the immunogenicity of the F3 epitope, F3-epitope-specific antibody levels in 81 patient sera infected with influenza virus were tested by ELISA. Based on the theory of the epitope vaccine, we designed an epitope peptide F3 (C-KAYSNCYPYDVPDY-G-KAYSNCYPYDVPDY), which contains the repeated F3 epitope KAYSNCYPYDVPDY (aa92-105) on HA (H3N2). The specificity and the titer of the antibodies induced by the epitope vaccine were determined by ELISA. The neutralizing activities of these anti-F3 antibodies were shown by inhibiting influenza virus infection of MDCK cells. RESULTS AND CONCLUSION: Comparison of HA sequences of 491 H3 subtype strains indicates that this epitope is highly conservative. Analysis of the sera from influenza virus-infected patients revealed a very low level of F3 epitope-specific antibodies, suggesting the poor immunogenicity of the F3 epitope on influenza virus. The epitope vaccine based on the F3 epitope induced high levels of F3 epitope-specific antibodies recognizing the epitope peptide F3 (antibody titer in antisera up to 1:25,600). Besides, the antisera could also recognize the natural HA in Western blotting. Interestingly, these antisera induced by the epitope vaccine could inhibit infection of MDCK cells by influenza virus (strain A/Wuhan/359/95) in the neutralization assay. These results suggest that the epitope vaccine can intensively increase the immunogenicity of neutralizing epitopes and may provide a new way to develop an effective vaccine against influenza virus.     

人流感病毒核蛋白的重组表达及其抗原性分析

目的 探索用原核表达的人甲3型流感病毒核蛋白(NP)免疫小鼠所获得的抗重组蛋白抗体检测甲型流感病毒的可行性.方法 用Clustal X, Antheprot等软件,对IFV-A3 NP进行分析,确定保守区并分析其抗原性.RT-PCR获得NP基因,分3段克隆NP基因到PET-28(c),并在B121中诱导表达.经Ni-Agarose亲和层析柱纯化后,免疫BALB/c鼠制备抗重组蛋白抗体.用Western Blot和免疫组化法检测抗重组蛋白抗体与病毒抗原的反应性.结果 重组质粒在BL21中高效表达,表达量为15-20 m9/L菌液.Western Blot检测显示,抗NP1、NP2、NP3多克隆抗体(1:2000)均对流感病毒NP具有反应性.免疫组化检测也显示,抗NP1、NP2、NP3多克隆抗体对IFV-A3、-Al感染的MDCK细胞有特异性染色,抗体滴度从1:640到1:1280.结论 重组表达的NP能够诱导产生高效价多克隆抗体,所产生的抗体与IFV-A3、A1具有交叉免疫反应性.通过生物信息学分析预测抗原性并制备多克隆抗体是可行的,并可望用于流感病毒感染的早期快速诊断的研究中.     

Epitope-vaccines: a new strategy to induce high levels of neutralizing antibodies against HIV-1

Based on the experimental evidence that gp120 subunit vaccine did not protect individuals from HIV-1 infection, we suggested that epitope-vaccines of HIV-1 gp41 may be a new strategy to induce high levels of neutralizing antibodies against HIV-1, and characterised immunogenicity of epitope-vaccines. Two epitopes, RILAVERYLKD-epitope (aa586-596) on the N-domain and ELDKWA-epitope (aa669-674) on the C-domain of gp41, were demonstrated by us and others to induce protective activity. After vaccination course, the RILAVERYLKD-dimer epitope-vaccine [C(RILAVERYLKDG)2-BSA] induced strong epitope-specific antibody response by about 1:25,600 dilution, and the ELDKWA-tetramer epitope-vaccine [C-(ELDKWAG)4-BSA] could yet induce strong antibody response to ELDKWA-epitope by 1:12,800-25,600 dilution of antisera in mice, while rgp41 subunit vaccine induced very weak antibody response to both epitopes (1:400). In rabbit experiments, the titres of ELDKWA-epitope-specific antibody induced by ELDKWA-epitope-vaccine [C-(ELDKWAG)4-BSA] reached to 1:6,400, while rgp41 subunit vaccine induced very weak antibody response to this epitope and to P1 and P2 peptides (1:400). Moreover, the ELDKWA-epitope-specific antibodies in mice and rabbit antisera induced by epitope-vaccine could very strongly interact with P2 peptide sequence-corresponding to the C-domain of gp41 (dilution by 1:25,600), and the RILAVERYLKD-epitope-specific antibodies in mice antisera induced by epitope-vaccine could also very strongly interact with P1 peptide sequence-corresponding to the N-domain of gp41 (dilution by 1:102,400). All these results provided experimental evidence that epitope-vaccine may be a new general strategy to induce high levels of neutralizing antibodies against HIV-1 or other viruses.     

