三个不同亚型HIV-1调节/辅助基因DNA疫苗的构建和免疫原性研究

目的 构建表达中国主要流行亚型HIV-1 tat-rev-integrase(c-half)-vif-nef(TRIVN)融合基因的DNA疫苗,并比较其免疫原性.方法 按人源密码子使用频率对HIV-1 CN54(B'/C重组亚型)与RL42(B'亚型)的tat、rev、integrase(C端144个氨基酸)、vif和nef基因序列进行优化,构建DNA疫苗.通过Western blot测定上述DNA疫苗与HIV-1 AE2f株来源的tat-rev-integrase(c-half)-vifnef融合基因DNA疫苗的体外表达效率;利用小鼠模型比较3个DNA疫苗单独免疫与混合免疫的免疫原性特征.结果 限制性酶切及DNA测序结果表明两个融合基因重组质粒构建正确;Western blot 检测结果显示:3个DNA疫苗的体外表达效率基本相当.小鼠免疫后ELISPOT检测结果显示:在总T细胞反应强度方面AE2f-TRIVN最强[(948.0±330.0)SFCs/106脾细胞],次之为混合DNA免疫组(500.0±155.0 SFCs/106脾细胞),再者为RL42-TRIVN[(195.1±44.0)SFCs/106脾细胞],CN54-TRIVN最弱[(89.5±17.0)SFCs/106脾细胞].T细胞反应分布情况显示:3个DNA疫苗单独免疫时,T细胞反应主要集中在Integrase和Vif蛋白上;而混合免疫可以部分改善针对Nef蛋白的免疫识别.结论 3个亚型TRIVN DNA疫苗中以AE2f-TRIVN的免疫原性最强;DNA疫苗混合免疫倾向于促进特异性T细胞反应在TRIVN融合抗原上的均匀分布.

Abstract：

0bjective To determine the immunogenicities of DNA vaccines expressing tat-rev-integrase(c-half)-vif-neffusion genes(TRIVN) derived from prevalent B', B'/C and AE recombinant subtypes of HIV-1 in China. Methods Two DNA vaccines were constructed by inserting the codon optimized tat-revintegrase(c-half)-vif-nef fusion genes derived from B' and B'/C subtype of HIV-1 into mammalian expression vector pSVI. 0. DNA vaccine containing tat-rev-integrase (c-half)-vif-nef fusion gene derived from HIV-1AE2f has been constructed previously. In vitro expression efficiencies of three DNA vaccines were determined by Western blot and their immunogenicities were compared by immunizing female BALB/c mice. IFN-γ ELISPOT assay was used to read out the specific T cell immunity. Results The constructed DNA vaccines were validated by restriction enzyme digestion and DNA sequencing. Western blot assay showed three constructed DNA vaccines could be expressed at a comparable level in vitro. After vaccination, AE-TRIVN mounted T cell immune responses at (948.0 ± 330.0) SFCs/106 splenocytes, followed by the mixed DNA vaccine[ (500.0 ± 155.0) SFCs/106 splenocytes ], RL-TRIVN r[ ( 195. 1 ± 44.0) SFCs/106 splenocytes ]and CN-TRIVN [ (89.5 ± 17.0) SFCs/106 splenocytes]. Interestingly, we observed that single DNA vaccination induced specific T cell responses predominantly targeting Integrase (C-half) and Vif, whereas the mixed DNA could significantly improve T cell responses against Nef. Conclusion AE-TRIVN was the most immunogenic among the three DNA vaccines and the mixed DNA vaccination could change the immunogenic hierarchy of T cell epitopes across the fusion genes vaccine.     

