Plant viral genes in DNA idiotypic vaccines activate linked CD4+ T-cell mediated immunity against B-cell malignancies

DNA delivery of tumor antigens can activate specific immune attack on cancer cells. However, antigens may be weak, and immune capacity can be compromised. Fusion of genes encoding activating sequences to the tumor antigen sequence facilitates promotion and manipulation of effector pathways. Idiotypic determinants of B-cell tumors, encoded by the variable region genes, are clone-specific tumor antigens. When assembled as single-chain Fv (scFv) alone in a DNA vaccine, immunogenicity is low. Previously, we found that fusion of a sequence from tetanus toxin (fragment C; FrC) promoted anti-idiotypic protection against lymphoma and myeloma. We have now investigated an alternative fusion gene derived from a plant virus, potato virus X coat protein, a primary antigen in humans. When fused to scFv, the self-aggregating protein generates protection against lymphoma and myeloma. In contrast to scFv-FrC, protection against lymphoma is mediated by CD4+ T cells, as is protection against myeloma. Plant viral proteins offer new opportunities to activate immunity against linked T-cell epitopes to attack cancer.     

Epitope-driven DNA vaccine design employing immunoinformatics against B-cell lymphoma: a biotech's challenge

DNA vaccination has been widely explored to develop new, alternative and efficient vaccines for cancer immunotherapy. DNA vaccines offer several benefits such as specific targeting, use of multiple genes to enhance immunity and reduced risk compared to conventional vaccines. Rapid developments in molecular biology and immunoinformatics enable rational design approaches. These technologies allow construction of DNA vaccines encoding selected tumor antigens together with molecules to direct and amplify the desired effector pathways, as well as highly targeted vaccines aimed at specific epitopes. Reliable predictions of immunogenic T cell epitope peptides are crucial for rational vaccine design and represent a key problem in immunoinformatics. Computational approaches have been developed to facilitate the process of epitope detection and show potential applications to the immunotherapeutic treatment of cancer. In this review a number of different epitope prediction methods are briefly illustrated and effective use of these resources to support experimental studies is described. Epitope-driven vaccine design employs these bioinformatics algorithms to identify potential targets of vaccines against cancer. In this paper the selection of T cell epitopes to develop epitope-based vaccines, the need for CD4(+) T cell help for improved vaccines and the assessment of vaccine performance against tumor are reviewed. We focused on two applications, namely prediction of novel T cell epitopes and epitope enhancement by sequence modification, and combined rationale design with bioinformatics for creation of new synthetic mini-genes. This review describes the development of epitope-based DNA vaccines and their antitumor effects in preclinical research against B-cell lymphoma, corroborating the usefulness of this platform as a potential tool for cancer therapy. Achievements in the field of DNA vaccines allow to overcome hurdles to clinical translation. In a scenario where the vaccine industry is rapidly changing from a mostly empirical approach to a rational design approach, these new technologies promise to discover and develop high-value vaccines, creating a new opportunity for future markets.     

Enhancing the potency of HBV DNA vaccines using fusion genes of HBV-specific antigens and the N-terminal fragment of gp96

BACKGROUND: Many clinical trials show that DNA vaccine potency needs to be greatly enhanced. We have reported that the N-terminal fragment of glycoprotein 96 (gp96) is able to produce an adjuvant effect for production of cytotoxic T-lymphocytes (CTLs) with hepatitis B virus (HBV)-specific peptides. Here, we report a new strategy for HBV DNA vaccine design using a partial gp96 sequence. MATERIALS AND METHODS: We linked the N-terminal 1-355aa (N355) of gp96 to HBV genes encoding for structural proteins, the major S and middle S2S envelope proteins and the truncated core HBcAg (1-149aa). ELISPOT, tetramer staining and intracellular IFN-gamma assay were performed to analyze the induced cellular immune responses of our DNA constructs in BALB/c mice and HLA-A2 transgenic mice. The relative humoral immune responses were analyzed in different IgG isotypes. RESULTS: The fusion genes induced 2- to 6-fold higher HBV-specific CD8(+) T cells as compared to the antigens alone. There was an approximate 10-fold decrease in the humoral immune responses with fusion genes based on HBV envelope proteins. Interestingly, the decreased humoral immune responses were not observed when antigens and plasmid encoding N355 were co-delivered. However, an approximate 20-fold higher antibody level was induced when linking N355 to a truncated HBcAg. Immunization by intramuscular injection resulted in predominantly IgG2a antibodies, which indicated that these vaccines preferentially prime Th1 responses. CONCLUSIONS: We constructed highly immunogenic fusions by linking the N-terminal fragment of gp96 to HBV antigens. Our results imply that the N-terminal fragment of gp96 may be used as a molecular adjuvant to enhance the potency of DNA vaccines.     

