【作者】 姜春来；

【导师】 李惟； 孔维；

【作者基本信息】 吉林大学 ， 生物化学和分子生物学， 2005， 博士

【摘要】 只有疫苗才能有效的控制艾滋病的流行。但传统的疫苗方案对HIV-1 来说并不可行,减毒活疫苗存在安全性的隐患;而灭活疫苗和亚单位疫苗又不能引起足够强的细胞免疫应答。在美国和泰国联合进行的艾滋病疫苗三期临床试验的失败也提示我们能同时诱导有效的细胞免疫和体液免疫才是艾滋病疫苗的目标。为了产生更好的细胞免疫应答,近期进入临床试验的疫苗多采用DNA疫苗或重组病毒疫苗。虽然DNA 疫苗可以引起细胞免疫,并具备生产方便,易于保存等优点,但其引起的免疫强度不够也限制了其更广泛的应用。目前主要采取两种方法增加其免疫强度:1)采用DNA 疫苗初免(prime)后用重组病毒疫苗加强免疫(boost)。此种免疫方案已在HIV-1,疟疾等疫苗中进行人体观察并得到令人满意的结果;2)应用免疫佐剂(如细胞因子)与DNA 疫苗共注射,也可达到效果。本实验以小鼠为动物模型,利用鼠的细胞因子质粒与HIV-1 DNA 疫苗共注射,然后以重组痘病毒疫苗(MVA)加强免疫。考察不同细胞因子或其组合对HIV-1 DNA 疫苗的加强作用。结果表明:1)以IL-2质粒与HIV-1 DNA 疫苗共注射小鼠,产生的细胞免疫强度高于以IL-2 蛋白与DNA 疫苗共注射所产生的;2)以IL-15,IL-18 和IFN-γ等细胞因子质粒分别与HIV-1 DNA 疫苗共注射小鼠,产生的免疫应答比单独使用DNA 疫苗的免疫应答强;3)以GM-CSF+IFN-γ和GM-CSF+IL-2 两种细胞因子组合与HIV-1 DNA 疫苗共注射小鼠引起的免疫应答比由其它细胞因子组合产生的要强;4)以MVA 先加强免疫后以病毒样颗粒(VLP)再加强免疫(re-boost)的免疫程序产生的免疫应答强于其它免疫程序所产生的免疫应答。更多还原

