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.     

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

Development of various vaccines for prostate cancer (PCa) is becoming an active research area. PCa vaccines are perceived to have less toxicity compared with the available cytotoxic agents. While various immune-based strategies can elicit anti-tumour responses, DNA vaccines present increased efficacy, inducing both humoural and cellular immunity. This immune activation has been proven effective in animal models and initial clinical trials are encouraging. However, to validate the role of DNA vaccination in currently available PCa management paradigms, strong clinical evidence is still lacking. This article provides an overview of the basic principles of DNA vaccines and aims to provide a summary of preclinical and clinical trials outlining the benefits of this immunotherapy in the management of PCa.     

DNA vaccines: progress and challenges

In the years following the publication of the initial in vivo demonstration of the ability of plasmid DNA to generate protective immune responses, DNA vaccines have entered into a variety of human clinical trials for vaccines against various infectious diseases and for therapies against cancer, and are in development for therapies against autoimmune diseases and allergy. They also have become a widely used laboratory tool for a variety of applications ranging from proteomics to understanding Ag presentation and cross-priming. Despite their rapid and widespread development and the commonplace usage of the term "DNA vaccines," however, the disappointing potency of the DNA vaccines in humans underscores the challenges encountered in the efforts to translate efficacy in preclinical models into clinical realities. This review will provide a brief background of DNA vaccines including the insights gained about the varied immunological mechanisms that play a role in their ability to generate immune responses.     

Inulin-derived adjuvants efficiently promote both Th1 and Th2 immune responses

There has been a recent resurgence of interest into new and improved vaccine adjuvants. This interest has been stimulated by the need for new vaccines to combat problematic pathogens such as SARS and HIV, and to counter potential bioterrorist attacks. A major bottleneck in vaccine development is the low immunogenicity of purified subunit or recombinant proteins, creating the need for safe human adjuvants with high potency. A major problem in the search for the ideal adjuvant is that adjuvants that promote cell-mediated (Th1) immunity (e.g. Freund's complete adjuvant) generally have unacceptable local or systemic toxicity that precludes their use in human vaccines. There is a need for a safe, non-toxic adjuvant that is able to stimulate both cell-mediated and humoral immunity. Inulin-derived adjuvants that principally stimulate the innate immune system through their ability to activate the alternative complement pathway have proven ability to induce both cellular and humoral immunity. With their excellent tolerability, long shelf-life, low cost and easy manufacture, they offer great potential for use in a broad range of prophylactic and therapeutic vaccines. Based on successful animal studies in a broad range of species, human trials are about to get underway to validate the use of inulin-based adjuvants in prophylactic vaccines against hepatitis B, malaria and other pathogens. If such trials are successful, then it is possible that inulin-derived adjuvants will one day replace alum as the adjuvant of choice in most human prophylactic vaccines.     

A review of the effectiveness of vaccine potency control testing

The use of potency control testing is a valuable tool for testing the actual relative strength of manufactured assembly lots of vaccine. Biological-based manufacturing methods are inherently variable and potency testing is a tool to ensure lot-to-lot consistency of commercial vaccines. A strong historical link to clinical efficacy has been established where correlation to efficacy and adequate test validation have been achieved. The link to immunogenicity and efficacy has traditionally been strongest with attenuated vaccines and toxoids. Control potency test failure does predict that a serial or batch of vaccine would most likely provide insufficient immunogenicity in typical field applications. Because of the complexity of pathogenic processes and associated immune responses, potency tests may not always directly predict the effectiveness of a vaccine. Thus, vaccines that pass control potency testing may not always provide adequate efficacy. This is particularly true of adjuvanted, inactivated vaccines. In the development of vaccine formulations and control tests for vaccines, the nature of the desired protective immune responses to the targeted pathogen (when known) should be considered. These considerations could provide better alternatives in the assays chosen as correlates of immunity and may more accurately predict efficacy and assure batch-to-batch consistency. Also, the effects of the dose and duration of antigen exposure as well as the nature of antigen presentation and generation of extrinsic cytokines could be characterised and correlated to vaccine potency as additional indicators of vaccine efficacy.     