Targeting the HA2 subunit of influenza A virus hemagglutinin via CD40L provides universal protection against diverse subtypes

The influenza viral hemagglutinin (HA) is comprised of two subunits. Current influenza vaccine predominantly induces neutralizing antibodies (Abs) against the HA1 subunit, which is constantly evolving in unpredictable fashion. The other subunit, HA2, however, is highly conserved but largely shielded by the HA head domain. Thus, enhancing immune response against HA2 could potentially elicit broadly inhibitory Abs. We generated a recombinant adenovirus (rAd) encoding secreted fusion protein, consisting of codon-optimized HA2 subunit of influenza A/California/7/2009(H1N1) virus fused to a trimerized form of murine CD40L, and determined its ability of inducing protective immunity upon intranasal administration. We found that mice immunized with this recombinant viral vaccine were completely protected against lethal challenge with divergent influenza A virus subtypes including H1N1, H3N2, and H9N2. Codon-optimization of HA2 as well as the use of CD40L as a targeting ligand/molecular adjuvant were indispensable to enhance HA2-specific mucosal IgA and serum IgG levels. Moreover, induction of HA2-specific T-cell responses was dependent on CD40L, as rAd secreting HA2 subunit without CD40L failed to induce any significant levels of T-cell cytokines. Finally, sera obtained from immunized mice were capable of inhibiting 13 subtypes of influenza A viruses in vitro. These results provide proof of concept for a prototype HA2-based universal influenza vaccine.     

Antibodies to HA and NA augment uptake of influenza A viruses into cells via Fc receptor entry

The hemagglutinin (HA) and neuraminidase (NA) of influenza A viruses induce antibodies which augment the uptake of influenza A virus by antigen presenting cells via Fc receptor entry. Antibody-dependent enhancement of uptake of virus by cells was mediated by Fc receptors because F(ab')2 preparations of lgG mixed with virus did not enhance virus uptake. The enhanced infection was measured using a fluorescent focus assay and was confirmed by dot-blot hybridization analysis. A 25-fold increase in the number of cells containing influenza antigens was detected when virus was mixed with subneutralizing concentrations of immune serum to the homologous virus before adding to neuraminidase-treated cells. Infection was also augmented using reassortant viruses which shared only the HA or the NA of the virus used to induce antibodies. Specific antisera to purified HA or NA also enhanced virus uptake. These results indicate that both the HA and the NA induce antibodies that enhance uptake of virus by Fc receptor bearing cells. In addition we determined that the drift of neutralizing antigens occurred more quickly than the drift of infection-enhancing antigens during the evolution of virus strains of the H3 subtype. The increase in the number of antigen presenting cells as a result of uptake of virus complexed with cross-reactive enhancing antibodies may affect the T cell responses to influenza infection.     

Elicitation of broadly neutralizing influenza antibodies in animals with previous influenza exposure

The immune system responds to influenza infection by producing neutralizing antibodies to the viral surface protein, hemagglutinin (HA), which regularly changes its antigenic structure. Antibodies that target the highly conserved stem region of HA neutralize diverse influenza viruses and can be elicited through vaccination in animals and humans. Efforts to develop universal influenza vaccines have focused on strategies to elicit such antibodies; however, the concern has been raised that previous influenza immunity may abrogate the induction of such broadly protective antibodies. We show here that prime-boost immunization can induce broadly neutralizing antibody responses in influenza-immune mice and ferrets that were previously infected or vaccinated. HA stem-directed antibodies were elicited in mice primed with a DNA vaccine and boosted with inactivated vaccine from H1N1 A/New Caledonia/20/1999 (1999 NC) HA regardless of preexposure. Similarly, gene-based vaccination with replication-defective adenovirus 28 (rAd28) and 5 (rAd5) vectors encoding 1999 NC HA elicited stem-directed neutralizing antibodies and conferred protection against unmatched 1934 and 2007 H1N1 virus challenge in influenza-immune ferrets. Indeed, previous exposure to certain strains could enhance immunogenicity: The strongest HA stem-directed immune response was observed in ferrets previously infected with a divergent 1934 H1N1 virus. These findings suggest that broadly neutralizing antibodies against the conserved stem region of HA can be elicited through vaccination despite previous influenza exposure, which supports the feasibility of developing stem-directed universal influenza vaccines for humans.     

