DNA疫苗研究进展

DNA疫苗是当前基因研究、疫苗应用领域的一项重要课题,DNA免疫技术在疾病控制中发展迅速。本文探讨了DNA疫苗的免疫机制、技术特点及影响因素,并对几种DNA疫苗在医学上的研究进展以及应用前景作一综述。     

如何增强DNA免疫诱导的抗病毒细胞免疫效应

为进一步增强DNA免疫诱导的特异性抗病毒细胞免疫,近年,国内外学者采用了多种策略来增强DNA免疫的效果。这些策略包括:DNA疫苗的载体优化、基因的转入、体内所表达抗原的处理及提呈、协同刺激分子和细胞因子的应用、多种疫苗的协同作用等,本文对几种较有效的增强策略作了简要阐述。     

DNA疫苗的研究与进展

DNA疫苗的研究是当前基因研究领域的一项重要课题,DNA免疫技术在疾病控制中正扮演着越来越重要的角色。本文探讨了DNA疫苗的免疫机制、影响因素,并对几种DNA疫苗在医学上的研究进展以及应用前景作一综述。     

真菌DNA免疫的研究进展

近些年来,真菌病的发生率明显上升,目前认为治疗真菌病除了抗真菌药外,还需使机体处于高免疫状态。DNA免疫被证实比传统疫苗能更有效地刺激细胞免疫功能。本文综述了近4年来真菌DNA免疫基础性研究的进展,概述了目前己研究的几种真菌DNA疫苗并分析了影响真菌DNA免疫的因素,讨论了增强真菌DNA免疫效应的策略。     

增强沙眼衣原体DNA疫苗免疫效应的研究进展

沙眼衣原体是引起人类非淋菌性尿道炎最主要的病原体,常引起严重的并发症,对人类健康威胁很大。DNA疫苗被认为是预防其感染最有潜力的候选疫苗,然而单纯使用裸DNA疫苗进行动物免疫得到的保护作用比较有限。本文综述了几种能够增强沙眼衣原体DNA疫苗免疫效应的方法,此外,其他病原体DNA疫苗研究中的经验值得借鉴。     

真菌DNA免疫的研究进展

近些年来，真菌病的发生率明显上升，目前认为治疗真菌病除了抗真菌药外，还需使机体处于高免疫状态。DNA免疫被证实比传统疫苗能更有效地刺激细胞免疫功能，本文综述了近4年来真菌DNA免疫基础性研究的进展，概述了目前已研究的几种真菌DNA疫苗并分析了影响真菌DNA免疫的因素，讨论了增强真菌DNA免疫效应的策略。     

增强沙眼衣原体DNA疫苗免疫效应的研究进展

沙眼衣原体是引起人类非淋菌性尿道炎最主要的病原体,常引起严重的并发症,对人类健康威胁很大.DNA疫苗被认为是预防其感染最有潜力的候选疫苗,然而单纯使用裸DNA疫苗进行动物免疫得到的保护作用比较有限.本文综述了几种能够增强沙眼衣原体DNA疫苗免疫效应的方法,此外,其他病原体DNA疫苗研究中的经验值得借鉴.     

分子佐剂在DNA疫苗中的应用

如何增强和提高DNA疫苗的免疫效果是新一代DNA疫苗分子设计和研究的关键问题,其中之一就是应用分子佐剂来优化DNA疫苗。分子佐剂可将以基因方式传递的免疫刺激分子与DNA疫苗共免疫,在体内表达相应的蛋白,增强T、B细胞对疫苗成分的体液及细胞免疫应答。目前研究较多的分子佐剂有:细胞因子如干扰素(IFN)、白细胞介素2(IL-2)、IL-12、粒细胞-巨噬细胞集落刺激因子(GM-CSF)等;共刺激分子如B7-2、CD40L等;补体佐剂如C3;免疫刺激序列如CpG、寡核苷酸(ODN)等。分子佐剂在体内与疫苗基因共表达,改善抗原的周边微环境,提高疫苗的免疫效果。因此,分子佐剂是目前DNA疫苗研究中的热点问题之一。     

分子佐剂在DNA疫苗中的应用

如何增强和提高DNA疫苗的免疫效果是新一代DNA疫苗分子设计和研究的关键问题,其中之一就是应用分子佐剂来优化DNA疫苗.分子佐剂可将以基因方式传递的免疫刺激分子与DNA疫苗共免疫,在体内表达相应的蛋白,增强T、B细胞对疫苗成分的体液及细胞免疫应答.目前研究较多的分子佐剂有:细胞因子如干扰素(IFN)、白细胞介素2(IL-2)、IL-12、粒细胞-巨噬细胞集落刺激因子(GM-CSF)等;共刺激分子如B7-2、CD40L等;补体佐剂如C3;免疫刺激序列如CpG、寡核苷酸(ODN)等.分子佐剂在体内与疫苗基因共表达,改善抗原的周边微环境,提高疫苗的免疫效果.因此,分子佐剂是目前DNA疫苗研究中的热点问题之一.     

