Development of DNA vaccines for foot-and-mouth disease, evaluation of vaccines encoding replicating and non-replicating nucleic acids in swine.

We have developed naked DNA vaccine candidates for foot-and-mouth disease (FMD), an important disease of domestic animals. The virus that causes this disease, FMDV, is a member of the picornavirus family, which includes many important human pathogens, such as poliovirus, hepatitis A virus, and rhinovirus. Picornaviruses are characterized by a small (7-9000 nucleotide) RNA genome that encodes capsid proteins, processing proteinases, and enzymes required for RNA replication. We have developed two different types of DNA vaccines for FMD. The first DNA vaccine, pP12X3C, encodes the viral capsid gene (P1) and the processing proteinase (3C). Cells transfected with this DNA produce processed viral antigen, and animals inoculated with this DNA using a gene gun produced detectable antiviral immune responses. Mouse inoculations with this plasmid, and with a derivative containing a mutation in the 3C proteinase, indicated that capsid assembly was essential for induction of neutralizing antibody responses. The second DNA vaccine candidate, pWRMHX, encodes the entire FMDV genome, including the RNA-dependent RNA polymerase, permitting the plasmid-encoded viral genomes to undergo amplification in susceptible cells. pWRMHX encodes a mutation at the cell binding site, preventing the replicated genomes from causing disease. Swine inoculated with this vaccine candidate produce viral particles lacking the cell binding site, and neutralizing antibodies that recognize the virus. Comparison of the immune responses elicited by pP12X3C and pWRMHX in swine indicate that the plasmid encoding the replicating genome stimulated a stronger immune response, and swine inoculated with pWRMHX by the intramuscular, intradermal, or gene gun routes were partially protected from a highly virulent FMD challenge.     

The potential of DNA vaccination against tumor-associated antigens for antitumor therapy

Conventional treatment approaches for malignant tumors are highly invasive and sometimes have only a palliative effect. Therefore, there is an increasing demand to develop novel, more efficient treatment options. Increased efforts have been made to apply immunomodulatory strategies in antitumor treatment. In recent years, immunizations with naked plasmid DNA encoding tumor-associated antigens have revealed a number of advantages. By DNA vaccination, antigen-specific cellular as well as humoral immune responses can be generated. The induction of specific immune responses directed against antigens expressed in tumor cells and displayed e.g., by MHC class I complexes can inhibit tumor growth and lead to tumor rejection. The improvement of vaccine efficacy has become a critical goal in the development of DNA vaccination as antitumor therapy. The use of different DNA delivery techniques and coadministration of adjuvants including cytokine genes may influence the pattern of specific immune responses induced. This brief review describes recent developments to optimize DNA vaccination against tumor-associated antigens. The prerequisite for a successful antitumor vaccination is breaking tolerance to tumor-associated antigens, which represent "self-antigens." Currently, immunization with xenogeneic DNA to induce immune responses against self-molecules is under intensive investigation. Tumor cells can develop immune escape mechanisms by generation of antigen loss variants, therefore, it may be necessary that DNA vaccines contain more than one tumor antigen. Polyimmunization with a mixture of tumor-associated antigen genes may have a synergistic effect in tumor treatment. The identification of tumor antigens that may serve as targets for DNA immunization has proceeded rapidly. Preclinical studies in animal models are promising that DNA immunization is a potent strategy for mediating antitumor effects in vivo. Thus, DNA vaccines may offer a novel treatment for tumor patients. DNA vaccines may also be useful in the prevention of tumors with genetic predisposition. By DNA vaccination preventing infections, the development of viral-induced tumors may be avoided.     

Enhancement of sindbis virus self-replicating RNA vaccine potency by linkage of herpes simplex virus type 1 VP22 protein to antigen

Recently, self-replicating and self-limiting RNA vaccines (RNA replicons) have emerged as an important form of nucleic acid vaccines. Self-replicating RNA eventually causes lysis of transfected cells and does not raise the concern associated with naked DNA vaccines of integration into the host genome. This is particularly important for development of vaccines targeting proteins that are potentially oncogenic. However, the potency of RNA replicons is significantly limited by their lack of intrinsic ability to spread in vivo. The herpes simplex virus type 1 protein VP22 has demonstrated the remarkable property of intercellular transport and provides the opportunity to enhance RNA replicon vaccine potency. We therefore created a novel fusion of VP22 with a model tumor antigen, human papillomavirus type 16 E7, in a Sindbis virus RNA replicon vector. The linkage of VP22 with E7 resulted in a significant enhancement of E7-specific CD8+ T-cell activities in vaccinated mice and converted a less effective RNA replicon vaccine into one with significant potency against E7-expressing tumors. These results indicate that fusion of VP22 to an antigen gene may greatly enhance the potency of RNA replicon vaccines.     

