Immunization against influenza A virus: comparison of conventional inactivated, live-attenuated and recombinant baculovirus produced purified hemagglutinin and neuraminidase vaccines in a murine model system

To simulate the 2003-2004 influenza season and compare available vaccination methods, immunologically naive mice were immunized with: influenza A virus hemagglutinin (rHA) and neuraminidase (rNA) from A/Panama/2007/99 H3N2 or A/Fujian/411/2002 H3N2 expressed by recombinant baculovirus, chromatographically purified, either as single antigens (rHA or rNA) or in combination (rHArNA); conventional inactivated monovalent (CIV) vaccines from each heterotypic strain; or a live-attenuated influenza (LAV) vaccine derived from the A/Panama/2007/99 strain. HA containing vaccines were highly immunogenic for the HA antigen, with no statistically significant differences among groups in the amount of homotypic anti-HA antibody induced. Little cross-reactive anti-HA antibody was induced by any vaccine, including LAV. Statistically, the greatest amount of anti-NA antibody was induced by the purified NA alone or in combination with purified HA; the least amount of anti-NA antibody was found in mice immunized with LAV or CIV. Immunization with vaccines immunogenic for both HA and NA resulted in an immune response to both surface glycoproteins that suppressed homotypic, closely related heterotypic infection and had a greater reduction in mPVT following an infectious challenge by a distantly related heterotypic strain. These studies suggest that vaccines immunogenic for both HA and NA offer an increased level of protection from influenza.     

Mucosal immunity induced by adenovirus-based H5N1 HPAI vaccine confers protection against a lethal H5N2 avian influenza virus challenge

Development of effective vaccines against highly pathogenic avian influenza (HPAI) H5N1 viruses is a global public health priority. Considering the difficulty in predicting HPAI H5N1 pandemic strains, one strategy used in their design includes the development of formulations with the capacity of eliciting broad cross-protective immunity against multiple viral antigens. To this end we constructed a replication-defective recombinant adenovirus-based avian influenza virus vaccine (rAdv-AI) expressing the codon-optimized M2eX-HA-hCD40L and the M1-M2 fusion genes from HPAI H5N1 human isolate. Although there were no significant differences in the systemic immune responses observed between the intramuscular prime-intramuscular boost regimen (IM/IM) and the intranasal prime-intramuscular boost regimen (IN/IM), IN/IM induced more potent CD8⁺ T cell and antibody responses at mucosal sites than the IM/IM vaccination, resulting in more effective protection against lethal H5N2 avian influenza (AI) virus challenge. These findings suggest that the strategies used to induce multi-antigen-targeted mucosal immunity, such as IN/IM delivery of rAdv-AI, may be a promising approach for developing broad protective vaccines that may be more effective against the new HPAI pandemic strains.     

2004年中国甲3亚型流感病毒(H3N2)抗原性及基因特性研究

目的阐明2004年中国流行的甲3亚型流感病毒血凝素抗原性及其基因变异情况。方法对2004年分离的甲3亚型毒株先进行单向血凝抑制试验及交叉血凝抑制试验;在此基础上选取不同时间、地点的甲3亚型流感毒株进行血凝素基因HA1区核苷酸序列测定并推导出其氨基酸序列,然后进行基因进化特性分析。结果单向血凝抑制实验结果表明,2004年共有52.3%毒株与A/Fujian/411/2002(H3N2)(20042005毒株)有4倍或以上的血凝抑制滴度差异,交叉血凝抑制实验结果表明,它们间的抗原比为4。HA1区核苷酸序列和氨基酸序列分析表明,我国从2004年2月分离的甲3亚型毒株开始出现了与A/Fujian/411/2002(H3N2)和A/Wellington/1/2004(H3N2)(2005年国际代表株)相比较,在其HA1蛋白分子上存在有4个氨基酸位点(159位Y>F,189位S>N,145位K>N,226位V>I)发生了替换。此类毒株首发于我国南方,然后到我国北方。结论我国2004年2月份以后所分离的甲3亚型流感毒株已经发生抗原性及基因特性的改变。     