【Abstract】 The human immunodeficiency virus-1 (HIV-1) is an +ss RNA retrovirus belonging to the lentivirus family that selectively infects and kills CD4+ T-cells and macrophages resulting in immune system failure. The spread of HIV-1 infection continues to be a major public health concern worldwide. Chemotherapeutic has been limited to cost and the strict nature of the administration regimens, and high mutation rate in virus. These restrictions support that there is a need for the development of effective vaccine, and only by using vaccine can control HIV-1 spread. Traditional vaccine is not available to control HIV-1 virus infection. Attenuated live vaccine posed an unacceptable risk of infection for a preventive HIV-1 vaccine due to the potential for reversion to wild-type virus or the potential for adverse effects of integration and persistence of the virus; while killed or subunit vaccine cannot elicit strong cellular immune response. More and more evidence show that cytotoxic T lymphocyte (CTL) responses play an important role in the protection against HIV viruses. Several groups have observed that in acute cases of HIV-1 infection, viral clearance was associated with specific CTL activity. HIV-1 specific CTLs were also found in a number of chronically exposed prostitutes in Gambia who continue to resist infection with HIV-1. Long-term non-progressor HIV-infected individuals, chronic low virus load and is related with high frequency CTL Cytotoxic T lymphocytes (CTL) not only recognize processed viral fragments presented on the infected cell surface and destroy virus-infected cells, but also target all viral gene products the are expressed during viral replication. Targeting viral proteins through the development of specific CTL responses could aid in lowering the viral load by interfering with viral assembly. Some type vaccines can elicit cellular immune response, such as DNA vaccine, recombinant virus vaccine and virus-like particle. DNA vaccine or nucleic acid immunization is an important vaccination strategy that delivers DNA constructs encoding for a specific immunogen onto the host. Intramuscular injection of DNA vaccine leads to uptake of the plasmid by host cells and expression of the protein Ag. The protein enters the Ag-processing pathways, resulting in strong and persistent humoral and cellular immune response. This vaccination technique is being explored as an immunization strategy against a variety of infectious diseases, including HIV-1 infection. Furthermore, we hypothesized that the anti-HIV immune responses induced by DNA vaccine constructs could be manipulated through the co-delivery of molecular adjuvant constructs. Molecular adjuvant consists of plasmid expression cassettes that encode for immunologically important molecules, such as cytokines, that play critical regulatory and signaling roles in the immune response. The diversity or virus and the uncertainty of the correlates of protection are obstacles in the development of an effective HIV vaccines as well as immunotherapy. This matter will be difficult to deal with once a successful vaccine is identified in one or more geographical areas. So we do some work to confirm that epidemic HIV-1 strain is B/C recombinant subtype in China GuangXi. According to this result, we design a combination vaccine ,i.e. DNA prime and MVA boost regime. For efficient priming with DNA, the immunogen needs to be highly expressed. The natural HIV-1 genome encodes multiple mRNAs derived from a single precursor RNA by a highly regulated splicing mechanism. The Rev protein of the virus is involved in promoting the nuclear export, stabilization and utilization of these mRNAs by the protein translational machinery in the cytoplasm. Rev binds to these mRNAs through a Rev response element (RRE).The structural genes of HIV-1 are also known to encode sequences called inhibitory/instability RNA sequences (INS), which inhibit RNA transport and protein expression. One of the problems in expressing HIVproteins has been the INS present throughout its genome. CODON OPTIMIZATION of genes encoding HIV-1, which results in the mutation of INS, makes these genes Rev-independent. In our study, we used codon-optimized sequences that were expressed in a Rev -independent manner. The immune responses elicited by DNA vaccination have afforded a degree of protection in nonhuman primate against challenges with nonpathogenic AIDS virus. But these immune responses have not been of a magnitude sufficient to protect against pathogenic viral challenges. We therefore were interested in exploring strategies for augmenting DNA vaccine-elicited immune response. We sought to explore the utility of cytokine plasmid as a vaccine adjuvant. Cytokine gene co-delivery was chosen because cytokines play a critical regulatory and signaling role in the development of an immune response. Th1 cytokine regulate the cellular or T cell-mediated arm of the immune response. We have reported that IL-2 plasmid can augment DNA vaccine-elicited immune responses in mice more effectively than IL-2 protein. In this study, we examined the modulation of immune responses to HIV-1 DNA vaccine using cytokine molecular adjuvants in mice. More specifically, Th1 cytokine such as IL-2, IL-12, IL-15, IL-18, IFN-gamma, as well as GM-CSF, were individually cloned into expression vectors under control of a CMV promoter. The cytokine plasmids alone or in combination were then injected into mice along with DNA vaccine constructs, followed by MVA vaccine boost. We analyzed the immunologic effects of the coinjection with these genetic adjuvant cassettes on the direction and magnitude of antigen-specific immune responses could be modulated by the coinjection of cytokine genes, and the use of this molecular adjuvant strategy may be used to enhance the potency of vaccine-induced immunity in mice, and perhaps ultimately in humans. In this work, we developed expression plasmid encoding for cytokine genes in an effort to analyze their ability to function as in vivo modulators of the immune responses induced by DNA vaccine constructs expression HIV-1 GagPol and Env proteins. We observed that both the magnitude and the direction of antigen-specific humoral and cellular responses can be modulated using the adjuvants. Specifically, coinjection with Th1 cytokine in our study all caused increased levels of antibodies. We also found that coinjection with cytokine genes drove the immune responses toward a more Th1-like phenotype.更多还原