Effective immunotherapy of cancer by DNA vaccination.

Direct injection of naked plasmid DNA either intramuscularly or intradermally induces strong, long-lived cell-mediated and humoral immune responses to the antigen encoded by the gene vaccine. In the present study, we used gene vaccination with naked plasmid DNA to induce prophylactic immune responses to tumor associated antigens. MAGE-1 (melanoma antigen 1) is an ideal candidate for cancer vaccines because it belongs to a family of genes that are expressed in a number of human tumors of various histological types but not in normal adult tissues except for the testis, and because both humoral and cell-mediated immune responses against MAGE-1 antigen were detected in tumor patients. Intradermal administration of plasmid DNA encoding MAGE-1 (pcMAGE1) induced anti-MAGE-1-specific antibody in BALB/c mice. In contrast, no detectable level of anti-MAGE-1 antibody was induced by intramuscular injection of pcMAGE1. Also, intradermal injection of pcMAGE1 was capable of generating CTLs reactive with MAGE-1-transfected murine tumor cells, M-MSV-MAGE1. Most of the mice (8 out of 10) immunized with pcMAGE1 rejected the challenge of M-MSV-MAGE1 tumor cells, compared with control animals most of which developed tumors. This suggests that intradermal DNA vaccination could provide a novel immunotherapy of cancer.     

DNA and adenovirus tumor vaccine expressing truncated survivin generates specific immune responses and anti-tumor effects in a murine melanoma model

Survivin is overexpressed in major types of cancer and is considered an ideal "universal" tumor-associated antigen that can be targeted by immunotherapeutic vaccines. However, its anti-apoptosis function raises certain safety concerns. Here, a new truncated human survivin, devoid of the anti-apoptosis function, was generated as a candidate tumor vaccine. Interleukin 2 (IL-2) has been widely used as an adjuvant for vaccination against various diseases. Meanwhile, the DNA prime and recombinant adenovirus (rAd) boost heterologous immunization strategy has been proven to be highly effective in enhancing immune responses. Therefore, the efficacy of a new cancer vaccine based on a truncated form of survivin, combined with IL-2, DNA prime, and rAd boost, was tested. As prophylaxis, immunization with the DNA vaccine alone resulted in a weak immune response and modest anti-tumor effect, whereas the tumor inhibition ratio with the DNA vaccine administered with IL-2 increased to 89?% and was further increased to nearly 100?% by rAd boosting. Moreover, complete tumor rejection was observed in 5 of 15 mice. Efficacy of the vaccine administered therapeutically was enhanced by nearly 300?% when combined with carboplatin. These results indicated that vaccination with a truncated survivin vaccine using DNA prime-rAd boost combined with IL-2 adjuvant and carboplatin represents an attractive strategy to overcoming immune tolerance to tumors and has potential therapeutic benefits in melanoma cancer.     

Enhancement of Sindbis virus self-replicating RNA vaccine potency by linkage of Mycobacterium tuberculosis heat shock protein 70 gene to an antigen gene

Recently, self-replicating RNA vaccines (RNA replicons) have emerged as an effective strategy for nucleic acid vaccine development. Unlike naked DNA vaccines, RNA replicons eventually cause lysis of transfected cells and therefore do not raise the concern of integration into the host genome. We evaluated the effect of linking human papillomavirus type 16 E7 as a model Ag to Mycobacterium tuberculosis heat shock protein 70 (HSP70) on the potency of Ag-specific immunity generated by a Sindbis virus self-replicating RNA vector, SINrep5. Our results indicated that this RNA replicon vaccine containing an E7/HSP70 fusion gene generated significantly higher E7-specific T cell-mediated immune responses in vaccinated mice than did vaccines containing the wild-type E7 gene. Furthermore, our in vitro studies demonstrated that E7 Ag from E7/HSP70 RNA replicon-transfected cells can be processed by bone marrow-derived dendritic cells and presented more efficiently through the MHC class I pathway than can wild-type E7 RNA replicon-transfected cells. More importantly, the fusion of HSP70 to E7 converted a less effective vaccine into one with significant potency against E7-expressing tumors. This antitumor effect was dependent on NK cells and CD8(+) T cells. These results indicated that fusion of HSP70 to an Ag gene may greatly enhance the potency of self-replicating RNA vaccines.     

