DNA vaccines

Within the last decade bacterial plasmids encoding foreign antigens have revolutionized vaccine design. Although no DNA vaccine has yet been approved for routine human or veterinary use, the potential of this vaccine modality has been demonstrated in experimental animal models. Plasmid DNA vaccination has shown efficacy against viral, bacterial and parasitic infections, modulated the effects of autoimmune and allergic diseases and induced control over cancer progression. With a better understanding of the basic immune mechanisms that govern induction of protective or curative immune responses, plasmid DNA vaccines and their mode of delivery are continuously being optimized. Because of the simplicity and versatility of these vaccines, various routes and modes of delivery are possible to engage the desired immune responses. These may be T or B effector cell responses able to eliminate infectious agents or transformed cells. DNA vaccines may also induce an immunoregulatory/modulatory or immunosuppressive (tolerizing) response that interferes with the differentiation, expansion or effector functions of B and T cells. In this sense a DNA vaccine may be thought of as a 'negative' vaccine. Pre-clinical and initial small-scale clinical trials have shown DNA vaccines in either of these modes to be safe and well tolerated. Although DNA vaccines induce significant immune responses in small animal trials their efficacy in humans has so far been less promising thus necessitating additional optimizations of this novel vaccine approach.     

Accelerated immune response to DNA vaccines

DNA vaccines offer considerable promise for improvement over conventional vaccines. For the crucial step of delivering DNA vaccines intracellularly, electroporation (EP) has proven to be highly effective. This method has yielded powerful humoral and cellular responses in various species, including nonhuman primates. In an attempt to further improve DNA vaccination we used micron-size gold particles (which do not bind or adsorb DNA) as a particulate adjuvant which was coinjected with DNA intramuscularly into mice, followed by EP of the target site. The presence of gold particles accelerated the antibody response significantly. Maximum titers against hepatitis B surface antigen (HBsAg) were reached after one boost in 6 weeks, whereas 8 weeks were required without particles. These immunizations were effective in protecting mice against tumor challenge with cancer cells expressing HBsAg as a surrogate cancer antigen. Computer modeling of electric fields and gene expression studies indicate that gold particles do not stimulate EP and subsequent antigen expression. The particles may act as an attractant for immune cells, especially antigen presenting cells. We conclude that particulate adjuvants combined with DNA vaccine delivery by EP reduces the immune response time and may increase vaccine efficacy. This method may become valuable for developing prophylactic as well as therapeutic vaccines. The rapid response may be of particular interest in countering bio-terrorism.     

Paradigm shifts in cancer vaccine therapy

Cancer vaccines constitute a unique therapeutic modality in that they initiate a dynamic process involving the host's immune response. Consequently, (a) repeated doses (vaccinations) over months may be required before patient clinical benefit is observed and (b) there most likely will be a "dynamic balance" between the induction and maintenance of host immune response elements to the vaccinations vs. host/tumor factors that have the potential to diminish those responses. Thus "patient response" in the form of disease stabilization and prolonged survival may be more appropriate to monitor than strictly adhering to "tumor response" in the form of Response Criteria In Solid Tumors (RECIST) criteria. This can be manifested in the form of enhanced patient benefit to subsequent therapies following vaccine therapy. This article will review these phenomena unique to cancer vaccines with emphasis on prostate cancer vaccines as a prototype for vaccine therapy. The unique features of this modality require the consideration of paradigm shifts both in the way cancer vaccine clinical trials are designed and in the way patient benefit is evaluated.     

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.     

