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。     

抗人禽流感病毒H5N1人源化单链抗体噬菌体文库的构建和筛选

目的 利用抗人禽流感病毒H5N1 IgG抗体阳性的人禽流感康复患者外周血淋巴细胞,构建人源化Fv段单链抗体(seFv)噬菌体文库,并筛选与禽流感病毒相关蛋白有结合活性的scFv抗体文库.方法 提取人外周血淋巴细胞总RNA,逆转录成cDNA,以其为模板,利用家族特异性IgG基因的引物,扩增重链和轻链的可变区基因,并用合成的连接子将轻链和重链基因连接成单链抗体片段后,重组到噬菌粒载体pCANTAB5E中.将重组噬菌粒载体电转化大肠杆菌TG1,酶切和PCR鉴定抗体库的重组率,通过测定噬菌体抗体库的滴度计算抗体库的库容,用特异性禽流感病毒相关蛋白筛选表达的单链抗体.结果 构建了源于人禽流感康复患者血清的scFv抗体文库,库容为3.75×104;筛选出与禽流感病毒相关蛋白有结合活性的scFv抗体文库.结论 成功构建了抗人禽流感病毒H5N1的人源scFv噬菌体抗体库,并筛选出特异性结合人禽流感病毒相关蛋白的单链抗体,为进一步制备快速检测试剂和治疗研究提供了基础数据.     

羊驼抗H5N1血凝素重链可变区-人IgGFc段嵌合抗体的制备和鉴定

本研究旨在制备羊驼抗H5N1禽流感病毒的重链抗体可变区-人Fc段嵌合体抗体制备,对所得嵌合抗体进行制备和功能鉴定,为临床应用奠定基础。用pET-22b表达载体构建抗H5N1禽流感病毒羊驼重链可变区(VHH)-人IgG1Fc嵌合基因,以包涵体形式表达VHH23-hFc嵌合抗体蛋白,采用优化的方法复性后,获得高纯度VHH23-hFc嵌合抗体,用ELISA法鉴定嵌合抗体亲和力、热稳定性和小鼠体内的半衰期。结果显示,透析复性后原核表达的抗H5N1禽流感病毒VHH23-hFc嵌合抗体亲和力为2.24×106 mol/L,具有较好免疫学活性,热稳定性也较好,小鼠体内半衰期达到35h,为下一步开展该抗体的体内外病毒中和试验奠定良好基础。     

噬菌体呈现抗体库的构建及抗Fas—Fab抗体的研究

目的构建噬菌体抗体库,获得具有功能的抗Fas-Fab噬菌体抗体。方法以Fas重组蛋白为抗原免疫Balb/c小鼠。取其脾细胞提取mRNA,采用RT-PCR方法扩增抗体基因,构建重链和κ链基因库,用重组Fas抗原对所构建的抗体库进行4轮筛选,并以ELISA法鉴定其功能。结果获得抗体重链Fd基因和κ链基因长度约700bp。构建的重链Fd基因为3.5×106的抗体重链基因库。构建的重链和κ链基因库的容量均为3.1×106。经VCSM13感染得到噬菌体的滴度为8.9×1016cFu/L的噬菌体抗体库,含有抗体重链和κ链基因的噬菌体占27％。用重组人Fas抗原进行4轮筛选,得到100％的富集,说明Fas重组抗原富集了抗Fas-Fab噬菌体抗体,经ELISA检测均有抗Fas抗体的特异性。结论制备的可溶性抗Fas-Fab抗体具有抗Fas抗体的特异性,为进一步的研究奠定了基础。     

羊驼非免疫重链单域抗体库的构建和鉴定

本研究旨在通过构建羊驼非免疫重链单域抗体库,完成抗体库多样性的鉴定,为进一步筛选抗原特异性重链抗体奠定基础。我们从未经免疫的羊驼外周血中分离外周血单个核细胞(PBMC),抽提RNA后,用RT-PCR方法特异性扩增羊驼重链抗体可变区(VHH)片段;并采用两步连接方法将重链抗体可变区片段与噬菌粒载体pCANTAB5E连接获得重组子,多次电转感受态大肠杆菌TG1后获得VHH抗体基因库;并采用稀释计数法测定抗体库库容量,随机挑取克隆测序验证抗体库多样性。结果显示,我们所构建的羊驼非免疫重链单域抗体库的库容量为1.5×109,随机克隆测序验证多样性良好,独立克隆所占比例为80%,并显示出和人源抗体较高的同源性。上述结果表明,我们已经成功构建获得大容量的羊驼非免疫重链单域抗体库,为进一步筛选抗原特异性重链抗体奠定基础。     