The ‘Co-Delivery’ Approach to Liposomal Vaccines: Application to the Development of influenza-A and hepatitis-B Vaccine Candidates

DNA vaccination with mammalian-expressible plasmid DNA encoding protein antigens is known to be an effective means to elicit cell-mediated immunity, sometimes in the absence of a significant antibody response. This may be contrasted with protein vaccination, which gives rise to antibody responses with little evidence of cell-mediated immunity. This has led to considerable interest in DNA vaccination as a means to elicit cell-mediated immune responses against conserved viral antigens or intracellular cancer antigens, for the purpose of therapeutic vaccination. However, almost all current vaccines are used prophylactically and work by producing antibodies rather than cell mediated immune responses. In the present study we have therefore explored the combination of DNA and protein forms of an antigen using two exemplary prophylactic vaccine antigens, namely inactivated influenza virion and hepatitis-B surface antigen. We studied the effects of various combinations of DNA and protein on the antibody response. Co-administration of soluble forms of DNA and protein representations of the same antigen gave rise to the same level of antibody response as if protein were administered alone. In contrast, we found that when these antigens are entrapped in the same liposomal compartment, that there was a strong synergistic effect on the immune response, which was much greater than when either antigen was administered alone, or in various other modes of combination (e.g. co-administration as free entities, also pooled liposomal formulations where the two materials were contained in separate liposomal vehicles in the same suspension). The synergistic effect of liposomally co-entrapped DNA and protein exceeded, markedly, the well known adjuvant effects of plasmid DNA and liposomes. We have termed this new approach to vaccination ‘co-delivery’ and suggest that it may derive from the simultaneous presentation of antigen via MHC class-I (DNA) and MHC class-II (protein) pathways to CD8+ and CD4+ cells at the same antigen presenting cell – a mode of presentation that would commonly occur with live viral pathogens. We conclude that co-delivery is a very effective means to generate protective antibody responses against viral pathogens.     

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.     

Induction of T cell-mediated immunity using a c-Myb DNA vaccine in a mouse model of colon cancer

Overexpression of the proto-oncogene c-Myb occurs in more than 80% of colorectal cancer (CRC) and is associated with aggressive disease and poor prognosis. To test c-Myb as a therapeutic target in CRC we devised a DNA fusion vaccine to generate an anti-CRC immune response. c-Myb, like many tumor antigens, is weakly immunogenic as it is a "self" antigen and subject to tolerance. To break tolerance, a DNA fusion vaccine was generated comprising wild-type c-Myb cDNA flanked by two potent Th epitopes derived from tetanus toxin. Vaccination was performed targeting a highly aggressive, weakly immunogenic, subcutaneous, syngeneic, colon adenocarcinoma cell line MC38 which highly expresses c-Myb. Prophylactic intravenous vaccination significantly suppressed tumor growth, through the induction of anti-tumor immunity for which the tetanus epitopes were essential. Vaccination generated anti-tumor immunity mediated by both CD4(+) and CD8(+) T cells and increased infiltration of immune effector cells at the tumor site. Importantly, no evidence of autoimmune pathology in endogenous c-Myb expressing tissues was detected as a consequence of breaking tolerance. In summary, these results establish c-Myb as a potential antigen for immune targeting in CRC and serve to provide proof of principle for the continuing development of DNA vaccines targeting c-Myb to bring this approach to the clinic.     

Chemical and biological conjugation strategies for the development of multivalent protein vaccine nanoparticles

The development of subunit vaccine platforms has been of considerable interest due to their good safety profile and ability to be adapted to new antigens, compared to other vaccine typess. Nevertheless, subunit vaccines often lack sufficient immunogenicity to fully protect against infectious diseases. A wide variety of subunit vaccines have been developed to enhance antigen immunogenicity by increasing antigen multivalency, as well as stability and delivery properties, via presentation of antigens on protein nanoparticles. Increasing multivalency can be an effective approach to provide a potent humoral immune response by more strongly engaging and clustering B cell receptors (BCRs) to induce activation, as well as increased uptake by antigen presenting cells and their subsequent T cell activation. Proper orientation of antigen on protein nanoparticles is also considered a crucial factor for enhanced BCR engagement and subsequent immune responses. Therefore, various strategies have been reported to decorate highly repetitive surfaces of protein nanoparticle scaffolds with multiple copies of antigens, arrange antigens in proper orientation, or combinations thereof. In this review, we describe different chemical bioconjugation methods, approaches for genetic fusion of recombinant antigens, biological affinity tags, and enzymatic conjugation methods to effectively present antigens on the surface of protein nanoparticle vaccine scaffolds.     