Antigenic determinants of influenza virus hemagglutinin. XII. the epitopes of a synthetic peptide representing the C-terminus of HA1

A synthetic peptide comprising the C-terminal 24 amino acids of the heavy chain (HA1) of influenza virus hemagglutinin was constructed and examined for antigenic and immunogenic activity. Monoclonal antibodies as well as polyclonal antisera raised against the synthetic peptide were able to bind to intact virus. This binding was greatly enhanced if the virus was first subjected to pH 5, suggesting that this treatment exposes the C-terminus of HA1. Using synthetic analogs of the native sequence it was shown that the epitope recognized by one of the monoclonal antibodies encompasses one or more of the C-terminal four amino acids of HA1 (residues 325-328), which are conserved within subtypes but differ between subtypes, while the other monoclonal antibody recognizes a different epitope which involves at least one of the five variable residues at positions 311-315.     

我国人群中一种新重配的H1N2亚型流感病毒株的发现

１９９６年１月太原铁路卫生防疫站从上感患者中分离到３株流感病毒。经血清学鉴定，它们不同于１９８９和１９９２年所发现的Ｈ１Ｎ２亚型毒株，其ＨＡ的抗原性类似于Ａ／ＰＲ／８／３４（Ｈ１Ｎ１）病毒，而明显不同于当前人群中流行的Ｈ１Ｎ１亚型毒株。病毒粒不同基因节段迁移率比较表明，它们的１～４基因节段迁移率接近于Ａ／ＰＲ／８／３４（Ｈ１Ｎ１）毒株，５～６基因节段迁移率类似于Ａ／武汉／３５９／９５（Ｈ３Ｎ２）病毒，而７～８两节段既不同于Ａ／ＰＲ／８／３４（Ｈ１Ｎ１），又不同于Ａ／武汉／３５９／９５（Ｈ３Ｎ２）病毒。故可认为它们是一种新重配的Ｈ１Ｎ２亚型毒株。     

Epitope-vaccine induces high levels of ELDKWA-epitope-specific neutralizing antibody

Based on the fact that mAb 2F5 recognizing ELDKWA-epitope on the C-domain of HIV-1 gp41 has significant neutralization potency against 90% of the investigated viruses of African, Asian, American and European strains, we attempted to characterise immunogenicity of the ELDKWA-epitope on an epitope-vaccine, and to produce ELDKWA-epitope-specific monoclonal antibodies (mAb) induced by the epitope-vaccine. The C-domain peptide (P2) and the ELDKWA-tetramer peptide [C-(ELDKWAG)₄] were conjugated with BSA or P24-EC (GPKEPFRDYVDRFYK, a peptide of HIV-1 gag-protein P24, proved to be a good carrier peptide to induce an immune response to the hapten on the conjugates[¹⁸])by different methods. After the vaccination course, two P2-BSA peptide-vaccines both induced a strong antibody response against the P2-peptide by about 1:12800-25600 dilution, and a weak antibody response against the ELDKWA-epitope (1:1600-3200). The P2-P24EC and P2 (conjugated with itself) peptide-vaccines could also induce a weak antibody response against the ELDKWA-epitope (1:1600-3200), while an rgpl60 subunit vaccine induced a very weak antibody response (1:400). Interestingly, the ELDKWA-tetramer epitope-vaccine [C-(ELDKWAGVBSA] could induce a strong antibody response against the ELDKWA-epitope (1:12800-25600), i.e. It increased the level of ELDKWA-antibody eight-fold, clearly better than the P2 peptide-vaccine, and much better than the rgp160 subunit vaccine, which indicates that the immunogenicities of the ELDKWA-epitope on the ELDKWA-tetramer peptide, the C-domain peptide and rgp160 are very different. These results suggest that the ELDKWA-epitope-vaccine may be a new strategy for inducing high levels of epitope-specific neutralizing antibodies against HIV- 1. Using hybridoma-technique, a mouse monoclonal antibody recognizing the ELDKWA-epitope on ELDKWA-peptide and C-domain peptide was produced by immunization with the C-(ELDKWAG)₄-BSA epitope-vaccine, which indicates a new way to produce an epitope-specific mAb, namely immunization with epitope-vaccine instead of a natural or recombinant protein immunogen.     