HIV-1 B''亚型gag-env融合基因的DNA疫苗构建和免疫原性分析

目的 构建HIV-1 B′亚型中国流行株gag和env融合基因的DNA疫苗，对其免疫原性进行研究。方法 根据已报道的HIV-1 B′亚型RIA2分离株gag和env基因的氨基酸序列按哺乳动物密码子使用频率进行优化并人工合成基因，插入真核表达载体pDRVISV1．0中，构建表达RL42 gag-env，融合蛋白的DNA疫苗，pSVRL／GE。用Western blot和抗gag p55抗体细胞内染色的方法体外检测pSVRUGE的表达效率。DNA疫苗pSVRL/GE免疫BALB／c小鼠后，用ELISPOT检测小鼠的细胞免疫反应。结果 限制性内切酶鉴定表明融合基因已成功插入pDRVISV1．0载体中，Western blot证实融合基因可有效表达融合蛋白；细胞内染色结果表明，pSVRL/GE转染的293T细胞中49．8％表达gag p55，荧光强度均值为924；而空载体pDRVISV1．0转染的293T细胞中非特异背景染色只有0．5％。经免疫的小鼠脾细胞体外用H-2^d限制性表位肽AMQMIKET刺激后，ELISPOT检测显示，pSVRUGE免疫小鼠每10^6脾细胞形成226个斑点（SD=140），而空载对照组每1驴脾细胞形成29个斑点（SD=16）（P〈0．05）。结论 所构建的DNA疫苗pSVRL/GE可高效表达相应抗原蛋白，并可有效激活机体的细胞免疫反应。     

表达HIV-1 B亚型env基因的质粒DNA及腺病毒载体疫苗的免疫原性研究

目的 构建表达中国B亚型HIV-1流行株env基因的DNA及重组腺病毒载体疫苗,将其用于预防或治疗HIV感染.方法 构建质粒DNA疫苗pVR-gp160及重组腺病毒载体疫苗rAdV-gp160.将这两种疫苗以不同的方式免疫BALB/c小鼠,分别采用ELISPOT方法 和ELISA方法 检测免疫小鼠中HIV-1 Gp120特异性细胞免疫反应及抗体反应.结果 DNA疫苗单独免疫及DNA疫苗初免/腺病毒疫苗加强免疫的联合免疫方案皆可诱导较高水平的Gp120特异性细胞免疫反应;而在体液免疫方面,各实验组产生的Gp120特异性抗体水平都较低.结论 所构建的DNA疫苗及rAdV疫苗能有效表达Gp160蛋白,并可有效激活机体的细胞免疫反应.     

不同基因融合方式对HIV-1AE2f Tat、Rev和Nef蛋白免疫原性的影响

目的 构建表达HIV-1 AE2f gp145-tat-rev-nef融合基因的DNA疫苗,并比较gp145-tat-rev-nef(AE-Gp145TRN)和tat-rev-integrase(c-half)(AE-TRIVN)两种不同基因融合方式对Tat、Rev和Nef蛋白免疫原性的影响.方法 按人源密码子使用频率对HIV-1 AE2f的gp145、tat、rev和nef基因序列进行优化并构建DNA疫苗.经Western blot进行体外表达检测后,利用小鼠模型比较AE-Gp145TRN与AE-TRIVN的免疫原性.结果 限制性酶切及DNA测序结果表明AE-Gp145TRN融合基因表达重组质粒构建正确;Western blot结果显示其能够在体外表达相应的融合蛋白.小鼠免疫后IFN-γ ELISPOT检测结果显示:AE-TRIVN所活化的针对Tat、Rev和Nef蛋白的总T细胞反应强度[(148±91)SFCs/106脾细胞]显著高于Gp145TRN[(55±28)SFCs/106脾细胞,P=0.017].T细胞反应分布情况显示:AE-TRIVN所活化的T细胞反应主要集中在Rev蛋白上;而Gp145TRN则可以显著提高对Nef蛋白的免疫识别.结论 AE-TRIVN与Gp145TRN在小鼠体内所活化的T细胞反应特征有明显区别,提示不同的抗原融合方式可能影响融合基因DNA疫苗的免疫结果.