DNA vaccination strategies for anti-tumour effective gene therapy protocols

After more than 15 years of experimentation, DNA vaccines have become a promising perspective for tumour diseases, and animal models are widely used to study the biological features of human cancer progression and to test the efficacy of vaccination protocols. In recent years, immunisation with naked plasmid DNA encoding tumour-associated antigens or tumour-specific antigens has revealed a number of advantages: antigen-specific DNA vaccination stimulates both cellular and humoral immune responses; multiple or multi-gene vectors encoding several antigens/determinants and immune-modulatory molecules can be delivered as single administration; DNA vaccination does not induce autoimmune disease in normal animals; DNA vaccines based on plasmid vectors can be produced and tested rapidly and economically. However, DNA vaccines have shown low immunogenicity when tested in human clinical trials, and compared with traditional vaccines, they induce weak immune responses. Therefore, the improvement of vaccine efficacy has become a critical goal in the development of effective DNA vaccination protocols for anti-tumour therapy. Several strategies are taken into account for improving the DNA vaccination efficacy, such as antigen optimisation, use of adjuvants and delivery systems like electroporation, co-expression of cytokines and co-stimulatory molecules in the same vector, different vaccination protocols. In this review we discuss how the combination of these approaches may contribute to the development of more effective DNA vaccination protocols for the therapy of lymphoma in a mouse model.     

DNA fusion vaccines enter the clinic

Induction of effective immune attack on cancer cells in patients requires conversion of weak tumor antigens into strong immunogens. Our strategy employs genetic technology to create DNA vaccines containing tumor antigen sequences fused to microbial genes. The fused microbial protein engages local CD4+ T cells to provide help for anti-tumor immunity, and to reverse potential regulation. In this review, we focus on induction of CD8+ T cells able to kill target tumor cells. The DNA vaccines incorporate tumor-derived peptide sequences fused to an engineered domain of tetanus toxin. In multiple models, this design induces strong CD8+ T-cell responses, able to suppress tumor growth. For clinical relevance, we have used “humanized” mice expressing HLA-A2, successfully inducing cytolytic T-cell responses against a range of candidate human peptides. To overcome physical restriction in translating to patients, we have used electroporation. Clinical trials of patients with cancer are showing induction of responses, with preliminary indications of suppression of tumor growth and evidence for clinically manageable concomitant autoimmunity.     

DNA vaccination against tumors

DNA vaccines have been used to generate protective immunity against tumors in a variety of experimental models. The favorite target antigens have been those that are frequently expressed by human tumors, such as carcinoembryonic antigen (CEA), ErbB2/neu, and melanoma-associated antigens. DNA vaccines have the advantage of being simple to construct, produce and deliver. They can activate all arms of the immune system, and allow substantial flexibility in modifying the type of immune response generated through codelivery of cytokine genes. DNA vaccines can be applied by intramuscular, dermal/epidermal, oral, respiratory and other routes, and pose relatively few safety concerns. Compared to other nucleic acid vectors, they are usually devoid of viral or bacterial antigens and can be designed to deliver only the target tumor antigen(s). This is likely to be important when priming a response against weak tumor antigens. DNA vaccines have been more effective in rodents than in larger mammals or humans. However, a large number of methods that might be applied clinically have been shown to ameliorate these vaccines. This includes in vivo electroporation, and/or inclusion of various immunostimulatory molecules, xenoantigens (or their epitopes), antigen-cytokine fusion genes, agents that improve antigen uptake or presentation, and molecules that activate innate immunity mechanisms. In addition, CpG motifs carried by plasmids can overcome the negative effects of regulatory T cells. There have been few studies in humans, but recent clinical trials suggest that plasmid/virus, or plasmid/antigen-adjuvant, prime-boost strategies generate strong immune responses, and confirm the usefulness of plasmid-based vaccination.     