抗戊型肝炎病毒噬菌体抗体库的构建与筛选

目的 :构建人抗戊型肝炎病毒 (HEV)噬菌体抗体库 ,筛选人源中和性抗HEV的单克隆抗体 (mAb)。方法 :取抗HEV抗体阳性的 6例HE患者静脉血 ,分离淋巴细胞 ,提取细胞总RNA后逆转录。用一组人IgGFab基因特异性引物 ,分别扩增IgGκ0轻链与重链Fd段基因。将κ轻链与Fd段基因先后克隆入噬菌体载体pComb3的相应位点 ,经电穿孔法转化大肠杆菌XL1 Blue ,再以辅助噬菌体VCSM13超感染 ,构建人抗HEV噬菌体抗体库。采用独特的 5轮筛选法 (逐渐降低抗原包被量 ,严格洗脱条件 ) ,以固相化的 4种含中和抗原表位的HEV代表株ORF2重组混合抗原 ,筛选人噬菌体抗体库 ,并以ELISA鉴定噬菌体抗体。结果 :经数次电转化构建了容量为1.9× 10 7重组率为 80 %的κ轻链基因库 ;容量为 1.8× 10 7重组率为 2 0 %的Fab基因库。以含中和抗原表位的HEV代表株ORF2重组混合抗原特异淘筛 5次 ,出现特异富集。ELISA鉴定第 5轮筛选产物 ,得到 4株与HEVORF2重组混合抗原具有较高亲和力的Fab噬菌体抗体 ,可能为中和抗体。结论 :成功地构建了人抗HEV噬菌体抗体库 ,并获得人源抗HEV特异性噬菌体抗体。     

抗D二聚体的人源性噬菌体抗体库的构建

目的：应用噬菌体展示技术构建人源性抗D二聚体噬菌体抗体抗体组合文库。 方法： 从不同人群外周血淋巴细胞中提取总RNA，经反转录后，以免疫球蛋白信号肽序列引物和家族特异性免疫球蛋白可变区基因引物,进行半套式PCR扩增人全套抗体基因片段，并克隆于pComb3H载体，电转化大肠杆菌XL1-Blu，在辅助噬菌体的超感染下，构建噬菌体抗体组合文库。 结果： 采用不同的引物进行重链、轻链Kappa和Lambda链的半套式PCR扩增，均能获得相应大小的PCR产物，其结果扩增率达到100％。经4次电转化构建了库容为2.8×108的抗体库,轻、重链基因的重组率为46％，经辅助噬菌体的超感染，得到噬菌体滴度为4.1×1017PFU /L的人源性噬菌体抗体库。 结论： 成功构建了含抗D二聚体的人源性噬菌体抗体库，为进一步筛选抗D二聚体的Fab噬菌体抗体奠定了基础。     

构建预设CDR3基因噬菌体抗体库筛选抗人整合素ανβ3 mAb的人源化Fab

目的:构建预设CDR3基因的噬菌体抗体库,通过抗原表位导向选择方法筛选抗人整合素ανβ3单克隆抗体(mAb)人源化Fab。方法:将鼠mAbLCDR3重组到人轻链可变区文库中并与人/鼠嵌合重链Fd基因配对构建杂合噬菌体抗体库,用固相人整合素ανβ3抗原筛选人源化轻链基因。再用所获人轻链基因与移植有鼠mAbHCDR3的人重链Fd基因配对构建人源噬菌体抗体库,筛选人源化Fab。结果:分别构建了库容为2.1×106和2×107的杂合噬菌体抗体库和人源噬菌体抗体库,筛选到3株人源Fab克隆。经间接ELISA及竞争抑制ELISA证实,能特异结合整合素ανβ3抗原,其中人源D5株Fab克隆的基因序列表明,人轻链可变区基因属VKIII亚群,人重链可变区基因属VH1亚群。结论:利用噬菌体抗体库技术,成功地进行了鼠抗人整合素ανβ3mAbE10人源化的改造,为进一步临床应用奠定了基础。     