我国不同艾滋病候选疫苗小鼠细胞免疫应答评价

目的 比较我国不同艾滋病(AIDS)候选疫苗小鼠细胞免疫应答水平.方法 将我国6种艾滋病候选疫苗按其各自免疫程序,免疫BALB/c小鼠,分离脾细胞,利用酶联免疫斑点法(ELISPOT)与胞内因子染色法(ICS)检测其对HIV特异多肽的细胞免疫应答水平.结果 6种艾滋病候选疫苗利用潜在T细胞表位(PTE)Gag、Env和Pol肽库和ELISPOT法检测IFN-γ的分泌情况,结果各疫苗的小鼠细胞免疫阳转率为70%～100%,各疫苗引起特异细胞免疫应答的反应肽库不同,强度亦不相同.1#和2#疫苗利用ELISPOT检测分泌Th1类细胞因子IFN-γ和IL-2的小鼠脾细胞数量,两疫苗的IFN-γ和IL-2检测结果均呈中度相关性(r1=0.62,P1＜0.01;r2=0.79,P2＜0.01).ELISPOT和ICS检测1#疫苗免疫后分泌IFN-γ的小鼠脾细胞数,结果两种方法的小鼠阳转率均为66.7%(10/15),ELISPOT阳性细胞数与ICS阳性细胞比例呈中度相关(r=0.55,P＜0.05).结论 我国不同AIDS候选疫苗引起的小鼠细胞免疫应答在广度和强度上存在差别.ELISPOT或ICS检测IFN-γ等Th1类细胞因子可作为疫苗诱导小鼠细胞免疫应答的评价指标.     

Interaction between a 1998 human influenza virus N2 neuraminidase and monoclonal antibody Mem5

Influenza virus constantly escapes antibody inhibition by introducing mutations that disrupt protein-protein interactions. Based on the structure of the complex between neuraminidase (NA) of influenza A/Memphis/31/98 (H3N2) and the Fab of a monoclonal antibody (Mem5) that binds and inhibits the Memphis/98 NA, we investigated the contribution made by individual amino acids of NA to the stability of the complex. We made mutations D147A, D147N, H150A, H197A, D198A, D198N, E199A, E199Q, K221R, A246K, D251N, and D251A. Binding of each mutant to NA was quantitated by NA inhibition assays and ELISA. Most of the mutant NAs were inhibited by Mem5 to the same extent as wild-type, but with lower affinity. The exceptions were E199A, E199Q, and K221R, in which binding was abrogated. The ELISA results confirmed a correlation between NA inhibition and binding. The Mem5 epitope is dominated by a few high-energy interactions as was found in the epitope on an avian subtype N9 NA that binds antibody NC41 and different to the more diffuse energy distribution in the NC10 epitope on N9 NA. Energetic dominance of a particular interaction, which is associated with potential for antibody escape mutations, may be associated with the absence of water molecules in the vicinity. Critical contacts in a dominant antigenic site are likely to mutate, allowing some predictions of antigenic drift.     

2004-2005年中国A(H1N1)亚型流感病毒抗原性及基因特性研究

目的 阐明2004-2005年中国流行的A（H1N1）亚型流感病毒血凝素抗原性及其基因变异情况.方法 对2004-2005年分离的A（H1N1）亚型毒株先进行单向血凝抑制试验;在此基础上选取不同时间、地点的A（H1N1）亚型流感毒株进行血凝素基因HA1区核苷酸序列测定并推导出其氨基酸序列,然后进行基因进化特性分析.结果 单向血凝抑制实验结果表明,2004年A（H1N1）亚型病毒株对鉴定血清的血凝抑制效价与A/Shanghai/1/1999（H1N1）毒株没有4倍差异;2005年分离的A（H1H1）亚型毒株中有62株（占6.2%）病毒与A/Shanghai/1/1999（H1N1）毒株本身的血凝抑制效价相比有4倍差异.HA1区核苷酸序列和氨基酸序列分析表明,我国2005年分离到的A（H1N1）亚型流感病毒株有以下位点发生变异,54 K＞R、90T＞K、101Y＞H、149R＞K、169V＞A、190D＞N、212R＞K、219K＞R、245W＞R、246Y＞F、258T＞N、318V＞A,其中54、190位氨基酸位于抗原决定簇.结论 我国2005年分离的A（H1N1）亚型流感毒株基因特性和抗原性已开始发生变异.     