DNA疫苗分子机制研究:TBK-1的作用

DNA疫苗的研究开辟了当今临床疾病预防和治疗领域新的模式.TANK结合激酶1(TANK-binding kinase-1,TBK-1)作为固有免疫应答蘑要的信号转导分子,在Toll样受体、RIG-Ⅰ介导的固有免疫应答中起着重要的作用,并能增强机体的适应性免疫应答.越来越多的研究表明,TBK-1在DNA疫苗诱导机体免疫应答过程中发挥着重要的作用.该文对相关的研究进行了综述.     

The hemagglutinin–neuraminidase gene of Newcastle Disease Virus: A powerful molecular adjuvant for DNA anti-tumor vaccination

Plasmid-encoded DNA vaccine is a novel and potentially powerful tool for cancer therapy. Since the strength of immune responses induced by DNA vaccine is usually rather low, a major goal in DNA vaccine development is to enhance vaccine-induced immunity. In this study, we investigated an approach based on the use of a viral surface protein with pleiotropic function as a potential immune enhancer. To this end, we prepared bicistronic DNA plasmids encoding the hemagglutinin–neuraminidase (HN) protein of Newcastle Disease Virus in addition to a tumor target antigen. We demonstrate a higher tumor antigen-specific T cell-mediated immune response and a lower humoral response upon vaccination with a bicistronic DNA plasmid with incorporated HN gene. In a prophylactic immunization tumor model with the surrogate tumor antigen beta-galactosidase (β-gal) and in a therapeutic immunization tumor model with the xenogeneic tumor antigen human Epithelial Cell Adhesion Molecule (hEpCAM), HN gene incorporation into the DNA vaccine led to better survival and tumor regression in mice. There was also cross protection in the therapeutic tumor model against a second challenge by the parental mouse mammary carcinoma cells in mice vaccinated with the bicistronic plasmids.     

Evaluation of protective immunity of Leptospira immunoglobulin like protein A (LigA) DNA vaccine against challenge in hamsters

We demonstrated earlier that immunization with recombinant Leptospira immunoglobulin like protein A (LigA) induced significant protection against virulent Leptospira interrogans serovar Pomona challenge in hamsters. However, the protective immune mechanism remains unclear. In the present study we demonstrated the protective efficacy of a LigA DNA vaccine and evaluated the immune mechanism underlying the protection against leptospirosis in hamsters. The LigA DNA vaccine was constructed in two truncated forms as the conserved portion (LigAcon) and a variable portion (LigAvar). Four-week-old hamsters were immunized three times at two-week intervals with vector alone or an equal amount of a recombinant construct containing either LigAcon or LigAvar. All animals were challenged intraperitoneally 2 weeks after the last immunization with a dose (LD50=10(8)) of virulent L. interrogans serovar Pomona. Prior to challenge, four animals were sacrificed, the spleen was removed aseptically, and splenocytes were assayed for lymphocyte proliferation and cytokine profiles in response to recall antigen. The protective efficacy was evaluated on the basis of survival and histopathological lesions in the kidney. The immuno-protective mechanism was assessed on the basis of Th1/Th2 profile of cytokines in immunized animals. Our results indicate that immunization with LigA DNA vaccine provides significant protection against leptospirosis. We suggest that immuno-protection is conferred by both humoral and cellular immunity as revealed by an increase in antibody titers during subsequent boosters, significant proliferation of lymphocytes and enhancement of both Th1 and Th2 cytokines. Taken together, the present study suggests that a LigA DNA vaccine is a promising candidate for prevention of leptospirosis.     

Vaccination of mice with DNA vaccine induces the immune response and partial protection against T. spiralis infection

Trichinellosis is a serious parasitic zoonosis which is widely distributed in the world. Pork still is the predominant source of outbreaks of human trichinellosis in many countries. Vaccines are urgently needed to prevent swine from Trichinella infection. The gene (TspE1) encoding a 31 kDa antigen of T. spiralis was cloned to a eukaryotic expression plasmid pcDNA3 as DNA vaccine. The ability of the DNA vaccine to express antigen in mammalian CHO cells was previously confirmed by indirect fluorescencent antibody test (IFAT) and Western blotting. To evaluate its immunogenicity and its host protective potential, BALB/c mice were immunized with the DNA vaccine by intramuscular injection and gene-gun delivery. The serum antibody response was assayed by IFAT, enzyme linked immunosorbant assay (ELISA) and Western blotting. The cellular immune response was investigated by splenocyte proliferation assay. Vaccine administration by either route induced both humoral and cellular immune response against TspE1, which provided the partial protection against challenge infection with T. spiralis, as shown by significant reduction of larval burden in muscles. Thus, DNA immunization may offer an attractive alternative strategy against swine trichinellosis.     