RNA复制子疫苗研究进展

最近兴起的RNA复制子疫苗,利用源自病毒的能够自主复制的RNA,其结构蛋白基因由外源抗原基因取代,保留了非结构蛋白(RNA复制酶)基因。RNA复制酶可使RNA载体在细胞质中高水平复制,并实现外源抗原基因的高水平表达,可同时诱导细胞免疫和体液免疫应答。大量双链RNA可诱导被感染细胞凋亡,宿主细胞的凋亡有利于免疫系统识别外源抗原。RNA复制子疫苗克服了传统疫苗和普通DNA疫苗存在的缺点,具有抗原表达效率高、安全性好、应用范围广等优点,因而被视为一种发展前景很好的疫苗形式。目前已对一些疾病模型基于复制子的治疗性和预防性疫苗进行了研究(涉及的对象包括病毒、肿瘤以及细菌毒素等),并对某些不足之处进行了改进。     

Electroporation as a "prime/boost" strategy for naked DNA vaccination against a tumor antigen

We have developed novel DNA fusion vaccines encoding tumor Ags fused to pathogen-derived sequences. This strategy activates linked T cell help and, using fragment C of tetanus toxin, amplification of anti-tumor Ab, CD4(+), and CD8(+) T cell responses is achievable in mice. However, there is concern that simple DNA vaccine injection may produce inadequate responses in larger humans. To overcome this, we tested electroporation as a method to increase the transfection efficiency and immune responses by these tumor vaccines in vivo in mice. Using a DNA vaccine expressing the CTL epitope AH1 from colon carcinoma CT26, we confirmed that effective priming and tumor protection in mice are highly dependent on vaccine dose and volume. However, suboptimal vaccination was rendered effective by electroporation, priming higher levels of AH1-specific CD8(+) T cells able to protect mice from tumor growth. Electroporation during priming with our optimal vaccination protocol did not improve CD8(+) T cell responses. In contrast, electroporation during boosting strikingly improved vaccine performance. The prime/boost strategy was also effective if electroporation was used at both priming and boosting. For Ab induction, DNA vaccination is generally less effective than protein. However, prime/boost with naked DNA followed by electroporation dramatically increased Ab levels. Thus, the priming qualities of DNA fusion vaccines, integrated with the improved Ag expression offered by electroporation, can be combined in a novel homologous prime/boost approach, to generate superior antitumor immune responses. Therefore, boosting may not require viral vectors, but simply a physical change in delivery, facilitating application to the cancer clinic.     

Mucosal adjuvants and delivery systems for protein-, DNA- and RNA-based vaccines

Almost all vaccinations today are delivered through parenteral routes. Mucosal vaccination offers several benefits over parenteral routes of vaccination, including ease of administration, the possibility of self-administration, elimination of the chance of injection with infected needles, and induction of mucosal as well as systemic immunity. However, mucosal vaccines have to overcome several formidable barriers in the form of significant dilution and dispersion; competition with a myriad of various live replicating bacteria, viruses, inert food and dust particles; enzymatic degradation; and low pH before reaching the target immune cells. It has long been known that vaccination through mucosal membranes requires potent adjuvants to enhance immunogenicity, as well as delivery systems to decrease the rate of dilution and degradation and to target the vaccine to the site of immune function. This review is a summary of current approaches to mucosal vaccination, and it primarily focuses on adjuvants as immunopotentiators and vaccine delivery systems for mucosal vaccines based on protein, DNA or RNA. In this context, we define adjuvants as protein or oligonucleotides with immunopotentiating properties co-administered with pathogen-derived antigens, and vaccine delivery systems as chemical formulations that are more inert and have less immunomodulatory effects than adjuvants, and that protect and deliver the vaccine through the site of administration. Although vaccines can be quite diverse in their composition, including inactivated virus, virus-like particles and inactivated bacteria (which are inert), protein-like vaccines, and non-replicating viral vectors such as poxvirus and adenovirus (which can serve as DNA delivery systems), this review will focus primarily on recombinant protein antigens, plasmid DNA, and alphavirus-based replicon RNA vaccines and delivery systems. This review is not an exhaustive list of all available protein, DNA and RNA vaccines, with related adjuvants and delivery systems, but rather is an attempt to highlight many of the currently available approaches in immunopotentiation of mucosal vaccines.     