H5N1禽流感病毒HA假病毒的构建及特性研究

目的 构建包膜蛋白为H5N1禽流感病毒HA蛋白的假病毒,对其生物学特性进行研究,并将其初步应用于H5N1禽流感病毒的血清检测.方法 将我国分离的高致病性H5N1禽流感病毒的HA基因插入真核表达质粒,得到pLP-HA,与假病毒构建体系的三种质粒pLP1,pLF2和pEmGFP,瞬时共转染人胚肾细胞293T,48 h收集假病毒上清,对其感染性,血凝活性进行测定,并应用于微量中和实验.同时,构建了优化HA基因的假病毒以及一株含有越南禽流感病毒HA基因的假病毒,进行比较.结果 电镜下观察到假病毒颗粒的存在;Western-Blot表明HA蛋白存在于假病毒颗粒中;HA假病毒与野生型活病毒的微量中和实验相比,两者结果具有很好的相关性.结论 成功构建了不同高致病性H5N1禽流感病毒HA蛋白的假病毒,所构建的假病毒可以应用于微量中和实验.研究发现不同禽流感病毒株HA蛋白假病毒的包装效率不同,并且真核表达优化基因并不能显著提高假病毒颗粒包装效率.     

Generation of the influenza B viruses with improved growth phenotype by substitution of specific amino acids of hemagglutinin

Variability in growth characteristics of influenza B viruses remains a serious limitation in the manufacture of inactivated influenza vaccines. Currently, serial passage in eggs is the strategy used in most instances for selection of high growth virus variants. In previous studies we found that adaptation of the strain B/Victoria/504/2000 to high growth in eggs was associated with changes only in hemagglutinin (HA). The high growth phenotype was associated with acquisition of either two (R162M and D196Y) or three (G141E, R162M and D196Y) amino acid (AA) substitutions, predicted to be near the receptor-binding domain of HA. In the present study we analyzed, using reverse genetics, the contribution to virus growth of each of these AA substitutions and determined their effect on antigenic properties. We found that G141E and R162M were most favorable for virus growth; however, only R162M could improve virus growth without antigenic alteration. Substitution D196Y had least effect on virus growth but substantially altered antigenic properties. Additional virus variants with AA substitutions at positions 126, 129, 137 and 141 were generated and characterized. The AA changes advantageous for growth of B/Victoria/504/2000 were also tested in the context of the HA of the B/Beijing/184/93, a virus with stable low-growth phenotype. All of the tested AA substitutions improved the replicative capabilities of the corresponding viruses, but only N126D and K129E had no effect on antigenicity. The results of our studies demonstrate that introduction of specific AA substitutions into viral HA can improve viral replicative efficiency while preserving the original antigenic properties.     

新型甲型H1N1流感病毒HA基因DNA疫苗诱导小鼠产生中和抗体

目的 探讨新型甲型流感病毒(2009H1N1)血凝素(HA)DNA疫苗诱导小鼠产生中和抗体特性.方法 构建2009H1N1或1918甲型流感病毒(1918H1N1)HA蛋白表达质粒2009HA和1918HA,采用25μg或200μg剂量2009HA质粒免疫小鼠,以2009HA或1918HA蛋白为包被抗原,测定小鼠血清中2009HA抗体总量或交叉反应抗体含量,分别用2009H1N1和1918H1N1两种假病毒(pp)测定抗体中和活性.结果 25 μg或200μg的2009HA质粒加强免疫小鼠后,4～16周内两组小鼠血清中2009HA总抗体水平以及对2009H1N1pp的中和抗体滴度相似(P>0.05),都含有与1918HA蛋白交叉反应抗体,对1918H1N1pp的交叉中和抗体滴度相似(P>0.05).结论 小剂量2009HA质粒DNA疫苗能够诱导小鼠产生持久的高水平中和抗体,对于预防新现流感病毒具有潜在应用价值.     