Immunity obtained by gene-gun inoculation of a rotavirus DNA vaccine to the abdominal epidermis or anorectal epithelium.

We have previously shown that gene-gun delivery of murine rotavirus DNA vaccines to the epidermis induced protection against rotavirus challenge in mice. In this study, we used a rotavirus group antigen (VP6)-specific DNA vaccine to compare epidermal immunization with immunization to the anorectal epithelium for efficacy in inducing protective immunity. The vaccine was administered into cells of the abdominal epidermis or anorectal epithelium of adult BALB/c mice with an Accell gene-gun (PowderJect, Inc). Vaccines administered by either route elicited rotavirus-specific ELISA antibodies and analysis of the IgG subtypes indicated Th2-type responses were generated by both routes of administration, in contrast to Th1-type responses generated by live rotavirus. Protection against virus challenge was obtained in mice inoculated by either route, as shown by significant reduction of virus excreted in stools. The protection obtained by immunization of the anorectal epithelium was greater than that for epidermal immunization at the same vaccine dose. These results suggest that mucosal immunization of DNA vaccines may be an effective means to generate protective immunity against mucosal pathogens.     

Unique immunogenicity of hepatitis B virus DNA vaccine presented by live-attenuated Salmonella typhimurium

A novel vaccine for hepatitis B virus (HBV) was designed by putting a naked DNA vaccine carrying hepatitis B surface antigen (HBsAg) into live-attenuated Salmonella typhimurium. Mucosal immunization by the oral route in mice showed significantly stronger cytotoxic T lymphocyte (CTL) response than recombinant HBsAg vaccination (P < 0.01 at an effector:target ratio of 100:1), while comparable to intramuscular naked DNA immunization at all effector:target ratios. Contrary to previous reports on naked DNA vaccines given intramuscularly, the IgG antibody response induced by the mucosal DNA vaccine is relatively weak when compared to recombinant HBsAg vaccine (P < 0.001 at day 21). These findings are supported by a high interferon-gamma but a low interleukin-4 level detected in the supernatant of splenic cell cultures obtained from mucosally immunized mice. As distinct to recombinant HBsAg vaccine which is effective for protection, oral mucosal DNA vaccine should be considered as a candidate for therapeutic immunization in chronic HBV infection, donor immunization before adoptive transfer of HBV-specific CTL to HBsAg positive bone marrow transplant recipients, and immunization of non-responders to recombinant HBsAg vaccine. This strongly cellular and relatively absent humoral response may make this vaccine a better candidate as a therapeutic vaccine for chronic HBV carriers than naked DNA vaccines, as the humoral response is relatively less important for the clearance of HBV from hepatocytes, but its presence may lead to side effects such as serum sickness and immune complex deposition in chronic HBV carriers.     

Molecular adjuvants for malaria DNA vaccines based on the modulation of host-cell apoptosis

Malaria represents a major global health problem but despite extensive efforts, no effective vaccine is available. Various vaccine candidates have been developed that provide protection in animal models, such as a gene gun-delivered DNA vaccine encoding the circumsporozoite protein (CSP) of Plasmodium berghei. A common shortcoming of most malaria vaccines is the requirement for multiple immunizations leaving room for improvement even for established vaccine candidates such as the CSP-DNA vaccine. In this study, we explored whether regulating apoptosis in DNA vaccine transfected host cells could accelerate the onset of protective immunity and provide significant protection after a single immunization. A pro-apoptotic gene (Bax) was used as a molecular adjuvant in an attempt to mimic the immunostimulatory apoptosis triggered by viral or virus-derived vaccines, while anti-apoptotic genes such as Bcl-XL may increase the life span of transfected cells thus prolonging antigen production. Surprisingly, co-delivery of either Bax or Bcl-XL greatly reduced CSP-DNA vaccine efficacy after a single immunization. Co-delivery of Bax for three immunizations still had a detrimental effect on protective immunity, while repeated co-delivery of Bcl-XL had no negative impact. The fine characterization of humoral and cellular immune response modulated by these two molecular adjuvants revealed a previously unknown effect, i.e., a shift in the Th-profile. These results demonstrate that pro- or anti-apoptotic molecules should not be used as molecular adjuvants without careful evaluation of the resulting immune response. This finding represents yet another example that strategies to enhance vaccine efficacy developed for other model systems such as viral diseases cannot easily be applied to any vaccine.     

