含HIV-1 gag基因的重组腺病毒5型与35型嵌合病毒免疫效果研究

目的探讨含HIV-1 gag基因的重组腺病毒5型与35型嵌合病毒(rAd5/F35)在BALB/c小鼠中的免疫效果。方法PCR,间接免疫荧光鉴定HIV-1gag基因在重组腺病毒(rAd5/F35-mod.gag)的正确插入和体外细胞水平的表达,用rAd5/F35-mod.gag以不同的方式免疫BALB/c小鼠,ELISA检测小鼠血清中的p24特异性抗体,细胞内细胞因子染色法检测小鼠的特异性细胞毒性T淋巴细胞(CTL)应答。结果重组腺病毒rAd5/F35-mod.gag在体外细胞水平可以很好的表达目的基因gag;在小鼠体内重组腺病毒rAd5/F35-mod.gag可诱导特异性的CTL应答和血清IgG抗体反应,用rAd5/F35单独免疫2次,所诱发的CTL和血清IgG反应最强。但Ad5初次感染后,产生的抗Ad5抗体可以抑制rAd5/F35疫苗的特异性免疫反应。结论重组腺病毒rAd5/F35-mod.gag单独免疫可在小鼠体内诱导特异性的CTL应答和血清IgG抗体反应。只改变Ad5纤突蛋白Fiber,不能避免抗Ad5抗体的抑制作用。     

重组嵌合腺病毒35型和重组痘苗病毒SIV疫苗在小鼠体内联合免疫的实验研究

目的:研究嵌合腺病毒35型载体和痘苗病毒载体SIVenvT疫苗通过不同策略联合免疫小鼠所诱导的细胞和体液免疫应答。方法:通过PCR和Western blot方法对表达SIV mac239株SIVenvT基因的重组嵌合腺病毒35型(rAd5F35-SIVenvT)和重组痘苗病毒安卡拉株(rMVA-SIVenvT)进行体外...     

重组猪IL-2对口蹄疫重组腺病毒免疫增强效果的研究

目的构建表达猪白细胞介素-2(poIL-2)的重组腺病毒,研究其作为佐剂对口蹄疫重组腺病毒免疫效果的影响。方法将poIL-2基因克隆到腺病毒的穿梭载体中,与腺病毒骨架载体共转化BJ5183感受态细胞,获得的重组病毒质粒通过脂质体转染HEK-293A细胞,获得表达poIL-2的重组腺病毒(rAd5poIL-2)。以rAd5poIL-2作为佐剂与表达PMDV VP基因的重组腺病毒(rAd5VP1)联合免疫小鼠,以单独免疫rAd5VP1的小鼠作为对照。通过测定淋巴细胞增殖指数、特异性抗体、IL-4和IFN-γ的水平来衡量rAd5poIL-2对rAd5VP1的免疫增强效果。结果经PCR鉴定,成功构建了rAd5poIL-2。MTT与抗体检测结果显示,rAd5poIL-2既能增强rAd5VP1诱导小鼠淋巴细胞增殖,又能促进rAd5VP1诱导小鼠特异性抗体的产生,细胞因子检测结果显示,rAd5poIL-2能促进rAd5VP1诱导Th1和Th2的分化。结论 rAd5poIL-2可望成为口蹄疫疫苗的候选佐剂。     

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.     

rAd5和rAAV2/1载体疫苗诱导载体特异性和外源基因特异性免疫反应的研究

目的 在小鼠体内比较携带相同外源基因的rAd5和rAAV2/1载体疫苗诱导的载体特异性和外源基因特异性免疫应答的差异.方法 分别用携带HIV-1 gag基因的rAd5和rAAV2/1疫苗免疫BALB/c小鼠,在免疫后不同时间点用Elispot方法和ELISA方法检测小鼠体内的rAd5或rAAV2/1载体特异性及HIV-1 gag特异性细胞免疫反应和抗体反应.结果 rAd5-gag能够诱导较强的gag特异性细胞免疫反应,显著高于针对Ad5载体的细胞免疫反应;而rAAV2/1 -gag诱导的gag特异性及AAV2/1载体特异性细胞免疫反应水平都较低.rAd5-gag诱导的P24特异性和Ad5特异性IgG抗体水平都较高,并且二者相当;与rAd5-gag相比rAAV2/1 -gag可诱导更高水平的P24 IgG抗体,而且rAAV2/1 -gag诱导的P24 igG高于AAV2/1载体特异性IgG水平.结论 rAd5载体可诱导强的外源基因特异性细胞免疫和抗体反应,针对载体的细胞免疫反应较弱而抗体反应较强;rAAV2/1载体可诱导强的外源基因特异性抗体反应,明显高于针对载体的抗体反应,外源基因特异性和载体特异性细胞免疫反应水平都很低.     