Prostate cancer vaccines: the long road to clinical application

Cancer vaccines as a modality of immune-based cancer treatment offer the promise of a non-toxic and efficacious therapeutic alternative for patients. Emerging data suggest that response to vaccination largely depends on the magnitude of the type I immune response generated, epitope spreading and immunogenic modulation of the tumor. Moreover, accumulating evidence suggests that cancer vaccines will likely induce better results in patients with low tumor burden and less aggressive disease. To induce long-lasting clinical responses, vaccines will need to be combined with immunoregulatory agents to overcome tumor-related immune suppression. Immunotherapy, as a treatment modality for prostate cancer, has received significant attention in the past few years. The most intriguing characteristics that make prostate cancer a preferred target for immune-based treatments are (1) its relative indolence which allows sufficient time for the immune system to develop meaningful antitumor responses; (2) prostate tumor-associated antigens are mainly tissue-lineage antigens, and thus, antitumor responses will preferentially target prostate cancer cells. But, also in the event of eradication of normal prostate epithelium as a result of immune attack, this will have no clinical consequences because the prostate gland is not a vital organ; (3) the use of prostate-specific antigen for early detection of recurrent disease allows for the initiation of vaccine immunotherapy while tumor burden is still minimal. Finally, for improving clinical outcome further to increasing vaccine potency, it is imperative to recognize prognostic and predictive biomarkers of clinical benefit that may guide to select the therapeutic strategies for patients most likely to gain benefit.     

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.     

Immunological characterization of peptide mimetics of carbohydrate antigens in vaccine design strategies.

Targeting antigens which cannot be readily addressed by genetic vectors is a major challenge in vaccine design. The inter-conversion of carbohydrate antigens into peptide mimetic forms provides a means to broaden the immune response to carbohydrate antigens. Peptides that mimic carbohydrate antigens offer new possibilities to augment immune responses to such antigens that include inducing carbohydrate reactive T-cell responses. Peptide mimeotopes can be formulated in a variety of ways that include multiple antigen peptides (MAP) and as DNA vaccines that prime for different antibody isotypes. On the immunological side we observe that: (i) depending on the immunogen formulation peptide mimetics can be processed by either CD5+ or CD5-B cells; (ii) peptide mimeotope immunization can induce cross-reactive responses to multiple carbohydrate forms; (iii) priming with peptide mimeotopes can enhance carbohydrate immune responses upon boosting and (iv) immunization with peptide mimeotopes can induce carbohydrate reactive T cells.     

Enhanced potency of individual and bivalent DNA replicon vaccines or conventional DNA vaccines by formulation with aluminum phosphate

DNA vaccines against botulinum neurotoxin (BoNTs) induce protective humoral immune responses in mouse model, but when compared with conventional vaccines such as toxoid and protein vaccines, DNA vaccines often induce lower antibody level and protective efficacy and are still necessary to increase their potency. In this study we evaluated the potency of aluminum phosphate as an adjuvant of DNA vaccines to enhance antibody responses and protective efficacy against botulinum neurotoxin serotypes A and B in Balb/c mice. The administration of these individual and bivalent plasmid DNA replicon vaccines against botulinum neurotoxin serotypes A and B in the presence of aluminum phosphate improved both antibody responses and protective efficacy. Furthermore, formulation of conventional plasmid DNA vaccines encoding the same Hc domains of botulinum neurotoxin serotypes A and B with aluminum phosphate adjuvant increased both antibody responses and protective efficacy. These results indicate aluminum phosphate is an effective adjuvant for these two types of DNA vaccines (i.e., plasmid DNA replicon vaccines and conventional plasmid DNA vaccines), and the vaccine formulation described here may be an excellent candidate for further vaccine development against botulinum neurotoxins.     

Strategies in cancer vaccines development

The recent definition of tumour-specific immunity in cancer patients and the identification of tumour-associated antigens have generated renewed enthusiasm for the application of immune-based therapies for the treatment of malignancies. Recent developments in cancer vaccines have also been based on an improved understanding of the cellular interactions required to induce a specific anti-tumour immune response. Consequently, a number of cancer vaccines have entered clinical trials. Targeting broad-spectrum tumour-associated antigens has emerged as a strategy to lower the risk of tumour escape due to the loss of specific nominal antigen. Amongst the most challenging of tumour-associated antigens to which to target in active specific immunotherapy applications are carbohydrate antigens. As carbohydrates are intrinsically T-cell-independent antigens, more novel approaches are perhaps needed to drive specific-T-cell-dependent immune responses to carbohydrate antigens. In this context peptide mimetics of core structures of tumour-associated carbohydrate antigens might be developed to augment immune responses to these broad-spectrum antigens.     