抗禽流感病毒轻链抗体库的构建

目的: 构建抗禽流感病毒 (AIV)抗体Fab轻链库,以便进一步构建完整AIV噬菌体抗体库。方法: 采用RT PCR技术, 从经禽流感灭活油乳剂疫苗免疫的罗曼蛋鸡的外周血淋巴细胞中扩增抗AIV抗体轻链基因, 将扩增的抗体轻链基因与载体PComb3HSS连接后, 电转化入大肠杆菌XLI Blue, 克隆筛选重组抗体Fab轻链基因的质粒, 并进行酶切鉴定和序列分析。结果: 扩增的AIV抗体轻链基因片段的大小约为 617bp。经克隆筛选鉴定和序列分析证明, 获得抗AIV抗体轻链库, 该轻链库库容量为 2. 5×106, 轻链基因的重组率为 50%。结论: 构建了库容量为 2. 5×106 的抗AIV抗体轻链基因库, 为下一步构建鸡抗AIV噬菌体抗体库和筛选特异性的AIVFab抗体奠定基础。     

抗呼吸道合胞病毒免疫噬菌体抗体库的构建及初步鉴定

目的: 构建小儿呼吸道合胞病毒感染患者人源性噬菌体抗体库, 搭建人源性抗体制备的技术平台, 为小儿呼吸道合胞病毒感染发病机制的研究、诊断、治疗和预防提供新的有效途径.方法: 从52例呼吸道合胞病毒感染患儿外周血淋巴细胞中提取总RNA, 并逆转录为cDNA.用PCR扩增轻链和重链Fd段(即重链的可变区和第一恒定区)基因, 并将扩增的轻链和重链基因片段克隆于pComb3x噬粒载体, 电转化XL1-Blue大肠杆菌, 经辅助噬菌体M13K07超感染后构建成Fab段噬菌体抗体库.对此抗体库双酶切进行鉴定, 并用呼吸道合胞病毒颗粒作抗原进行初步筛选.结果: 经过重轻链基因的重组, 成功构建一免疫噬菌体抗体基因库, 共有2.6×106个不同的克隆菌, 其中70%的克隆均含有轻链和重链Fd 基因.因此, 所构建的噬菌体抗体库的库容量为1.8×106, 经初步筛选, 抗体库得到了不同程度的富集.结论: 利用基因重组技术和噬菌体展示技术, 成功构建小儿呼吸道合胞病毒感染患者人源性免疫噬菌体抗体库, 为进一步的研究奠定了基础.     

Comparison of protection against H5N1 influenza virus in mouse offspring provided by maternal vaccination with HA DNA and inactivated vaccine

H5N1 influenza virus is one of the viruses that can potentially cause an influenza pandemic. Protection of newborns against influenza virus infection could be effectively provided by maternal immunization. In this study, female mice were immunized with H5N1 HA DNA vaccine or inactivated whole-virion vaccine, and the protection provided by maternal antibodies in their offspring against a lethal homologous influenza virus challenge was compared. The results showed that maternal antibodies, whether induced by a DNA vaccine or an inactivated vaccine, could completely protect offspring aged 1-4 weeks from a lethal influenza virus challenge. Breast-feeding was the major route of transfer for maternal antibodies. Milk-derived antibodies were able to effectively protect the offspring aged 1-4 weeks from lethal influenza virus infection, whereas maternal antibodies transferred through the placenta only partially protected the offspring 1-2 weeks of age. The milk- and placenta-transferred IgG2a antibody levels in offspring from their mothers, whether vaccinated with DNA vaccine or inactivated vaccine, were higher than the IgG1 levels. Our results indicated that maternal vaccination with HA DNA, as well as with whole-virion inactivated vaccine, could offer effective protection to offspring against H5N1 influenza virus infection.     

Long-Term Immunogenicity of an Inactivated Split-Virion 2009 Pandemic Influenza A H1N1 Virus Vaccine with or without Aluminum Adjuvant in Mice

In 2009, a global epidemic of influenza A(H1N1) virus caused the death of tens of thousands of people. Vaccination is the most effective means of controlling an epidemic of influenza and reducing the mortality rate. In this study, the long-term immunogenicity of influenza A/California/7/2009 (H1N1) split vaccine was observed as long as 15 months (450 days) after immunization in a mouse model. Female BALB/c mice were immunized intraperitoneally with different doses of aluminum-adjuvanted vaccine. The mice were challenged with a lethal dose (10× 50% lethal dose [LD₅₀]) of homologous virus 450 days after immunization. The results showed that the supplemented aluminum adjuvant not only effectively enhanced the protective effect of the vaccine but also reduced the immunizing dose of the vaccine. In addition, the aluminum adjuvant enhanced the IgG antibody level of mice immunized with the H1N1 split vaccine. The IgG level was correlated to the survival rate of the mice. Aluminum-adjuvanted inactivated split-virion 2009 pandemic influenza A H1N1 vaccine has good immunogenicity and provided long-term protection against lethal influenza virus challenge in mice.     