IL-18和HIV-1 gag-gp120嵌合基因DNA疫苗联合免疫小鼠的免疫应答

目的 :探讨IL 18和HIV 1gag gp12 0嵌合基因的DNA疫苗联合免疫小鼠的免疫应答。方法 :构建含IL 18的真核表达质粒pVAXIL18,将他与表达HIV 1gag gp12 0嵌合基因的核酸疫苗质粒pVAXGE共同肌注免疫BALB/c小鼠 ,检测免疫小鼠脾特异性CTL杀伤活性和血清抗体滴度。结果 :联合免疫组小鼠脾特异性CTL杀伤活性和血清抗体水平均显著高于单独免疫组 (P <0 .0 5 ) ,空白质粒对照组 (P <0 .0 1)和PBS对照组 (P <0 .0 1)。结论 :IL 18和HIV 1gag gp12 0嵌合基因的DNA疫苗联合免疫可诱导小鼠产生特异性细胞和体液免疫 ,且IL 18发挥了免疫佐剂的作用。     

含HIV-1 gp120基因的重组腺相关病毒和重组腺病毒疫苗联合免疫的研究

目的 探讨含HIV-1 gp120基因的重组腺相关病毒(rAAV)和重组腺病毒(rAdV)疫苗在BALB／c小鼠中联合免疫的效果。方法 将密码子优化的HIV-1 gp120基因分别插入腺相关病毒(AAV)和腺病毒(AdV)载体质粒，构建含该基因的rAVV和rAdV载体疫苗。将两种疫苗以不同的联合方式免疫BALB／c小鼠，ELISA检测小鼠血清中的gp120特异性抗体，细胞内细胞因子染色法检测小鼠的特异性细胞毒性T淋巴细胞(CTL)应答。结果 两种重组病毒均可表达目的基因gp120；在小鼠体内两种重组病毒联合免疫可诱导特异性的CTL应答和血清1gG抗体反应，但用rAAV初免2次，再用rAdV加强3次所诱发的CTL和血清1gG反应最强。结论 rAAV和rAdV疫苗联合免疫可在小鼠体内诱导特异性的CTL应答和血清1gG抗体反应。     

rAd5和rAAV2／1载体疫苗诱导载体特异性和外源基因特异性免疫反应的研究

目的 在小鼠体内比较携带相同外源基因的rAd5和rAAV2/1载体疫苗诱导的载体特异性和外源基因特异性免疫应答的差异.方法 分别用携带HIV-1 gag基因的rAd5和rAAV2/1疫苗免疫BALB/c小鼠,在免疫后不同时间点用Elispot方法和ELISA方法检测小鼠体内的rAd5或rAAV2/1载体特异性及HIV-1 gag特异性细胞免疫反应和抗体反应.结果 rAd5-gag能够诱导较强的gag特异性细胞免疫反应,显著高于针对Ad5载体的细胞免疫反应;而rAAV2/1 -gag诱导的gag特异性及AAV2/1载体特异性细胞免疫反应水平都较低.rAd5-gag诱导的P24特异性和Ad5特异性IgG抗体水平都较高,并且二者相当;与rAd5-gag相比rAAV2/1 -gag可诱导更高水平的P24 IgG抗体,而且rAAV2/1 -gag诱导的P24 igG高于AAV2/1载体特异性IgG水平.结论 rAd5载体可诱导强的外源基因特异性细胞免疫和抗体反应,针对载体的细胞免疫反应较弱而抗体反应较强;rAAV2/1载体可诱导强的外源基因特异性抗体反应,明显高于针对载体的抗体反应,外源基因特异性和载体特异性细胞免疫反应水平都很低.     