基因工程疫苗的病毒载体

病毒基因工程疫苗是以活病毒为载体将一段外源基因导入机体细胞内,并使外源基因维持较高水平的表达。通过使用复制型或复制缺陷型载体能使表达的抗原诱生机体产生相应的体液抗体,并能引起机体产生细胞介导的免疫反应及粘膜免疫反应。本文主要介绍有可能用于基因工程疫苗的DNA及RNA病毒载体构建及其应用。     

Progress in vaccine development against Helicobacter pylori

Based on the very high prevalence of diseases caused by Helicobacter pylori, particularly in the developing world, and the rapid emergence of antibiotic resistance among clinical isolates, there is a strong rationale for an effective vaccine against H. pylori. In this review we describe recent promising candidate vaccines and prophylactic or therapeutic immunization strategies for use against H. pylori, as well as studies to identify immune responses that are related to protection in experimental animals. We also describe identification of different types of immune responses that may be related to protection against symptoms based on comparisons of H. pylori-infected patients with duodenal ulcers or gastric cancer and asymptomatic carriers. We conclude that there is still a strong need to clarify the main protective immune mechanisms against H. pylori as well as to identify a cocktail of strong protective antigens, or recombinant bacterial strains that express such antigens, that could be administered by a regimen that gives rise to effective immune responses in humans.     

细胞因子作为DNA疫苗佐剂的研究进展

细胞因子是机体细胞(主要指免疫细胞)产生的一类具有广泛生物学活性的异质性肽类调节因子,在体内能激活免疫活性细胞,对免疫应答的产生和调节有重要作用。近年来,大量研究表明细胞因子可作为DNA疫苗佐剂来增强疫苗的免疫效果。简要综述了细胞因子作为DNA疫苗免疫佐剂的研究进展。     

Vaccines for colorectal cancer: an update

Colorectal cancer (CRC) is known as the third most common and fourth leading cancer associated death worldwide. The occurrence of metastasis has remained as a critical challenge in CRC, so that distant metastasis (mostly to the liver) has been manifested in about 20%-25% of patients. Several screening approaches have introduced for detecting CRC in different stages particularly in early stages. The standard treatments for CRC are surgery, chemotherapy and radiotherapy, in alone or combination. Immunotherapy is a set of novel approaches with the aim of remodeling the immune system battle with metastatic cancer cells, such as immunomodulatory monoclonal antibodies (immune checkpoint inhibitors), adoptive cell transfer (ACT) and cancer vaccine. Cancer vaccines are designed to trigger the intense response of immune system to tumor-specific antigens. In two last decades, introduction of new cancer vaccines and designing several clinical trials with vaccine therapy, have been taken into consideration in colon cancer patients. This review will describe the treatment approaches with the special attention to vaccines applied to treat colorectal cancer.     

HBV包膜-核心蛋白融合基因DNA疫苗诱导小鼠持久的细胞免疫应答

构建编码HBV包膜-核心蛋白融合基因的DNA疫苗pSC、pSS1S2C和编码HBV包膜蛋白或核心蛋白基因的DNA疫苗pHBs、pHBc,分别肌肉注射免疫BALB/c小鼠,检测小鼠的血清抗体、T细胞增殖和细胞毒性T淋巴细胞反应,比较融合基因DNA疫苗与单基因DNA疫苗诱生免疫应答的强度,发现融合基因DNA疫苗诱生抗体的效率明显不及单基因DNA疫苗,但其能诱导更强、更持久的细胞免疫应答,表明HBV包膜-核心蛋白融合基因DNA疫苗对于治疗慢性乙型肝炎可能比单基因DNA疫苗更为有效.     

Peptide‐based therapeutic cancer vaccine: Current trends in clinical application

The peptide‐based therapeutic cancer vaccines have attracted enormous attention in recent years as one of the effective treatments of tumour immunotherapy. Most of peptide‐based vaccines are based on epitope peptides stimulating CD8⁺ T cells or CD4⁺ T helper cells to target tumour‐associated antigens (TAAs) or tumour‐specific antigens (TSAs). Some adjuvants and nanomaterials have been exploited to optimize the efficiency of immune response of the epitope peptide to improve its clinical application. At present, numerous peptide‐based therapeutic cancer vaccines have been developed and achieved significant clinical benefits. Similarly, the combination of peptide‐based vaccines and other therapies has demonstrated a superior efficacy in improving anti‐cancer activity. We delve deeper into the choices of targets, design and screening of epitope peptides, clinical efficacy and adverse events of peptide‐based vaccines, and strategies combination of peptide‐based therapeutic cancer vaccines and other therapies. The review will provide a detailed overview and basis for future clinical application of peptide‐based therapeutic cancer vaccines.     