Vaccination with a DNA vaccine based on human PSCA and HSP70 adjuvant enhances the antigen-specific CD8+ T-cell response and inhibits the PSCA+ tumors growth in mice

BACKGROUND: DNA vaccines have been shown to be an effective approach to induce antigen-specific cellular and humoral immunity. However, the lower immune intensity in clinical trials limits the application of DNA vaccine. Here we intend to develop a new DNA vaccine based on prostate stem-cell antigen (PSCA), which has been suggested as a potential target for prostate cancer therapy, and enhance the DNA vaccine potency with heat shock proteins (HSPs) as adjuvant. METHODS: A series of DNA plasmids encoding human PSCA, human HSP70 and their conjugates was constructed and injected into male mice intramuscularly (i.m.). To evaluate the immune responses and therapeutic efficacy of these plasmids, major histocompatibility complex (MHC)-restricted PSCA and HSP70-specific epitopes were predicted and a mouse model with a human PSCA-expressing tumor was constructed. RESULTS: The result showed that mice vaccinated with PSCA-HSP plasmids generated the strongest PSCA-specific CD8+ T-cell immune response, but the CD4+ TH1 and TH2 cell immune responses were similar with those vaccinated with other HSP-adjuvant PSCA plasmids or only PSCA DNA. The immunity of HSP70 was also observed and the mice i.m. injected with PSCA+ HSP mixed plasmids generated the lowest anti-HSP antibodies. Furthermore, these vaccinations inhibited the growth of PSCA-expressing tumors and prolonged mouse survival. CONCLUSIONS: These observations emphasize and extend the potential of the human HSP70 gene as adjuvant for DNA vaccines, and the vaccine based on PSCA and HSP70 is of potential value for treating prostate cancer.     

Immunogenicity of DNA and Recombinant Sendai Virus Vaccines Expressing the HIV-1 gag Gene

Combinations of DNA and recombinant-viral-vector based vaccines are promising AIDS vaccine methods because of their potential for inducing cellular immune responses. It was found that Gag-specific cytotoxic lymphocyte (CTL) responses were associated with lowering viremia in an untreated HIV-1 infected cohort. The main objectives of our studies were the construction of DNA and recombinant Sendal virus vector (rSeV) vaccines containing a gag gene from the prevalent Thailand subtype B strain in China and trying to use these vaccines for therapeutic and prophylactic vaccines. The candidate plasmid DNA vaccine pcDNA3.1( )-gag and recombinant Sendai virus vaccine (rSeV-gag) were constructed separately. It was verified by Western blotting analysis that both DNA and rSeV-gag vaccines expressed the HIV-1 Gag protein correctly and efficiently. Balb/c mice were immunized with these two vaccines in different administration schemes. HIV-1 Gag-specific CTL responses and antibody levels were detected by intracellular cytokine staining assay and enzyme-linked immunosorbant assay (ELISA) respectively. Combined vaccines in a DNA prime/rSeV-gag boost vaccination regimen induced the strongest and most long-lasting Gag-specific CTL and antibody responses. It maintained relatively high levels even 9 weeks post immunization. This data indicated that the prime-boost regimen with DNA and rSeV-gag vaccines may offer promising HIV vaccine regimens.     

Plasmid-DNA lipid and polymeric nanovaccines: a new strategic in vaccines development

Vaccination is the most effective and least expensive technique used for human diseases prevention and eradication. The need for more vaccine doses and the rapid establishment of facilities for the development of new vaccines are stimulating significate changes in the vaccine industry, which is gradually moving towards cell culture production. One approach is the third generation of vaccines, which are based on the use of plasmid DNA (pDNA) containing transgenes that encode an antigen capable of mimicking intracellular pathogenic infection and triggering both humoral and cellular immune responses. Plasmid DNA vaccination has distinct advantages over other vaccine technologies in terms of safety, ease of fabrication and stability. The effectiveness of pDNA vaccines against viruses, bacteria, parasites and cancer cells has been demonstrated in preclinical and clinical assays. Furthermore, currently there are a few veterinary pDNA vaccines in the market. The application of a simple formulation of naked pDNA as a vaccine is attractive, but a low transfection efficiency is often obtained. The use of nanoparticles to increase transfection efficiency is an approach that has been tested clinically. This review provides a summary of vaccine production, advances and major challenges associated with pDNA lipid and polymeric nanovaccines applications.     