Development and application of reference antisera against 15 hemagglutinin subtypes of influenza virus by DNA vaccination of chickens

Reference antisera were produced against 15 influenza hemagglutinin (HA) subtypes using DNA vaccination to produce a high-quality polyclonal serum to the HA protein without antibodies to other influenza viral proteins. The HA gene from each of 15 different HA subtypes of influenza virus was cloned into a eukaryotic expression vector and injected intramuscularly, together with a cationic lipid, into 3- to 4-week-old specific-pathogen-free chickens. Birds were boostered twice at 4-week intervals after the initial injection, and in general, antibody titers increased after each boost. The antisera were successfully applied in the hemagglutination inhibition test, which is the standard method for the classification of the HA subtypes of influenza virus. We also demonstrated the HA specificity of the antisera by Western blot and immunodot blot analysis. DNA vaccination also provides a safer alternative for the production of HA-specific antibodies, since it is produced without the use of live virus.     

Comparing the antibody responses against recombinant hemagglutinin proteins of avian influenza A (H5N1) virus expressed in insect cells and bacteria

The hemagglutinin (HA) of influenza A virus plays an essential role in mediating the entry of the virus into host cells. Here, recombinant full-length HA5 protein from a H5N1 isolate (A/chicken/hatay/2004(H5N1)) was expressed and purified from the baculovirus-insect cell system. As expected, full-length HA5 elicits strong neutralizing antibodies, as evaluated in micro-neutralization tests using HA5 pseudotyped lentiviral particles. In addition, two fragments of HA5 were expressed in bacteria and the N-terminal fragment, covering the ectodomain before the HA1/HA2 polybasic cleavage site, was found to elicit neutralizing antibodies. But the C-terminal fragment, which covers the remaining portion of the ectodomain, did not. Neutralizing titer of the anti-serum against the N-terminal fragment is only four times lower than the anti-serum against the full-length HA5 protein. Using a novel membrane fusion assay, the abilities of these antibodies to block membrane fusion were found to correlate well with the neutralization activities.     

IMMUNOGENICITY AND SPECIFICITY OF THE CANDIDATE MULTI-EPITOPE-VACCINES AGAINST HIV-1

The failure of some candidate HIV-1 vaccines may result from inducing very weak neutralization activity against representative primary viral isolates. Based on our hypothesis that epitope-vaccine may be a new strategy to induce high levels of neutralizing antibodies against HIV-1, we designed two candidate multi-epitope-vaccines, EP1 [C-G-(ELDKWA-GPGRAFY)₂-K] and EP2 (CG-GPGRAFY-G-ELDKWA-G-RILAVERYLKD), containing three neutralizing epitopes (GPGRAFY, ELDKWA and RILAVERYLKD) on HIV-1 envelope protein, and expected them to induce epitope-specific antibodies of predefined epitope-specificity. The two peptides were conjugated to carrier protein bovine serum albumin (BSA) and used for immunization of rabbits. Proteins were purified from the rabbit sera induced by both candidate multi-epitope-vaccines (EP1-BSA and EP2-BSA) through affinity chromatography with epitope-peptide-conjugated sepharose-column, and identified as antibodies in silver-staining and immunoblotting. These antibodies were demonstrated to recognize three neutralizing epitopes on peptides and the recombinant gp41 in ELISA-assay and immunoblotting. These results indicated that both candidate multi-epitope-vaccines could induce high levels of antibodies of predefined epitope-specificity which recognized a few of neutralizing epitopes on peptides and protein, providing experimental evidence for the new strategy to develop an effective neutralizing-antibody-based multi-epitope-vaccine against HIV-1.     

Isolation of cross-reactive,subtype-specific monoclonal antibodies against influenza virus HA1 and HA2 hemagglutinin subunits

Summary The large (HA1) and small (HA2) subunits of influenza virus A/Vict/3/75 hemagglutinin were purified in denatured form by preparative electrophoresis. Both polypeptides were used to immunize mice from which monoclonal antibodies were obtained. These antibodies reacted not only with the corresponding hemagglutinin subunit but also with purified virions. When tested by radioimmunoassay against a panel of human viruses, most anti-HA1 and -HA2 antibodies behaved as subtype-specific, whereas anti-HA antibodies, raised against purified virus, were more restricted. The anti-subunit antibodies were negative in hemagglutination-inhibition and neutralization tests. The interest of these antibodies as reagents for research and diagnosis is discussed.     