Abstract：

Objective To construct DNA vaccine expressing HIV-1 AE2f gp145-tat-rev-nef fusion gene( AE-Gp145TRN) and to compare the immunogenicities of DNA vaccines expressing Tat, Rev and Nef in gene fusion formulations of tat-rev-integrase(c-half)-vif-nef( AE-TRIVN) and AE-Gpl45TRN. Methods DNA vaccine was constructed by inserting the codon optimized HIV-1 AE2( gp145-tat-rev-nef fusion gene into mammalian expression DNA vector. In vitro expression efficiency of the constructed DNA vaccine was determined by Western blot and the immunogenicities of AE-Gpl45TRN and AE-TRIVN were compared by immunizing female BALB/c mice. IFN-r ELISPOT assay was used to read out the specific T cell immunity. Results Western blot assay showed the constructed DNA vaccine could be expressed efficiently in vitro. After vaccination, AE-TRIVN mounted significantly higher T cell responses against Tat, Rev and Nef[(148±91)SFCs/106 splenocytes]than Gpl45TRN[(55±28) SFCs/106 splenocytes]. Specific T cell responses elicited by AE-TRIVN predominantly targeting Rev, whereas Gpl45TRN could significantly enhance T cell responses against Nef. Conclusion AE-TRIVN and Gpl45TRN induced distinct T cell response modalities, which implied different gene fusion formulations may affect the immunogenicity of specific DNA vaccines.     

以肿瘤抗原BAP31为靶点的基因疫苗构建及其诱导小鼠免疫反应的研究

目的: 构建重组表达载体P-BAP31和P-L-BAP31基因疫苗并接种小鼠, 评价其诱导的体液和细胞免疫应答.方法: 利用分子克隆技术, 构建重组质粒P-BAP31和P-L-BAP31, 同时构建P-BAP31/EGFP和P-L-BAP31/EGFP重组质粒, 并将其以脂质体瞬时转染HeLa细胞, 通过荧光显微镜观察目的蛋白的表达;将重组质粒P-BAP31和P-L-BAP31免疫C57BL/6小鼠, 采用间接ELISA检测免疫小鼠血清中特异性抗体效价;通过酶联免疫斑点实验(ELISPOT)检测免疫脾细胞在受到BAP31抗原刺激后产生IFN-γ和IL-4细胞因子的频率;通过乳酸脱氢酶(LDH释放)试验检测小鼠特异性细胞毒性T淋巴细胞(CTL)的应答.结果: 酶切及测序结果表明, 成功构建了针对BAP31肿瘤抗原的P-BAP31和P-L-BAP31基因疫苗.通过荧光显微镜观察重组质粒转染的HeLa细胞, 可见EGFP报告基因表达的绿色荧光.ELISA检测免疫小鼠血清中特异性抗体效价发现, 带LAMP组明显高于不带LAMP组(P<0.05), 且两者均高于空质粒(P-LAMP)及正常对照(N)组(P<0.01).ELISPOT检测脾细胞分泌细胞因子IFN-γ和IL-4的频率, 带LAMP组与其他组相比显著提高(P<0.01).LDH释放试验显示, 带LAMP组的特异性CTL活性高于不带LAMP组, 且均高于对照组(P<0.01).结论: 构建了针对肿瘤抗原BAP31的全长基因疫苗, 以其免疫C57BL/6小鼠可诱导特异性体液和细胞免疫反应, 其中, P-L-BAP31基因疫苗各项免疫反应均优于P-BAP31.     

人偏肺病毒DNA疫苗的构建和小鼠免疫反应的研究

目的 构建人偏肺病毒(hMPV)DNA疫苗,小鼠免疫后评价其细胞和体液免疫水平.方法 利用PCR方法,从hMPV的cDNA中扩增融合蛋白FATM(缺失跨膜区)基因和基质蛋白M基因,构建DNA疫苗pcDNA3.1His-FATM和pcDNA3.1His-M,瞬时转染后用Western Blot和间接免疫荧光方法检测F、M蛋白表达.疫苗肌内注射免疫小鼠,ELISA和ELISPOT方法分别检测血清IgG抗体和小鼠脾细胞CTL水平.结果 Western Blot和间接免疫荧光(IFA)方法证明构建的疫苗可表达FATM和M蛋白.peDNA3.1His-FATM单独免疫小鼠,血清抗体滴度为1:44;与pcDNA3.1His-M联合免疫后,血清抗体滴度为1:64.ELISPOT检测证明,联合免疫组小鼠脾细胞产生IFN-γ的效应CD8+T细胞数为42±8.9,高于单独免疫组32±7.4的水平.结论 DNA疫苗peDNA3.1His-F△TM可以诱导产生特异性的体液和细胞免疫,与pcDNA3.1His-M联合免疫,可以提高免疫水平.     