A combination of intradermal jet-injection and electroporation overcomes in vivo dose restriction of DNA vaccines

ABSTRACT: BACKGROUND: The use of optimized delivery devices has been shown to enhance the potency of DNA vaccines. However, further optimization of DNA vaccine delivery is needed for this vaccine modality to ultimately be efficacious in humans. METHODS: Herein we evaluated antigen expression and immunogenicity after intradermal delivery of different doses of DNA vaccines by needle or by the Biojector jet-injection device, with or without the addition of electroporation (EP). RESULTS: Neither needle injection augmented by EP nor Biojector alone could induce higher magnitudes of immune responses after immunizations with a high dose of a DNA vaccine as compared to immunizations with a considerably lower dose. Biojector delivery followed by EP, however, overcame this observed dose restriction and induced significantly higher cellular and humoral immune responses after immunization with a high dose of DNA. Furthermore, a close correlation between in vivo antigen expression and cell-mediated immune responses was observed. CONCLUSIONS: These results show that two optimized DNA vaccine delivery devices can act together to overcome dose restrictions of plasmid DNA vaccines.     

Technical and regulatory hurdles for DNA vaccines

DNA vaccines have been widely used in laboratory animals and non-human primates over the last decade to induce antibody and cellular immune responses. This approach has shown some promise, in models of infectious diseases of both bacterial and viral origin as well as in tumour models. Clinical trials have shown that DNA vaccines appear safe and well tolerated, but need to be made much more potent to be candidates for preventive immunisation of humans. This review describes recent work to improve the delivery of plasmid DNA vaccines and also to increase the immunogenicity of antigens expressed from the DNA vaccine plasmids, including various formulations and molecular adjuvants. Because DNA vaccines are relatively new and represent a novel vaccine technology, certain safety issues, such as the potential for induction of autoimmune disease and integration into the host genome, must be examined carefully. If potency can be improved and safety established, plasmid DNA vaccines offer advantages in speed, simplicity, and breadth of immune response that may be useful for the immunisation of humans against infectious diseases and cancers.     

DNA vaccine strategies: candidates for immune modulation and immunization regimens

DNA vaccine strategies can differ greatly, with significant effects on the outcome of immunization. In this article, we discuss plasmid design strategies and vaccine regimens. Effectiveness against a pathogen can be affected by the choice of antigen and inclusion of multiple antigens. Gene expression and the resulting immune response can be improved by gene modification and choice of promoters. In designing vaccine regimens, one must consider further dose, timing of doses, adjuvants, and routes of vaccination. Many vaccines are enhanced by combining DNA with other vaccines in "prime-boost" regimens, in which the second vaccine is often a recombinant viral vector or purified protein subunit. Prime-boost vaccines including DNA can elicit immune responses that differ in magnitude, quality, and balance of cellular and humoral responses from those elicited by single components and thus provide further enhancement for DNA immunizations.     