猪型(H1N1)流感病毒血凝素和神经氨酸酶基因来源的研究

目的 研究2002年我国内地从猪群中分离的猪型(H1N1)毒株HA和NA基因来源。及其使猪致病的原因。方法 用PCR扩增目的基因，用P^GEM-T Easy Vector，4℃过夜连接，重组质粒转入DH-10B细菌，筛选阳性菌落，酶切鉴定，送六合通公司自动测序，并作进化树分析。结果 3株猪型(H1N1)病毒的HA和NA基因属猪型(H1N1)流感病毒，而不同于其他禽或人的H1N1亚型流感病毒。2002年猪型毒株由1991年猪型毒株演变而来。近来我国内地猪群中猪型毒株活动增强，其对猪能致病是由于病毒粒HA和NA蛋白抗原性发生变异所造成。结论 3株猪型病毒的HA和NA基因来源于猪型(H1N1)毒株。近来猪型毒株对猪具有致病性和活动增强是由于其HA和NA蛋白分子上氨基酸序列发生替换所造成。     

1992-2012年中国H3N2亚型流感病毒选择压力分析

目的 分析1992-2012年中国H3N2亚型流感病毒所受选择压力,并探讨选择压力与各个抗原类毒株流行情况的关系.方法 对1992-2012年中国的H3N2亚型流感病毒(n=1206)进行抗原类流行和整体选择压力分析,同时对这21年来所有毒株的HA1进行位点正选择压力分析.结果 表明所有H3N2亚型流感病毒在这21年间共有4个位点受到正选择作用,整体选择压力的变化随着不同抗原类毒株的交替而变化,21年间共发现3个选择压力谷底年,与新抗原类的出现相关.结论 整体选择压力从谷底年上升时,新的抗原类出现并在人群中流行,此时是更换疫苗株的最佳时机.     

抗H5N1禽流感病毒羊驼重链单域抗体的制备和功能鉴定

目的:获得具有中和活性、高特异性和稳定性的抗H5N1禽流感病毒血凝素蛋白(HA)的羊驼重链单域(VHH)抗体。方法:利用pET-22b表达载体诱导表达抗H5N1禽流感病毒HA VHH抗体蛋白,以包涵体形式表达的VHH抗体蛋白采用最优复性方法进行复性后,获得高纯度的VHH抗体,分别采用ELISA法鉴定VHH抗体的亲和力和热稳定性,采用血凝抑制实验鉴定抗体的特异性和体外中和活性。结果:经复性的抗H5N1禽流感HA VHH抗体对H5N1禽流感病毒HA具有良好的特异性。通过对三种不同复性方法比较,利用柱上复性的VHH23抗体具有较好的热稳定性,亲和力为9.1×10-7mol/L,同时对H5N1禽流感病毒HA具有良好的体外中和活性。结论:实验结果表明通过原核表达获得具有较好中和活性、特异性及稳定性的抗H5N1禽流感病毒VHH抗体,为进一步开展抗体的体内病毒中和试验奠定良好基础。     

H3N2流感病毒体外细胞传代引起的变异

目的 了解用不同来源MDCK细胞分离H3N2流感病毒时,HA和NA片段氨基酸变异情况.方法 选取H3N2流感病毒核酸阳性的咽拭子标本13份,分别在三种不同来源的MDCK细胞中传5代.对标本中病毒及细胞传代的病毒进行HA和NA基因序列测定,分析病毒传代后HA和NA片段变异情况.结果 与咽拭子中的病毒序列相比,MDCK 5代病毒和MDCK-parent 5代病毒均发生了HA和/或NA的突变.9株MDCK 5代病毒HA发生了P221L/P/H突变;8株病毒发生了NA片段的148位或151位的突变.2株MDCK-parent 5代病毒HA221位为多态性.13株病毒MDCK-parent传代后均发生了NA多态性,大多发生在151位.在MDCK-SIAT传5代后的病毒HA均无突变,3株有NA突变.结论 在对流感病毒进行抗原性分析前对病毒进行细胞传代会发生病毒的适应性突变.H3N2流感病毒在MDCK、MDCK-parent中传代后,会引起HA和/或NA突变;MDCK-SIAT传代,病毒变异明显减少.     