In elderly persons live attenuated influenza A virus vaccines do not offer an advantage over inactivated virus vaccine in inducing serum or secretory antibodies or local immunologic memory.

In a double-blind, randomized trial, 102 healthy elderly subjects were inoculated with one of four preparations: (i) intranasal bivalent live attenuated influenza vaccine containing cold-adapted A/Kawasaki/86 (H1N1) and cold-adapted A/Bethesda/85 (H3N2) viruses; (ii) parenteral trivalent inactivated subvirion vaccine containing A/Taiwan/86 (H1N1), A/Leningrad/86 (H3N2), and B/Ann Arbor/86 antigens; (iii) both vaccines; or (iv) placebo. To determine whether local or systemic immunization augmented mucosal immunologic memory, all volunteers were challenged intranasally 12 weeks later with the inactivated virus vaccine. We used a hemagglutination inhibition assay to measure antibodies in sera and a kinetic enzyme-linked immunosorbent assay to measure immunoglobulin G (IgG) and IgA antibodies in sera and nasal washes, respectively. In comparison with the live virus vaccine, the inactivated virus vaccine elicited higher and more frequent rises of serum antibodies, while nasal wash antibody responses were similar. The vaccine combination induced serum and local antibodies slightly more often than the inactivated vaccine alone did. Coadministration of live influenza A virus vaccine did not alter the serum antibody response to the influenza B virus component of the inactivated vaccine. The anamnestic nasal antibody response elicited by intranasal inactivated virus challenge did not differ in the live, inactivated, or combined vaccine groups from that observed in the placebo group not previously immunized. These results suggest that in elderly persons cold-adapted influenza A virus vaccines offer little advantage over inactivated virus vaccines in terms of inducing serum or secretory antibody or local immunological memory. Studies are needed to determine whether both vaccines in combination are more efficacious than inactivated vaccine alone in people in this age group.     

Protective and cross-protective mucosal immunization of mice by influenza virus type A with bacterial adjuvant.

Mucosal immunization by inactivated viruses often fails to evoke a sufficient immune response. Intensive efforts have been made to enhance the response by suitable adjuvants. We used the G+ nonpathogenic delipidated bacterium Bacillus firmus with pronounced immunostimulatory properties as an adjuvant for immunizing mice with inactivated influenza virus type A. BALB/c mice were immunized intratracheally with inactivated influenza A H1N1 and H3N2 viruses. The production of antibodies in sera and secretions was determined by the ELISA. The local situation in the lungs was assessed histologically and by testing the cytokine expression. The protective and cross-protective effect against infection was tested in in vivo experiments after infection with influenza virus A H1N1. B. firmus as adjuvant increased both systemic and mucosal antibody responses, improved protection against homologous virus and induced cross-protection against virus H1N1 after immunization with virus H3N2.     

Protection against multiple influenza A virus subtypes by intranasal administration of recombinant nucleoprotein

Vaccination is a cost-effective way to control the influenza epidemic. Vaccines based on highly conserved antigens can provide protection against different influenza A strains and subtypes. In this study, the recombinant nucleoprotein (rNP) of the A/PR/8/34 (H1N1) influenza virus strain was effectively expressed using a prokaryotic expression system and then purified with a nickel-charged Sepharose affinity column as a candidate component for an influenza vaccine. The rNP was administered intranasally three times at 3-week intervals to female BALB/c mice in combination with an adjuvant (cholera toxin B subunit containing 0.2% of the whole toxin). Twenty-one days after the last immunization, the mice were challenged with homologous or heterologous influenza viruses at a lethal dose. The results showed that intranasal immunization of 10 μg rNP with adjuvant completely protected the immunized mice against the homologous influenza virus, and immunization with 100 μg rNP in combination with adjuvant provided good cross-protection against heterologous H5N1 and H9N2 avian influenza viruses. The results indicate that such a vaccine administered intranasally can induce mucosal and cell-mediated immunity, thus having the potential to control epidemics caused by new emerging influenza viruses.     