SIV DNA vaccine co-administered with IL-12 expression plasmid enhances CD8 SIV cellular immune responses in cynomolgus macaques

Current evidence suggests that a strong induced CD8 human immunodeficiency virus type 1 (HIV-1)-specific cell mediated immune response may be an important aspect of an HIV vaccine. The response rates and the magnitude of the CTL responses induced by current DNA vaccines in humans need to be improved and cellular immune responses to DNA vaccines can be enhanced in mice by co-delivering DNA plasmids expressing immune modulators. Two reported to work well in the mouse systems are interleukin (IL)-12 and CD40L. We sought to compare these molecular adjuvants in a primate model system. The cDNA for macaque IL-12 and CD40L were cloned into DNA vectors. Groups of cynomolgus macaques were immunized with 2 mg of plasmid expressing SIVgag alone or in combination with either IL-12 or CD40L. CD40L did not appear to enhance the cellular immune response to SIVgag antigen. However, more robust results were observed in animals co-injected with the IL-12 molecular adjuvant. The IL-12 expanded antigen-specific IFN-gamma positive effector cells as well as granzyme B production. The vaccine immune responses contained both a CD8 component as well a CD4 component. The adjuvanted DNA vaccines illustrate that IL-12 enhances a CD8 vaccine immune response, however, different cellular profiles.     

DNA vaccines: a ray of hope

Vaccines represent the most commonly employed immunologic intervention in medicine today. DNA vaccination or genetic immunization is a rapidly developing technology that offers new approaches for the prevention of disease. This method of vaccination provides a stable and long-lived source of the protein vaccine, and it is a simple, robust, and effective means of eliciting both antibody- and cell-mediated immune responses. Furthermore, DNA vaccines have a number of potential advantages such as they can address several diseases in one vaccine, they are cheap and easy to produce and have no special cold storage requirement because they are extremely stable. It has proven to be a generally applicable technology in various preclinical animal models of infectious and noninfectious diseases, and several DNA vaccines have now entered phase I/II, human clinical trials. There are several hurdles that need to be overcome on the road to the use of DNA vaccines widely. These include the technical challenges of improving delivery and/or potency so that low doses of DNA can achieve the efficacy of conventional vaccines.     

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.     

Biomarkers for the development of cancer vaccines: current status

Significant improvements in our knowledge of tumor immunology have resulted in more sophisticated vaccine approaches for the treatment of cancer. However, research into biomarkers that correlate with the clinical outcome of immunotherapy has lagged behind vaccine development. To this extent, very few immunological or other markers exist that can be used in clinical trials for immunotherapy. In this review, we discuss the current status of biomarker development specifically for the monitoring and development of cancer vaccines. This includes immunological biomarkers (measurement of T-cell and cytokine responses), autoimmunity as a correlate for treatment outcome, and the possible development of multiple biomarkers using high-throughput proteomics technologies. The generation of such biomarkers will allow us to make clinical decisions about patient treatment at an earlier stage and should aid in shortening the development time for vaccines.     

Removing N-terminal sequences in pre-s1 domain enhanced antibody and B-cell responses by an HBV large surface antigen DNA vaccine

Although the use of recombinant hepatitis B virus surface (HBsAg) protein vaccine has successfully reduced global hepatitis B infection, there are still a number of vaccine recipients who do not develop detectable antibody responses. Various novel vaccination approaches, including DNA vaccines, have been used to further improve the coverage of vaccine protection. Our previous studies demonstrated that HBsAg-based DNA vaccines could induce both humoral and CMI responses in experimental animal models. However, one form of the the HBsAg antigen, the large S antigen (HBs-L), expressed by DNA vaccine, was not sufficiently immunogenic in eliciting antibody responses. In the current study, we produced a modified large S antigen DNA vaccine, HBs-L(T), which has a truncated N-terminal sequence in the pre-S1 region. Compared to the original HBs-L DNA vaccine, the HBs-L(T) DNA vaccine improved secretion in cultured mammalian cells and generated significantly enhanced HBsAg-specific antibody and B cell responses. Furthermore, this improved HBsL DNA vaccine, along with other HBsAg-expressing DNA vaccines, was able to maintain predominantly Th1 type antibody responses while recombinant HBsAg protein vaccines produced in either yeast or CHO cells elicited mostly Th2 type antibody responses. Our data indicate that HBsAg DNA vaccines with improved immunogenicity offer a useful alternative choice to recombinant protein-based HBV vaccines, particularly for therapeutic purposes against chronic hepatitis infection where immune tolerance led to poor antibody responses to S antigens.