恙虫病东方体Karp株47kD蛋白基因的表达及DNA免疫初步研究

目的　观察恙虫病东方体Karp株相对分子质量 (Mr)为 47× 10 3 蛋白基因的DNA免疫效果。方法　将恙虫病东方体Karp株Mr 为 47× 10 3 的蛋白基因插入真核表达载体pcDNA3.1( +) ,构建重组质粒pcDNA3.1 47。在证明该重组质粒可在哺乳动物COS7细胞中表达的基础上 ,单独和将其与Mr 为 40× 10 3 重组蛋白联合免疫小鼠。在每次加强免疫之后 10d ,检测小鼠体液和细胞免疫反应。结果　质粒DNA和蛋白联合免疫组所诱导的抗体水平和脾淋巴细胞增殖反应均高于重组质粒DNA和蛋白单独免疫组 ,而第二次加强免疫后重组质粒诱导脾淋巴细胞的增殖情况则显著弱于其它免疫组 (P <0 .0 5 )。结论　重组质粒pcDNA3 .1 47和重组Mr 为 47× 10 3 蛋白在刺激小鼠免疫反应上具有相互加强作用。     

表达丙型肝炎病毒NS3和Core融合蛋白基因的DNA疫苗免疫方案优化

目的 本研究旨在构建一种包含丙型肝炎病毒(HCV)保守区基因的新型DNA疫苗,并在小鼠模型中使用电转技术优化其免疫原性.方法 首先,我们构建了包含HCV非结构蛋白NS3和核心蛋白Core部分基因序列的DNA疫苗,并证实了其表达;然后采用不同的体内电转方式于第0、4周分别免疫BALB/c小鼠,比较分析不同免疫方案的体液免疫(特异性IgG与抗体亚类)与细胞免疫应答(IFN-γ ELISPOT)的效果.结果 使用电转技术可显著增强新型DNA疫苗免疫原性,采用皮内注射加卡钳电极电转的方式产生最强NS3特异性T细胞免疫反应.结论 包含HCV保守区基因的新型DNA疫苗可通过优化电转技术增强免疫应答效果.这为我们下一步优化HCV DNA疫苗的免疫方案提供了依据.     

SARS冠状病毒spike基因DNA疫苗的初步研究

目的　构建SARS病毒spike基因片段真核表达质粒DNA疫苗;检测spike基因片段的免疫原性;筛选SARS病毒疫苗构建的理想靶抗原。方法　采用RT PCR从SARS冠状病毒北京0 1(BJ0 1)株cDNA获取了spike基因cDNA的全长D12、编码N末端氨基酸的cDNA片段D14和编码C末端氨基酸的cDNA片段D2 3;构建重组真核表达质粒pcDNA3 /D12、pcDNA3 D14、pcDNA3 D2 /3;用3种重组质粒和pcDNA3空质粒载体(对照)DNA皮下注射免疫Wistar大鼠;ELISA检测免疫后大鼠血清S蛋白特异性抗体IgG的产生;同位素51 Cr实验检测特异性CTL应答;ELISPOT检测单细胞水平IFN -γ分泌。结果　pcDNA3 /D2 3疫苗免疫组大鼠血清有S蛋白阳性抗体IgG产生、抗体滴度>10 0 0 ;脾淋巴细胞可诱导特异的CTL应答和IFN γ分泌,pcDNA3 /D2 3疫苗组与其它组之间统计分析P <0 .0 5。结论SARS冠状病毒S蛋白的C末端具有较强的免疫原性,是疫苗构建的理想候选靶抗原,本研究结果为SARS病毒的疫苗研究提供了资料。     