Antibodies elicited by influenza virus hemagglutinin fail to bind to synthetic peptides representing putative antigenic sites

A number of peptides of the hemagglutinin (HA) of X-31 influenza virus have been synthesised. The amino acid sequences of some of these peptides represent regions of HA which have been postulated [Wiley et al., Nature, Lond. 289, 373-378 (1981)] to form the antigenic sites of this molecule. Animals were immunized with free peptide or peptide conjugated to a carrier and the resulting antisera examined for their capacities to bind to homologous peptide, whole HA, reduced and alkylated HA, and intact virus. Not all peptides examined in this way were immunogenic. Only antibodies raised against the C-terminus of HA1 peptide displayed binding to virus. This antiserum bound to the intact HA but not to the reduced and alkylated form of the molecule. These results raise questions as to the feasibility of using synthetic peptides of the influenza HA in short linear sequences to elicit neutralising antibody.     

Epitope-vaccine induces high levels of ELDKWA-epitope-specific neutralizing antibody

Based on the fact that mAb 2F5 recognizing ELDKWA-epitope on the C-domain of HIV-1 gp41 has significant neutralization potency against 90% of the investigated viruses of African, Asian, American and European strains, we attempted to characterise immunogenicity of the ELDKWA-epitope on an epitope-vaccine, and to produce ELDKWA-epitope-specific monoclonal antibodies (mAb) induced by the epitope-vaccine. The C-domain peptide (P2) and the ELDKWA-tetramer peptide [C-(ELDKWAG)₄] were conjugated with BSA or P24-EC (GPKEPFRDYVDRFYK, a peptide of HIV-1 gag-protein P24, proved to be a good carrier peptide to induce an immune response to the hapten on the conjugates[¹⁸])by different methods. After the vaccination course, two P2-BSA peptide-vaccines both induced a strong antibody response against the P2-peptide by about 1:12800-25600 dilution, and a weak antibody response against the ELDKWA-epitope (1:1600-3200). The P2-P24EC and P2 (conjugated with itself) peptide-vaccines could also induce a weak antibody response against the ELDKWA-epitope (1:1600-3200), while an rgpl60 subunit vaccine induced a very weak antibody response (1:400). Interestingly, the ELDKWA-tetramer epitope-vaccine [C-(ELDKWAGVBSA] could induce a strong antibody response against the ELDKWA-epitope (1:12800-25600), i.e. It increased the level of ELDKWA-antibody eight-fold, clearly better than the P2 peptide-vaccine, and much better than the rgp160 subunit vaccine, which indicates that the immunogenicities of the ELDKWA-epitope on the ELDKWA-tetramer peptide, the C-domain peptide and rgp160 are very different. These results suggest that the ELDKWA-epitope-vaccine may be a new strategy for inducing high levels of epitope-specific neutralizing antibodies against HIV- 1. Using hybridoma-technique, a mouse monoclonal antibody recognizing the ELDKWA-epitope on ELDKWA-peptide and C-domain peptide was produced by immunization with the C-(ELDKWAG)₄-BSA epitope-vaccine, which indicates a new way to produce an epitope-specific mAb, namely immunization with epitope-vaccine instead of a natural or recombinant protein immunogen.     

IMMUNOGENICITY AND SPECIFICITY OF THE CANDIDATE MULTI-EPITOPE-VACCINES AGAINST HIV-1

The failure of some candidate HIV-1 vaccines may result from inducing very weak neutralization activity against representative primary viral isolates. Based on our hypothesis that epitope-vaccine may be a new strategy to induce high levels of neutralizing antibodies against HIV-1, we designed two candidate multi-epitope-vaccines, EP1 [C-G-(ELDKWA-GPGRAFY)₂-K] and EP2 (CG-GPGRAFY-G-ELDKWA-G-RILAVERYLKD), containing three neutralizing epitopes (GPGRAFY, ELDKWA and RILAVERYLKD) on HIV-1 envelope protein, and expected them to induce epitope-specific antibodies of predefined epitope-specificity. The two peptides were conjugated to carrier protein bovine serum albumin (BSA) and used for immunization of rabbits. Proteins were purified from the rabbit sera induced by both candidate multi-epitope-vaccines (EP1-BSA and EP2-BSA) through affinity chromatography with epitope-peptide-conjugated sepharose-column, and identified as antibodies in silver-staining and immunoblotting. These antibodies were demonstrated to recognize three neutralizing epitopes on peptides and the recombinant gp41 in ELISA-assay and immunoblotting. These results indicated that both candidate multi-epitope-vaccines could induce high levels of antibodies of predefined epitope-specificity which recognized a few of neutralizing epitopes on peptides and protein, providing experimental evidence for the new strategy to develop an effective neutralizing-antibody-based multi-epitope-vaccine against HIV-1.     