DNA encoding a single mycobacterial antigen protects against leprosy infection

The continuing incidence of leprosy infection around the world and the inability of Mycobacterium bovis bacille Calmette-Guérin (BCG) to protect certain populations clearly indicates that an improved vaccine against leprosy is needed. The immuno dominant 35 kDa protein, shared by Mycobacterium leprae and Mycobacterium avium, but not Mycobacterium tuberculosis or BCG, is recognised by >90% of leprosy patients, making it an ideal candidate antigen for a subunit vaccine. Immunization of outbred Swiss Albino mice with a DNA-35 vaccine stimulated specific T cell activation and IFN-gamma production. DNA-35 immunization induced significant levels of protection against M. leprae footpad infection, comparable to that produced by BCG. Therefore, DNA immunization with the 35 kDa antigen is effective against M. leprae infection and genetic immunization with a combination of antigens holds the potential for an improved vaccine against leprosy.     

Immunization with a portion of rickettsial outer membrane protein A stimulates protective immunity against spotted fever rickettsiosis.

Two approaches for presentation of a part of the rickettsial outer membrane protein A (OmpA) of Rickettsia rickettsii, namely (1) recombinant Mycobacterium vaccae (rMV) or (2) recombinant DNA vaccine, stimulated protective immunity against a lethal challenge with the closely related bacterium, R. conorii, in mice. After primary immunization with rMV and booster immunization with homologous recombinant protein, 67 and 55% of mice were protected against challenge in two experiments. DNA vaccination with booster recombinant protein immunization protected six out of eight animals from a lethal challenge. Production of IFN-gamma by antigen-exposed T-lymphocytes of DNA vaccine recipients indicated that cellular immunity had been stimulated.     

DNA prime/MVTT boost regimen with HIV-1 mosaic Gag enhances the potency of antigen-specific immune responses

HIV-1 diversity and latent reservoir are the major challenges for the development of an effective AIDS vaccine. It is well indicated that Gag-specific CD8⁺ T cells serve as the dominant host immune surveillance for HIV-1 control, but it still remains a challenge for vaccine design to induce broader and stronger cytotoxic T cell immunity against the virus. Genetic variation of the HIV-1 gag gene across different clades is one of the reasons for the reduction of antigenic epitope coverage. Here, we report an immunization strategy with heterologous vaccines expressing a mosaic Gag antigen aimed to increase antigenic breadth against a wider spectrum of HIV-1 strains. Priming using a DNA vaccine via in vivo electroporation, followed by boosting with a live replication-competent modified vaccinia TianTan (MVTT) vectored vaccine, elicited greater and broader protective Gag-specific immune responses in mice. Compared to DNA or MVTT homologous immunization, the heterologous DNA/MVTT vaccination resulted in higher frequencies of broadly reactive, Gag-specific, polyfunctional, long-lived cytotoxic CD8⁺ T cells, as well as increased anti-Gag antibody titer. Importantly, the DNA/MVTT heterologous vaccination induced protection against EcoHIV and mesothelioma AB1-Gag challenges. In summary, the stronger protective Gag-specific immunity induced by the heterologous regimen using two safe vectors shows promise for further development to enhance anti-HIV-1 immunity. Our study has important implications for immunogen design and the development of an effective HIV-1 heterologous vaccination strategy.     

Co-expression of granulocyte-macrophage colony-stimulating factor with antigen enhances humoral and tumor immunity after DNA vaccination

Granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to enhance humoral and tumor immunity resulting from DNA immunization. The genes encoding GM-CSF and antigen were cloned onto the same plasmid backbone, but separate promoters drove expression of each gene. beta-Galactosidase was used as the model antigen to generate antibody responses while the human tumor antigen, MAGE-1, was used to monitor tumor resistance. Immunization with a DNA vaccine co-expressing GM-CSF and beta-gal resulted in higher antigen-specific IgG responses than immunization with antigen encoding plasmid alone or co-inoculated with GM-CSF expressing plasmid. Similarly, DNA vaccines expressing both MAGE-1 antigen and GM-CSF were more effective in protecting against B16-MAGE-1 melanoma. However, both GM-CSF co-expressing DNA vaccines and co-inoculation with plasmids encoding the cytokine or antigen enhanced the generation antigen-specific IFN-gamma and IL-6 responses. These results demonstrate that co-expressing both GM-CSF and antigen on a DNA vaccine enhances humoral and tumor immune responses.