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。     

含密码子优化型HIV-1 gp120基因重组腺病毒的构建及其免疫效果研究

目的 构建能表达野生型和密码子优化型人免疫缺陷病毒Ⅰ型(HIV-1)B亚型中国流行株gp120基因的非复制型腺病毒。方法 按哺乳动物细胞偏好的密码子对HIV-1B亚型中国流行株Ch gp42的gp120基因进行优化，合成优化基因。将野生型和密码子优化的gp120基因插入穿梭质粒，再与腺病毒骨架质粒pAdEasy-1共转化E．coli BJ5183，获得重组子，转染293细胞后获得重组病毒。分别以两种重组腺病毒疫苗免疫小鼠，ELISA检测小鼠血清中的特异性抗体，乳酸脱氢酶法检测小鼠细胞毒性T淋巴细胞(CTL)反应。结果 获得两株重组腺病毒rAd-wt．gp120和rAd．mod．gp120，能正确表达Gp120。rAd-mod．gp120比rAd-wt．gp120蛋白表达水平明显提高。重组腺病毒免疫小鼠后能产生HIV-1特异性的抗体及CTL反应，rAd-mod．gp120组明显优于rAd-wt．gp120组。结论 成功构建了表达野生型和密码子优化的HIV-1 gp120基因的重组腺病毒，能诱导HIV-1特异性体液和细胞免疫反应。     

Immunogenicity of the recombinant adenovirus type 5 vector with type 35 fiber containing HIV-1 gag gene

The immune efficiency of a recombinant adenovirus type 5 with type 35 fiber containing HIV-1 gag gene (rAd5/F35-mod.gag) was investigated in BALB/c mice, in which the rAd5/F35-mod.gag was firstly identified with PCR, then transfected to 293 cells and the in vitro expression level of Gag protein was determined by Western blotting and indirect immuno-fluorescent assay. Mice were immunized with intramuscular injections of rAd5/F35-mod.gag, rAd5-mod.gag or DNA and were boosted after 3 weeks. To test the effect of pre-existing anti-viral immunity on immunization, mice were also injected with Ad5-GFP vector and then immunized 4 and 7 weeks later with Ad5/F35-mod. gag vector. The P24-specific IgG antibody in sera of immunized mice was determined by ELISA and the specific cytotoxic T lymphocyte (CTL) response was assayed by intracellular cytokine staining. It was demonstrated that the rAd5/F35-mod. gag vector could express efficiently the HIV Gag protein in 293 cells in vitro and induce strong HIV-specific immune responses in vivo. The strongest CTL and serum IgG response occurred when mice were immunized twice with injection of rAd5/F35 alone, but the anti-Ad5 antibody after primary infection with adenovirus could inhibit the specific immune responses induced by rAd5/F35 vector. It is concluded that single immunization with recombinant adenovirus rAd5/F35-mod. gag can induce specific CTL and serum IgG antibody responses in mice, but the immunogenicity of rAd5/F35 is comparably weaker than that of rAd5.     

Inverted terminal repeat sequences of adeno-associated virus enhance the antibody and CD8(+) responses to a HIV-1 p55Gag/LAMP DNA vaccine chimera

The immune responses to an HIV-1 p55Gag vaccine encoded as a DNA chimera with the lysosomal associated membrane protein-1 (LAMP) have been examined for the effect of the addition of the inverted terminal repeat (ITR) sequences of the adeno-associated virus (AAV) to the DNA plasmid construct, and of packaging the LAMP/gag gene as a recombinant AAV vector (rAAV). DNA plasmids encoding Gag and the LAMP/Gag protein chimera were constructed in two vectors, the pcDNA3.1 and a corresponding plasmid containing the ITR sequences (pITR) flanking the expression elements of the plasmid, and the pITR LAMP/gag DNA plasmid was encapsidated in the rAAV vector. Human 293 cells transfected in vitro with LAMP/gag plasmids either in pcDNA3.1 or pITR produced much Gag protein in cell extracts (1.6 and 2.2 ng of Gag/mg of protein, respectively). The immune responses of mice to immunization with these constructs were examined under three protocols: DNA prime/DNA boost, DNA prime/rAAV boost, and a single rAAV immunization. The results demonstrated that under DNA prime/DNA boost protocol, the "naked" DNA vaccines encoding the LAMP/gag chimera, either as pcDNA3.1 or pITR DNA plasmid constructs, elicited strong CD4(+) T cell responses. In contrast, significantly higher levels of CD8(+) and antibody responses were observed with the pITR-DNA constructs. Immunization with the rAAV vector under the DNA prime/rAAV boost protocol resulted in sustained T cell responses and a markedly increased antibody response, predominantly of the IgG(1) isotype resulting from the activation of the Th2 subset of CD4(+) T cells, that was sustained for at least 5 months after immunization.     