插入IL-2基因优化HBV DNA疫苗的研究

目的：观察IL-2基因的插入对DNA疫苗免疫效应的影响，探讨优化DNA疫苗设计、提高DNA疫苗兔疫效应的途径。方法：采用PCR产物直接克隆和重组DNA技术构建了人IL-2和HBsAg融合基因的真核表达载体pcDNA3．1 -S/IL-2，通过脂质体基因转移技术导入Cos-7细胞中检测其瞬间表达，并经肌肉注射免疫C57Bl/6小鼠，以检测和比较它们的细胞和体液免疫应答。结果：通过酶切、PCR及测序证实已正确完整地插入 IL-2基因，成功构建了 pcDNA3．1 -S/IL-2重组质粒。体外转染Cos-7后可见基因的表达和分泌，pcDNA3．1 -S/IL-2可以提高免疫小鼠的抗 HBs抗体滴度和脾淋巴细胞诱生的IL-2的生物活性水平，增加 HBsAg特异性的脾淋巴细胞增殖指数。结论：IL-2和 HBsAg基因的融合表达对 DNA疫苗的免疫反应起协同和增强作用，提示IL-2基因插入可能是增强DNA疫苗的免疫效应的可行途径。     

HIV-2 gag rFPV与rDNA疫苗的联合免疫研究

目的 探讨HIV-2核心蛋白基因gag重组DNA疫苗与重组鸡痘病毒进行联合免疫引起小鼠的免疫应答，为研究HIV-2基因重组疫苗的免疫策略提供实验基础。方法 大量制备并纯化HIV-2 gag重组DNA疫苗和重组鸡痘病毒，以肌肉注射的方式免疫BALB／c小鼠，ELISA法检测小鼠血清HIV-2抗体，流式细胞仪测定CD4^＋、CD8^＋T淋巴细胞亚类数量，乳酸脱氢酶（LDH）释放法检测脾CTL对HIV-2靶细胞的杀伤活性。结果 重组DNA疫苗和重组鸡痘病毒单独免疫及二者联合免疫均刺激小鼠产生HIV-2特异性抗体，脾T细胞亚类数量增加，并产生针对HIV-2靶细胞的特异性CTL杀伤活性，但联合免疫组在各项指标上均高于单独免疫组。结论 以HIV-2gag重组DNA疫苗进行基础免疫、以HIV-2gag重组鸡痘病毒进行加强免疫能诱导小鼠产生更强的特异性细胞和体液免疫应答。     

H3N8型马流感血凝素核酸疫苗的初步研究

目的研发一种具有良好免疫原性的H3N8型马流感血凝素(hemagglutinin,HA)核酸疫苗。方法根据马A型流感病毒A/Equine/Xinjiang/3/08(H3N8)的HA蛋白序列,经密码子优化后化学合成能够表达相应蛋白的基因片段,并将该片段克隆到核酸疫苗载体pJW4303中,构建带有天然信号肽的H3HA核酸疫苗,命名为H3HA/XJ3-08-wt;进一步对HA基因进行修饰,以人组织纤维蛋白酶原激活剂(human tissue plasminogen activator,tPA)取代H3HA天然信号肽,命名为H3HA/XJ3-08-tPA,或切除部分HA的基因片段使之只表达HA蛋白的胞外域,命名为H3HA/XJ3-08-dTM。用这3种重组质粒分别转染293T细胞,蛋白表达经蛋白质印迹检测得到确认。采用电转录法免疫新西兰白兔。ELISA方法检测免疫后兔血清中抗H3HA抗体滴度,血凝抑制实验(hemagglutination inhibition,HI)检测保护性抗体水平。结果 3种H3HA核酸疫苗均可在293T细胞中高效表达,并能够在新西兰白兔体内诱导产生特异性抗H3HA抗体,HI检测到不同水平的保护性抗体。其中以H3HA/XJ3-08-tPA的免疫原性最强。结论新构建的H3N8型马流感血凝素核酸疫苗具有良好的免疫原性,为此类疫苗的进一步研发奠定了基础。     