嵌合人偏肺病毒(hMPV)表位的重组流感病毒免疫保护效果

目的评价重组嵌合表达人偏肺病毒(hMPV)表位的流感病毒的免疫应答及免疫保护效果。方法用8质粒系统拯救出的在NS基因中嵌合hMPV表位的重组流感病毒免疫BALB/c小鼠,检测其刺激机体产生的针对hMPV和流感病毒的抗体滴度,及脾细胞分泌细胞因子含量。免疫后小鼠用hMPV和流感病毒进行攻击,对攻击后小鼠肺组织和鼻甲骨中病毒含量采用数字PCR方法进行测定,同时对肺组织进行HE染色,评价其对肺部损伤的保护作用。结果重组流感病毒株rFLU/hMPV/B(嵌入了hMPV的B细胞表位)免疫小鼠后,产生了针对hMPV和流感病毒的特异性抗体。rFLU/hMPV/CTL+Th(嵌入了CTL表位/Th细胞表位)免疫小鼠后,产生了针对流感病毒的特异性抗体及hMPV特异的细胞毒性T淋巴细胞反应(IFN-γ分泌增多)。rFLU/hMPV/B和rFLU/hMPV/CTL+Th引起了平衡的Th1/Th2免疫应答。用hMPV和流感病毒攻击重组病毒免疫后小鼠,肺组织病理HE染色结果显示重组病毒免疫后小鼠肺病变明显减轻,肺组织和鼻甲骨中hMPV和流感病毒含量也明显减少。结论重组的2株嵌合有hMPV B细胞表位和CTL表位/Th细胞表位的重组流感病毒株,可刺激机体产生针对hMPV和/或流感病毒体液免疫应答,嵌合有hMPV CTL表位/Th细胞表位的重组流感病毒株还可刺激机体产生hMPV特异性的细胞免疫应答,对hMPV和流感病毒攻击也有一定保护作用,该重组病毒株有潜在疫苗应用价值。     

2000～2002年我国流行的甲3(H3N2)亚型流感病毒抗原性及基因特性的研究

目的 了解2000～2002年我国流行的甲3流感病毒HA基因突变及其抗原变异情况。方法 鸡胚传代流感病毒，收获尿囊液作为抗原性分析抗原并提取病毒的RNA，进行逆转录—聚合酶链反应(RT-PCR)，扩增产物用纯化试剂盒纯化后测序，用MegAlign软件进行基因种系发生树分析。结果 与A/武汉/359/1995(H3N2)、A／Sydney／5／1997(H3N2)相比，2000～2002年我国分离到的甲3亚型流感病毒的血凝素重链区氨基酸序列存在差异。2001～2002年分离到的甲3毒株与2000年分离出的毒株的血凝素蛋白重链区(HA2)氨基酸序列有4个位点差异，它们分别位于83、186、202和222位，其中83和186分别位于抗原决定簇E和B区，其余均位于受体结合位点(RBS)的左臂。结论 2000～2002年分离到的甲3亚型流感病毒的基因特性发生突变并导致其抗原性发生漂移。     

Development of subtype-specific and heterosubtypic antibodies to the influenza A virus hemagglutinin after primary infection in children

Children undergoing primary infection with an H1N1 or H3N2 influenza A virus developed subtype-specific hemagglutination inhibition antibodies and enzyme-linked immunosorbent assay antibodies to purified hemagglutinin (HA) of the infecting virus subtype. They also developed lower titered ELISA antibodies to the noninfecting H1 or H3 HA and to H8 (an avian strain) HA. Thus, after primary infection with an influenza A virus, children develop enzyme-linked immunosorbent assay, but not hemagglutination inhibition, antibodies reactive with heterosubtypic HAs. These heterosubtypic antibodies could influence the response to infection with other wild-type or attenuated vaccine strains of influenza A virus.