Efficacy of inactivated vaccines against H5N1 avian influenza infection in ducks

The current Asian H5N1 highly pathogenic avian influenza virus has spread over much of Asia and into Europe and Africa. As well as affecting village and commercial chicken operations in many South East Asian countries, it differs from past H5 avian influenza viruses in that it causes morbidity and mortalities in other domesticated birds, such as ducks and turkeys and in wild water birds. Effective vaccines that can prevent infection, as well as disease, and be used in a variety of avian species are needed for field use. In this report, a bivalent H5N9+H7N1 oil emulsion vaccine is compared, in ducks, to a monovalent H5N3 oil emulsion vaccine that has been derived by reverse genetics with an H5 from A/chicken/Vietnam/C58/04. While both vaccines protected against morbidity, the monovalent vaccine provided effective protection, with no evidence of shedding of the challenge virus and no serological response to the H5N1 challenge virus.     

甲3型流感病毒M基因披甲RNA的研制

目的 研制内含甲3型流感病毒M基因RNA的披甲RNA.方法 将MS2噬菌体基因组中编码成熟酶蛋白、包膜蛋白和pac位点的DNA片段克隆到表达质粒pET30b中,构建重组质粒pAR-1.将甲3型流感病毒M基因连接到pAR-1 pac位点的下游,诱导表达出内含M基因RNA的披甲RNA AR-2,并进行纯化、定量和稳定性研究.结果 成功构建和表达了耐核糖核酸酶、内含M基因RNA的披甲RNA,每1 ml LB培养基可诱导表达出约8.9×1011个拷贝的AR-2.结论 制备的AR-2稳定,无生物传染危险性,可作为甲型流感病毒M基因核酸检测的标准品和质控品.     

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。     

Mismatched hemagglutinin and neuraminidase specificities in recent human H3N2 influenza viruses

The hemagglutinin (HA) of influenza viruses initiates infection by binding to sialic acid on the cell surface via alpha2,6 (human) or alpha2,3 (avian) linkage. The influenza neuraminidase (NA) can cleave both alpha2,3- and alpha2,6-linked sialic acids, but all influenza NAs have a marked preference for the non-human alpha2,3 linkage. Recent H3N2 influenza viruses have lost the ability to agglutinate chicken red blood cells. To determine if changes in HA specificity or affinity correlate with NA specificity or activity, we examined red cell binding and elution of a series of H3N2 viruses. We found that the NA activity of many influenza viruses does not release binding by their HA. In some egg-adapted strains, lack of elution correlates with low levels of viral NA activity, and these elute rapidly when bacterial NA is added. However, a Fujian-like virus, A/Oklahoma/323/03, does not elute by its own NA or with Vibrio cholerae sialidase, and it binds to red cells pre-treated with V. cholerae sialidase. It elutes after addition of the broad specificity Micromonospora viridifaciens sialidase. Human glycophorin inhibits A/Oklahoma/323/03 hemagglutination 6-fold better than fetuin. We conclude that specific forms of sialic acid are used as receptor by recent human H3N2 influenza viruses, perhaps involving branched alpha2,6 sialic acid or alpha2,8 sialic acid structures on O-linked carbohydrates. The virus itself has no O-linked glycans, so even though the NA is not able to cleave receptors on cells, the viruses will not self-aggregate. It will be important to monitor efficacy of neuraminidase inhibitors in case there are NA-resistant receptors in the human respiratory tract that allow the viruses to be less dependent on NA activity.     