Comparison of different delivery systems of DNA vaccination for the induction of protection against tuberculosis in mice and guinea pigs

The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy and toxicity induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs.     

Induction of potent immune responses by cationic microparticles with adsorbed human immunodeficiency virus DNA vaccines

The effectiveness of cationic microparticles with adsorbed DNA at inducing immune responses was investigated in mice, guinea pigs, and rhesus macaques. Plasmid DNA vaccines encoding human immunodeficiency virus (HIV) Gag and Env adsorbed onto the surface of cationic poly(lactide-coglycolide) (PLG) microparticles were shown to be substantially more potent than corresponding naked DNA vaccines. In mice immunized with HIV gag DNA, adsorption onto PLG increased CD8(+) T-cell and antibody responses by approximately 100- and approximately 1,000-fold, respectively. In guinea pigs immunized with HIV env DNA adsorbed onto PLG, antibody responses showed a more rapid onset and achieved markedly higher enzyme-linked immunosorbent assay and neutralizing titers than in animals immunized with naked DNA. Further enhancement of antibody responses was observed in animals vaccinated with PLG/DNA microparticles formulated with aluminum phosphate. The magnitude of anti-Env antibody responses induced by PLG/DNA particles was equivalent to that induced by recombinant gp120 protein formulated with a strong adjuvant, MF-59. In guinea pigs immunized with a combination vaccine containing HIV env and HIV gag DNA plasmids on PLG microparticles, substantially superior antibody responses were induced against both components, as measured by onset, duration, and titer. Furthermore, PLG formulation overcame an apparent hyporesponsiveness of the env DNA component in the combination vaccine. Finally, preliminary data in rhesus macaques demonstrated a substantial enhancement of immune responses afforded by PLG/DNA. Therefore, formulation of DNA vaccines by adsorption onto PLG microparticles is a powerful means of increasing vaccine potency.     

Experimental vaccines against measles in a world of changing epidemiology

Vaccination with the current live attenuated measles vaccine is one of the most successful and cost-effective medical interventions. However, as a result of persisting maternal antibodies and immaturity of the infant immune system, this vaccine is poorly immunogenic in children <9 months old. Immunity against the live vaccine is less robust than natural immunity and protection less durable. There may also be some concern about (vaccine) virus spread during the final stage of an eventual measles eradication program. Opinions may differ with respect to the potential threat that some of these concerns may be to the World Health Organisation goal of measles elimination, but there is a consensus that the development of new measles vaccines cannot wait. Candidate vaccines are based on viral or bacterial vectors expressing recombinant viral proteins, naked DNA, immune stimulating complexes or synthetic peptides mimicking neutralising epitopes. While some of these candidate vaccines have proven their efficacy in monkey studies, aerosol formulated live attenuated measles vaccine are evaluated in clinical trials.     

猪瘟DNA疫苗研究进展

自1990年DNA疫苗问世以来,已有许多研究者构建了不同类型的DNA疫苗。这些载体能诱发机体产生不同程度的特异性体液免疫和(或)细胞免疫。研究者们也在猪瘟DNA疫苗研究方面做出了很多努力并取得了一定的成果。以下从猪瘟DNA疫苗的构建和评价、佐剂在猪瘟DNA疫苗中的应用、猪瘟DNA疫苗与其他疫苗的联合应用以及目前猪瘟DNA疫苗存在的问题和解决途径等方面做了比较全面的阐述。     

Superior induction of T cell responses to conserved HIV-1 regions by electroporated alphavirus replicon DNA compared to that with conventional plasmid DNA vaccine