HIV-1 DNA vaccines

HIV-1 was among the original DNA vaccine targets and HIV DNA vaccines are now in human trials. Lack of strong correlates of protective immunity makes vaccine design difficult; however, DNA vaccines have the potential to be an ideal vaccine and therapeutic approach against HIV-1. DNA vaccines induce conformational-dependent antibodies, mimic live vaccines but without the pathogenic potential, and can easily be made polyvalent. Genes which encode important CTL and antibody epitopes can be included while those that confer pathogenicity, virulence, antibody enhancement or represent non-conserved epitopes can be excluded. In our hands pre-treatment of muscles with bupivacaine or cardiotoxin did not offer any advantage over no muscle pre-treatment or gene gun inoculation of skin although gene gun immunization seem to favour a Th2 type response. As DNA vaccine candidates we have compared vaccines encoding native HIV MN gp160 with Rev-independent synthetic genes encoding MNgp160 and MNgp120 using mammalian high expression codons. In these experiments the gene encoding secreted gp120 gave highest antibody neutralizing titers. High and fast antibody responses could also be obtained by transferring the HIV-1 MN V3 loop to the secreted HBsAg as a fusion gene vaccine. Thus, in the case of HIV-1 MN genes encoding secreted surface glycoproteins may be preferred instead of membrane bound envelopes. CTL responses were induced in all cases. However, in order to meet the high diversity of HIV and HLA types our approach is to include many CTL epitopes in a multivalent minigene vaccine. We found that gene gun DNA vaccination with minimal epitopes could induce specific CTL. Flanking sequences influenced the CTL response but was not needed. DNA vaccines encoding known and computer predicted CTL epitopes are now being developed.     

Experimental Leishmania major infection suppresses HIV-1 DNA vaccine induced cellular immune response

The AIDS epidemic in the developing world represents a major global crisis and an effective vaccine is imperative. However, many parasites are common in developing countries and can result in a state of chronic immune activation that is polarized towards a Th2 profile and which can potentially impair responses to vaccines or other infectious challenges. In this study we demonstrate that experimental Leishmania major infection of BALB/c mice inhibits responses to a DNA-based HIV-1 gag vaccine. L. major infection in BALB/c results in a polarized Th2 immune response. In this study na?ve BALB/c mice immunized with the HIV-1 gag DNA vaccine mounted a cellular immune response against the vaccine antigen, HIV-1 gag. CD8+ T lymphocytes were able to respond in vitro to HIV-1 gag stimulation and secrete interferon (IFN)-gamma. However, L. major-infected, vaccinated BALB/c mice had a significantly reduced number of IFN-gamma-producing CD8+ T cells following in vitro stimulation with gag antigen. These data suggest that parasitic infection, which results in a Th2 profile, reduces the efficacy of DNA vaccines that are designed to induce antiviral CD8+ T cell responses.     

HIV-1 DNA vaccine efficacy is enhanced by coadministration with plasmid encoding IFN-alpha

Numerous strategies have been employed in an attempt to improve the immunogenicity and efficacy of nucleic acid vaccines. In the present study, the immunogenicity in the induction of humoral and cellular immune responses to HIV-1 DNA vaccine expressing a chimeric gene of gag and gp120 and the adjuvant effect of IFN-alpha on HIV-1 DNA vaccine were studied in a murine model. The DNA vaccine plasmid pVAX1-gag-gp120 and eukaryotic expression plasmid pVAX1-IFN were constructed by inserting the chimeric gene of gag and gp120 of HIV-1 and IFN-alpha into the downstream of CMV promoter of eukaryotic expression vector pVAX1, respectively. In vitro expression detected by RT-PCR and Western blotting showed that the genes of interest could be expressed in transfected HeLa cells. After BALB/c mice were immunized by three intramuscular inoculations of the HIV-1 DNA vaccine plasmids alone or in combination with IFN-alpha expression plasmids, the different levels of anti-HIV-1 humoral and cellular responses were measured comparable to the control groups immunized with pVAX1-IFN, parent plasmid pVAX1 or PBS. The percentage of CD3+CD4+ and CD3+CD8+ subgroups of spleen T lymphocytes and the specific cytotoxicity activities of splenic CTLs in the coinoculation group were significantly higher than those in the separate inoculation group, and an enhancement of antibody response was also observed in the coinoculation group compared with the separate inoculation group. Take together, coadministration of HIV-1 DNA vaccine plasmids and IFN-alpha expression plasmids can elicit stronger humoral and cellular immune responses in mice than HIV-1 DNA vaccine plasmids alone, and IFN-alpha can be an effective immunological adjuvant in DNA vaccination against HIV-1.