表达HIV-1 gag基因的重组AAV2/1和Ad5疫苗免疫原性比较

目的 在小鼠体内比较表达HIT-1 gag基因的重组AAV2/1和Ad5疫苗的免疫原性.方法 分别用rAAV2/1-gag或rAd5-gag单独免疫BALB/c小鼠一次或两次,于免疫后不同时间点用CFSE染色法和胞内细胞因子染色法检测Gag特异性细胞免疫应答,用ELISA方法检测Gag特异性抗体反应.结果 单次免疫结果显示,在所有检测点rAd5-gag所诱导的Gag特异性CTL应答都比rAAV2/1-gag强,抗体反应在免疫后4周无明显差异.两次免疫后4周的检测结果表明,rAd5-gag所诱导的Gag特异性CTL应答比rAAV2/1-gag强,但抗体反应比rAAV2/1-gag组弱.结论 rAd5-gag诱导了很好的HIV-1特异性细胞和抗体反应,而rAAV2/1-gag诱导的HIV-1特异性抗体反应很强,但细胞免疫应答较弱.     

Mucosal prior to systemic application of recombinant adenovirus boosting is more immunogenic than systemic application twice but confers similar protection against SIV-challenge in DNA vaccine-primed macaques

We investigated the immunogenicity and efficacy of a bimodal prime/boost vaccine regimen given by various routes in the Simian immunodeficiency virus (SIV) rhesus monkey model for AIDS. Twelve animals were immunized with SIV DNA-vectors followed by the application of a recombinant adenovirus (rAd5) expressing the same genes either intramuscularly (i.m.) or by oropharyngeal spray. The second rAd5-application was given i.m. All vaccinees plus six controls were challenged orally with SIVmac239 12 weeks post-final immunization.Both immunization strategies induced strong SIV Gag-specific IFN-γ and T-cell proliferation responses and mediated a conservation of CD4⁺ memory T-cells and a reduction of viral load during peak viremia following infection. Interestingly, the mucosal group was superior to the systemic group regarding breadth and strength of SIV-specific T-cell responses and exhibited lower vector specific immune responses. Therefore, our data warrant the inclusion of mucosal vector application in a vaccination regimen which makes it less invasive and easier to apply.     

DNA vaccine encoding human immunodeficiency virus-1 Gag, targeted to the major histocompatibility complex II compartment by lysosomal-associated membrane protein, elicits enhanced long-term memory response

Antigen presentation by major histocompatibility complex type II (MHC II) molecules and activation of CD4+ helper T cells are critical for the generation of immunological memory. We previously described a DNA vaccine encoding human immunodeficiency virus-1 p55Gag as a chimera with the lysosome-associated membrane protein (LAMP/gag). The LAMP/gag chimera protein traffics to the MHC II compartment of transfected cells and elicits enhanced immune responses as compared to a DNA vaccine encoding native gag not targeted to the MHC II compartment. We have now investigated the long-term responses of immunized mice and show that the LAMP/gag DNA vaccine promotes long-lasting B cell- and CD4+ and CD8+ T-cell memory responses induced by DNA encoding non-targeted Gag decay rapidly and elicit very low or undetectable levels of gag DNA is sufficient to generate T-cell memory. Following this initial priming immunization with LAMP/gag DNA, booster immunizations with native gag DNA or the LAMP/gag chimera are equally efficient in eliciting B- and T-cell secondary responses, results in accordance with observations that secondary expansion of CD8+ cells in the boost phase does not require additional CD4+ help. These findings underscore the significance of targeting DNA-encoded vaccine antigens to the MHC II processing compartments for induction of long-term immunological memory.     