Induction of cytotoxic T-lymphocyte and antibody responses against highly pathogenic avian influenza virus infection in mice by inoculation of apathogenic H5N1 influenza virus particles inactivated with formalin

We investigated whether a vaccine derived from an apathogenic reassortant type A H5N1 influenza strain could induce immune responses in vivo that mediated protection from highly pathogenic avian influenza virus infection in mice. After two subcutaneous immunizations with formalin-inactivated H5N1 whole virus particles (whole particle vaccine), significant killing specific for cells presenting a nucleoprotein peptide from the vaccine strain of the virus was observed. Similar vaccination with viruses treated with ether and formalin, which are commonly used for humans as ether-split vaccines, induced little or no cytotoxic T-cell response. Furthermore, whole particle vaccines of the apathogenic H5N1 strain were more effective than ether-split vaccines at inducing antibody production able to neutralize a highly pathogenic H5N1 strain. Finally, whole particle vaccines of H5N1 protected mice against infection by an H5N1 highly pathogenic avian influenza virus more effectively than did ether-split vaccines. These results suggest that formalin-inactivated virus particles of apathogenic strains are effective for induction of both cytotoxic T-lymphocyte and antibody responses against highly pathogenic avian influenza viruses in vivo, resulting in protection from infection by a highly pathogenic H5N1 virus.     

Intranasal Immunization with a Formalin-Inactivated Human Influenza A Virus Whole-Virion Vaccine Alone and Intranasal Immunization with a Split-Virion Vaccine with Mucosal Adjuvants Show Similar Levels of Cross-Protection

The antigenicity of seasonal human influenza virus changes continuously; thus, a cross-protective influenza vaccine design needs to be established. Intranasal immunization with an influenza split-virion (SV) vaccine and a mucosal adjuvant induces cross-protection; however, no mucosal adjuvant has been assessed clinically. Formalin-inactivated intact human and avian viruses alone (without adjuvant) induce cross-protection against the highly pathogenic H5N1 avian influenza virus. However, it is unknown whether seasonal human influenza formalin-inactivated whole-virion (WV) vaccine alone induces cross-protection against strains within a subtype or in a different subtype of human influenza virus. Furthermore, there are few reports comparing the cross-protective efficacy of the WV vaccine and SV vaccine-mucosal adjuvant mixtures. Here, we found that the intranasal human influenza WV vaccine alone induced both the innate immune response and acquired immune response, resulting in cross-protection against drift variants within a subtype of human influenza virus. The cross-protective efficacy conferred by the WV vaccine in intranasally immunized mice was almost the same as that conferred by a mixture of SV vaccine and adjuvants. The level of cross-protective efficacy was correlated with the cross-reactive neutralizing antibody titer in the nasal wash and bronchoalveolar fluids. However, neither the SV vaccine with adjuvant nor the WV vaccine induced cross-reactive virus-specific cytotoxic T-lymphocyte activity. These results suggest that the intranasal human WV vaccine injection alone is effective against variants within a virus subtype, mainly through a humoral immune response, and that the cross-protection elicited by the WV vaccine and the SV vaccine plus mucosal adjuvants is similar.     

Comprehensive serological analysis of two successive heterologous vaccines against H5N1 avian influenza virus in exotic birds in zoos

In 2005, European Commission directive 2005/744/EC allowed controlled vaccination against avian influenza (AI) virus of valuable avian species housed in zoos. In 2006, 15 Spanish zoos and wildlife centers began a vaccination program with a commercial inactivated H5N9 vaccine. Between November 2007 and May 2008, birds from 10 of these centers were vaccinated again with a commercial inactivated H5N3 vaccine. During these campaigns, pre- and postvaccination samples from different bird orders were taken to study the response against AI virus H5 vaccines. Sera prior to vaccinations with both vaccines were examined for the presence of total antibodies against influenza A nucleoprotein (NP) by a commercial competitive enzyme-linked immunosorbent assay (cELISA). Humoral responses to vaccination were evaluated using a hemagglutination inhibition (HI) assay. In some taxonomic orders, both vaccines elicited comparatively high titers of HI antibodies against H5. Interestingly, some orders, such as Psittaciformes, which did not develop HI antibodies to either vaccine formulation when used alone, triggered notable HI antibody production, albeit in low HI titers, when primed with H5N9 and during subsequent boosting with the H5N3 vaccine. Vaccination with successive heterologous vaccines may represent the best alternative to widely protect valuable and/or endangered bird species against highly pathogenic AI virus infection.