Evaluation of the VP22 protein for enhancement of a DNA vaccine against anthrax

BACKGROUND: Previously, antigens expressed from DNA vaccines have been fused to the VP22 protein from Herpes Simplex Virus type I in order to improve efficacy. However, the immune enhancing mechanism of VP22 is poorly understood and initial suggestions that VP22 can mediate intercellular spread have been questioned. Despite this, fusion of VP22 to antigens expressed from DNA vaccines has improved immune responses, particularly to non-secreted antigens. METHODS: In this study, we fused the gene for the VP22 protein to the gene for Protective Antigen (PA) from Bacillus anthracis, the causative agent of anthrax. Protective immunity against infection with B. anthracis is almost entirely based on a response to PA and we have generated two constructs, where VP22 is fused to either the N- or the C-terminus of the 63 kDa protease-cleaved fragment of PA (PA63). RESULTS: Following gene gun immunisation of A/J mice with these constructs, we observed no improvement in the anti-PA antibody response generated. Following an intraperitoneal challenge with 70 50% lethal doses of B. anthracis strain STI spores, no difference in protection was evident in groups immunised with the DNA vaccine expressing PA63 and the DNA vaccines expressing fusion proteins of PA63 with VP22. CONCLUSION: VP22 fusion does not improve the protection of A/J mice against live spore challenge following immunisation of DNA vaccines expressing PA63.     

Highly potent mRNA based cancer vaccines represent an attractive platform for combination therapies supporting an improved therapeutic effect

Direct vaccination with mRNA encoding tumor antigens is a novel and promising approach in cancer immunotherapy. CureVac's mRNA vaccines contain free and protamine-complexed mRNA. Such two-component mRNA vaccines support both antigen expression and immune stimulation. These self-adjuvanting RNA vaccines, administered intradermally without any additional adjuvant, induce a comprehensive balanced immune response, comprising antigen specific CD4+ T cells, CD8+ T cells and B cells. The balanced immune response results in a strong anti-tumor effect and complete protection against antigen positive tumor cells. This tumor inhibition elicited by mRNA vaccines is a result of the concerted action of different players. After just two intradermal vaccinations, we observe multiple changes at the tumor site, including the up-regulation of many genes connected to T and natural killer cell activation, as well as genes responsible for improved infiltration of immune cells into the tumor via chemotaxis. The two-component mRNA vaccines induce a very fast and boostable immune response. Therefore, the vaccination schedules can be adjusted to suit the clinical situation. Moreover, by combining the mRNA vaccines with therapies in clinical use (chemotherapy or anti-CTLA-4 antibody therapy), an even more effective anti-tumor response can be elicited. The first clinical data obtained from two separate Phase I/IIa trials conducted in PCA (prostate cancer) and NSCLC (non-small cell lung carcinoma) patients have shown that the two-component mRNA vaccines are safe, well tolerated and highly immunogenic in humans.     

Biotechnology approaches to produce potent,self-adjuvanting antigen-adjuvant fusion protein subunit vaccines

Traditional vaccination approaches (e.g. live attenuated or killed microorganisms) are among the most effective means to prevent the spread of infectious diseases. These approaches, nevertheless, have failed to yield successful vaccines against many important pathogens. To overcome this problem, methods have been developed to identify microbial components, against which protective immune responses can be elicited. Subunit antigens identified by these approaches enable the production of defined vaccines, with improved safety profiles. However, they are generally poorly immunogenic, necessitating their administration with potent immunostimulatory adjuvants. Since few safe and effective adjuvants are currently used in vaccines approved for human use, with those available displaying poor potency, or an inability to stimulate the types of immune responses required for vaccines against specific diseases (e.g. cytotoxic lymphocytes (CTLs) to treat cancers), the development of new vaccines will be aided by the availability of characterized platforms of new adjuvants, improving our capacity to rationally select adjuvants for different applications. One such approach, involves the addition of microbial components (pathogen-associated molecular patterns; PAMPs), that can stimulate strong immune responses, into subunit vaccine formulations. The conjugation of PAMPs to subunit antigens provides a means to greatly increase vaccine potency, by targeting immunostimulation and antigen to the same antigen presenting cell. Thus, methods that enable the efficient, and inexpensive production of antigen-adjuvant fusions represent an exciting mean to improve immunity towards subunit antigens. Herein we review four protein-based adjuvants (flagellin, bacterial lipoproteins, the extra domain A of fibronectin (EDA), and heat shock proteins (Hsps)), which can be genetically fused to antigens to enable recombinant production of antigen-adjuvant fusion proteins, with a focus on their mechanisms of action, structural or sequence requirements for activity, sequence modifications to enhance their activity or simplify production, adverse effects, and examples of vaccines in preclinical or human clinical trials.