Improved SIV DNA vaccine can be effectively used as a boost for Ad5 in prime-boost immunization strategy

In the study under evaluation, optimized SIV DNA were used to boost T-cell responses induced by a highly immunogenic SIV Ad5-prime in Chinese rhesus macaques. A regular prime-boost regimen (SIV DNA-prime and rAd boost) and naive macaques were used as the control. After vaccination, the animals were challenged intrarectally with SIVmac251, and partial protection was observed in the macaques immunized by the Ad5-prime DNA-boost regimen. SIV-specific T-cell responses in the enzyme-linked immunospot assay were significantly higher in the Ad5-prime DNA-boost, compared with the responses in the control macaques. Viral control correlated with the generation of HLA-DR+ T cells 2 weeks after the viral challenge. Further studies using prime and boost strategies and alternative routes of vaccination (including a simultaneous approach) are warranted to fully explore the potential of prime and boost regimens for HIV-1 vaccine development.     

Preclinical evaluation of cellular immune responses elicited by a polyvalent DNA prime/protein boost HIV-1 vaccine

While DNA vaccines have been shown to prime cellular immune responses, levels are often low in nonhuman primates or humans. Hence, efforts have been directed toward boosting responses by combining DNA with different vaccination modalities. To this end, a polyvalent DNA prime/protein boost vaccine, consisting of codon optimized HIV-1 env (A, B, C, E) and gag (C) and homologous gp120 proteins in QS-21, was evaluated in rhesus macaques and BALB/c mice. Humoral and cellular responses, detected following DNA immunization, were increased following protein boost in macaques and mice. In dissecting cellular immune responses in mice, protein-enhanced responses were found to be mediated by CD4+ and CD8+ T cells with a Th1 cytokine bias. Our study reveals that, in addition to augmenting humoral responses, protein boosting of DNA-primed animals augments cellular immune responses mediated by CD8+ CTL, CD4+ T-helper cells and Th1 cytokines; thus, offering much promise in controlling HIV-1 in vaccinees.     

DNA prime Listeria boost induces a cellular immune response to SIV antigens in the rhesus macaque model that is capable of limited suppression of SIV239 viral replication

DNA vaccines and recombinant Listeria monocytogenes that express and secrete SIV Gag and Env antigens were combined in a nonhuman primate prime-boost immunogenicity study followed by a challenge with SIV239. We report that recombinant DNA vaccine delivered intramuscularly, and recombinant L. monocytogenes delivered orally each individually have the ability to induce CD8+ and CD4+ T cell immune responses in a nonhuman primate. Four rhesus monkeys were immunized at weeks 0, 4, 8, and 12 with the pCSIVgag and pCSIVenv DNA plasmids and boosted with SIV expressing L. monocytogenes vaccines at weeks 16, 20, and 28. Four rhesus monkeys received only the L. monocytogenes vaccines at weeks 16, 20, and 28. A final group of monkeys served as a control group. Blood samples were taken before vaccination and 2 weeks post each injection and analyzed by ELISPOT for CD4+ and CD8+ T cell responses. Moderate vaccine induced SIV-specific cellular immune responses were observed following immunization with either DNA or L. monocytogenes vectors. However, the SIV antigen-specific immune responses were significantly increased when Rhesus macaques were primed with SIV DNA vaccines and boosted with the SIV expressing L. monocytogenes vectors. In addition, the combined vaccine was able to impact SIV239 viral replication following an intrarectal challenge. This study demonstrates for the first time that oral L. monocytogenes can induce a cellular immune response in a nonhuman primate and is able to enhance the efficacy of a DNA vaccine as well as provide modest protection against SIV239 challenge.     

Adjuvant activity of the catalytic A1 domain of cholera toxin for retroviral antigens delivered by GeneGun

Most DNA-encoded adjuvants enhance immune responses to DNA vaccines in small animals but are less effective in primates. Here, we characterize the adjuvant activity of the catalytic A1 domain of cholera toxin (CTA1) for human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) antigens in mice and macaques delivered by GeneGun. The inclusion of CTA1 with SIVmac239 Gag dramatically enhanced anti-Gag antibody responses in mice. The adjuvant effects of CTA1 for the secreted antigen HIV gp120 were much less pronounced than those for Gag, as the responses to gp120 were high in the absence of an adjuvant. CTA1 was a stronger adjuvant for Gag than was granulocyte-macrophage colony-stimulating factor (GM-CSF), and it also displayed a wider dose range than GM-CSF in mice. In macaques, CTA1 modestly enhanced the antibody responses to SIV Gag but potently primed for a recombinant Gag protein boost. The results of this study show that CTA1 is a potent adjuvant for SIV Gag when delivered by GeneGun in mice and that CTA1 provides a potent GeneGun-mediated DNA prime for a heterologous protein boost in macaques.