Vaccination using "naked" DNA is a highly attractive strategy for induction of pathogen-specific immune responses; however, it has been only weakly immunogenic in humans. Previously, we constructed DNA-launched Semliki Forest virus replicons (DREP), which stimulate pattern recognition receptors and induce augmented immune responses. Also, in vivo electroporation was shown to enhance immune responses induced by conventional DNA vaccines. Here, we combine these two approaches and show that in vivo electroporation increases CD8(+) T cell responses induced by DREP and consequently decreases the DNA dose required to induce a response. The vaccines used in this study encode the multiclade HIV-1 T cell immunogen HIVconsv, which is currently being evaluated in clinical trials. Using intradermal delivery followed by electroporation, the DREP.HIVconsv DNA dose could be reduced to as low as 3.2 ng to elicit frequencies of HIV-1-specific CD8(+) T cells comparable to those induced by 1 μg of a conventional pTH.HIVconsv DNA vaccine, representing a 625-fold molar reduction in dose. Responses induced by both DREP.HIVconsv and pTH.HIVconsv were further increased by heterologous vaccine boosts employing modified vaccinia virus Ankara MVA.HIVconsv and attenuated chimpanzee adenovirus ChAdV63.HIVconsv. Using the same HIVconsv vaccines, the mouse observations were supported by an at least 20-fold-lower dose of DNA vaccine in rhesus macaques. These data point toward a strategy for overcoming the low immunogenicity of DNA vaccines in humans and strongly support further development of the DREP vaccine platform for clinical evaluation.     

肿瘤DNA疫苗佐剂研究进展

对近年来在增加肿瘤DNA疫苗免疫原性、提高肿瘤DNA疫苗效力方面所取得的进展予以综述。简要阐述了以下几种较有效的增强肿瘤DNA疫苗效力策略的机制和进展：（1）以细胞因子表达质粒为佐剂;（2）以质粒编码的趋化因子、协同刺激分子、共刺激分子、补体为佐剂;（3）以CPGODN为佐剂;（4）其他一些佐剂。     

An improved RNA amplification procedure results in increased yield of autologous RNA transfected dendritic cell-based vaccine

Use of antigen encoding RNA transfected Dendritic cells in the field of cancer immunotherapy has been well established. The use of RNA overcomes limitations inherent to other autologous DC-based vaccines as it does not require specific HLA haplotypes, identification and characterization of antigens, and captures the broadest antigen repertoire. RNA offers yet another advantage-it could be amplified minimizing the requirement of tumor mass for autologous vaccine production, and will afford the opportunity to treat patients with minimal tumor burden. The original procedure described for RNA amplification resulted in a proportion of RNA transcribed in the antisense orientation. This study also demonstrates that the presence of double-stranded RNA correlates with the presence of antisense RNA. Alternative design of oligonucleotides that removes sequence redundancy eliminates the formation of both antisense and double-stranded RNA species. We provide further evidence that amplified RNA containing antisense and double-stranded RNA species results in lower recovery of DCs post-transfection and maturation, presumably through sequence-specific gene silencing. The removal of the double-stranded species from amplified RNA results in higher recovery of mature autologous amplified RNA transfected dendritic cells. Higher DC yield will allow for reduction of cost of vaccine manufacturing and prolonged treatment of a patient.     

A novel combination immunotherapy for cancer by IL-13Rα2-targeted DNA vaccine and immunotoxin in murine tumor models

Optimum efficacy of therapeutic cancer vaccines may require combinations that generate effective antitumor immune responses, as well as overcome immune evasion and tolerance mechanisms mediated by progressing tumor. Previous studies showed that IL-13Rα2, a unique tumor-associated Ag, is a promising target for cancer immunotherapy. A targeted cytotoxin composed of IL-13 and mutated Pseudomonas exotoxin induced specific killing of IL-13Rα2(+) tumor cells. When combined with IL-13Rα2 DNA cancer vaccine, surprisingly, it mediated synergistic antitumor effects on tumor growth and metastasis in established murine breast carcinoma and sarcoma tumor models. The mechanism of synergistic activity involved direct killing of tumor cells and cell-mediated immune responses, as well as elimination of myeloid-derived suppressor cells and, consequently, regulatory T cells. These novel results provide a strong rationale for combining immunotoxins with cancer vaccines for the treatment of patients with advanced cancer.     

肿瘤基因疫苗的研究进展

基因疫苗技术自从20世纪90年代问世以来被迅速应用到传染病、免疫缺陷、肿瘤等重大疾病的预防和治疗的研究中,有一部分已经进入临床试验阶段.肿瘤基因疫苗可以打破免疫耐受,增强免疫原性,诱导机体产生针对肿瘤的体液和细胞反应,既有预防又有治疗肿瘤的作用.能够防治肿瘤的基因疫苗发展迅猛,主要包括与肿瘤相关抗原（TAAs）有关的全长、表位、独特型（Id）和融合DNA疫苗,能够自主复制的RNA疫苗,与树突细胞（DCs）相关的肿瘤基因疫苗等.肿瘤基因疫苗的分子作用机制及其存在的弊端也日益成为关注的问题.