Comparison of hemagglutination inhibition assay, an ELISA-based micro-neutralization assay and colorimetric microneutralization assay to detect antibody responses to vaccination against influenza A H1N1 2009 virus

The hemagglutination inhibition (HI) assay has been the main method used to investigate immune responses to vaccination against influenza H1N1 (2009) virus. However microneutralization tests (MNT) have been shown to be more sensitive and more specific. In this study, the three methods of choice: (i) the HI assay, (ii) an ELISA-based conventional MNT and (iii) a colorimetric MNT in terms of their ability to detect antibody responses in serum pairs collected from 43 healthy individuals before and 21 days after vaccination were compared. The colorimetric MNT was established yielding intra- and inter-run imprecisions of 7.5% and 12.4%, respectively. Testing of antisera to seasonal influenza viruses demonstrated the assay to be specific for antibodies to influenza H1N1 (2009) virus. A good correlation between the three methods was found, being highest for the ELISA-MNT and the colorimetric MNT (r = 0.714 for geometric mean titers (GMT) and r = 0.695 for titer increases). Similar rates of fourfold titer increases were detected: 95.3% in the ELISA-MNT vs. 93.0% in colorimetric MNT and 95.3% in HI assay. The ELISA-based MNT demonstrated the highest titer range leading to the highest postvaccination GMT and the highest titer increase (>50-fold). The lowest GMTs were measured with the HI assay, while the colormetric MNT detected the highest GMT in prevaccination sera. Taken together, similar seroconversion rates were obtained with the three assays. The ELISA-MNT appeared to be the best method to compare absolute pre- and postvaccination GMTs. The colorimetric MNT, being less labour-intensive than the ELISA-MNT, seems to be a suitable tool in vaccination studies.     

含H5N1-HA基因重组腺病毒疫苗的构建及其诱导免疫应答的初步探讨

目的 构建包含H5N1-HA基因的重组腺病毒疫苗并探讨其免疫效果.方法 用Admax系统构建包含H5N1-HA基因的重组腺病毒疫苗,并用PCR、Western-Blot等方法对重组病毒疫苗进行鉴定;疫苗免疫小鼠后,通过HI实验和ELISPOT实验检测其体液免疫和细胞免疫反应,评价其免疫效果.结果 成功得到了含有H5N1-HA基因的重组腺病毒疫苗;基因表达鉴定表明,HA基因能够在细胞中进行表达;血凝抑制实验结果显示小鼠产生的针对HA抗体滴度在1:320和1:640之间;ELISPOT结果显示实验组和对照组(PBS)相比斑点数量差异有统计学意义(P<0.05),以上免疫结果表明重组腺病毒载体疫苗可以诱导小鼠产生良好的特异性体液和细胞免疫反应.结论 含H5NA-HA的重组腺病毒疫苗可以诱导小鼠产生良好的免疫反应,为研制人禽流感疫苗打下基础.     

Distinct host range of influenza H3N2 virus isolates in Vero and MDCK cells is determined by cell specific glycosylation pattern

Influenza A viruses were isolated in Vero, MDCK cells and chicken embryos. In contrast to MDCK-derived variants all H3N2 isolates obtained in Vero cells neither agglutinated chicken erythrocytes nor grew in chicken eggs. These host range differences of H3N2 Vero and MDCK isolates were noticed even in the absence of amino acid substitutions in the HA1 molecule. Evaluation of HA glycosylation pattern by treatment with endoglycosidases H and F revealed that Vero-variants contained more oligosaccharides of the high mannose type than did the corresponding MDCK-isolates. Removal of some mannose residues from the non-reducing termini of the carbohydrates by exomannosidase treatment resulted in the ability of Vero-isolates to agglutinate chicken erythrocytes. Glycosylation pattern and properties of H3N2 viruses grown in Vero cells were close to those of viruses grown in human kidney epithelial cells, whereas the H1N1 variants isolated from Vero, MDCK cells or eggs did not differ in agglutination properties, carbohydrate composition or ability to infect eggs.