The “A, B and C” of Her-2 DNA vaccine development

INTRODUCTION: The development of Her-2 DNA vaccine has progressed through three phases that can be categorized as phase "A": the pursuit of Her-2 as a tumor-associated "antigen", phase "B": tilting the "balance" between tumor immunity and autoimmunity and phase "C": the on-going "clinical trials". MATERIALS AND METHODS: In phase "A", a panel of human ErbB-2 or Her-2 plasmids were constructed to encode non-transforming Her-2 derivatives. The immunogenicity and anti-tumor activity of Her-2 DNA vaccines were tested in human Her-2 transgenic mice with or without the depletion of regulatory T cells (Tregs). However, Treg depletion or other immune modulating regimens may increase the risk of autoimmunity. In phase "B", the balance between tumor immunity and autoimmunity was assessed by monitoring the development of experimental autoimmune thyroiditis (EAT). To test the efficacy of Her-2 DNA vaccines in cancer patients, clinical trials have been initiated in phase "C". RESULTS AND CONCLUSIONS: Significant anti-Her-2 and anti-tumor activity was observed when Her-2 transgenic mice were electro-vaccinated after Treg depletion. Susceptibility to EAT was also enhanced by Treg depletion and there was mutual amplification between Her-2 immunity and EAT development. Although Tregs regulate both EAT and Her-2 immunity, their effector mechanisms may differ. It may be possible to amplify tumor immunity with improved strategies that can by-pass undue autoimmunity. Critical information will be revealed in the next decade to expedite the development of cancer vaccines.     

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.     

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.     

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.     

Differential segregation of protective immunity by encoded antigen in DNA vaccine against pseudorabies virus

A murine model immunized with plasmid DNA vaccine expressing three glycoproteins pCIgB, pCIgC and pCIgD were used to examine the relative potency of major glycoproteins as well as the contribution of immunological parameters in providing protective immunity against the pseudorabies virus (PrV). Among the three glycoprotein-encoded plasmid DNA vaccines, pCIgB produced the strongest response of PrV-specific IgG in the sera. pCIgB and pCIgD also induced a contrast pattern of immunity that was biased to the Th1 and Th2 types, respectively. pCIgC showed the potent inducer of CD8+ T-cell-mediated CTL activity against PrV. In addition, a cocktail vaccination of all three glycoprotein-encoded plasmid DNA vaccines induced the production of both cytokine types, Th1 and Th2 with levels that were the same as that of each immunogen. With regard to protective efficacy, pCIgB induced the most effective protection against a virulent virus challenge and a cocktail vaccination appeared to offer complete protection against a 5 LD50 challenge, but not a 10 LD50 one. pCIgD induced protection that was same as pCIgB, but pCIgC offered no effective protection. These results show the relative potency of the three glycoprotein-encoded PrV DNA vaccines in inducing protective immunity against PrV infection. The results in this study support previous results showing the importance of Th1-type CD4+ T cells and their antibodies in conferring protection.     

Optimizing the efficacy of epitope-directed DNA vaccination

An increasing number of clinical trials has been initiated to test the potential of prophylactic or curative vaccination with tumor Ag-encoding DNA vaccines. However, in the past years it has become apparent that for many Ags and in particular for tumor Ags the intracellular processing and presentation are suboptimal. To improve epitope-directed DNA vaccines we have developed a murine model system in which epitope-specific, DNA vaccine-induced T cell immunity can be followed by MHC tetramer technology directly ex vivo. We have used this well-defined model to dissect the parameters that are crucial for the induction of strong cytotoxic T cell immunity using two independent model Ags. These experiments have led to a set of five guidelines for the design of epitope-directed DNA vaccines, indicating that carboxyl-terminal fusion of the epitope to a carrier protein of foreign origin is the most favorable strategy. DNA vaccines that are based on these guidelines induce high-magnitude CD8(+) T cell responses in >95% of vaccinated animals. Moreover, T cell immunity induced by this type of optimized DNA vaccine provides long-term protection against otherwise lethal tumor challenges.     

Live recombinant vectors for AIDS vaccine development

Live recombinant vectors entered the AIDS vaccine field with the realization that live attenuated HIV vaccines posed too great a safety risk, and that subunit vaccines elicited antibodies which lacked the breadth or potency needed to induce sterilizing immunity. Vectored vaccines provided a means to bring the cellular arm of the immune system into play by mimicking natural viral infection. By delivering antigens within host cells, processing and presentation could occur for induction of cellular immune responses. This recombinant vector approach, either alone or combined with other strategies, has produced impressive results. Recombinants have been generated from DNA and RNA viruses and bacteria. With few exceptions, each vector poses some risk, yet each possesses unique features that make it attractive. In addition to safety, key considerations in vector selection have included previous success as a vaccine against the wild-type agent or other pathogens; ability to induce potent, persistent immune responses; ability to target mucosal inductive sites and antigen presenting cells; lack of integration into the host genome; presence of pre-existing immunity in people; ease of mucosal administration; cloning capacity; ease of engineering and production; and stability of the final product. Here we up-date the status of several live recombinant vectors that have shown good potential in pre-clinical studies. Some have progressed to human clinical trials, and others will shortly. The abundance of vectors, coupled with the complexity arising from use of combination regimens with other vaccine types and heterologous vectors, will necessitate selection of the most promising candidates for large-scale efficacy trials in people. The sooner comparative studies can be designed and implemented in which live recombinant vectors containing the same inserted genes are evaluated head-to-head, the closer we will be to an eventual vaccine.     

Recent advances in mRNA-based vaccine for cancer therapy; bench to bedside

The messenger RNA (mRNA) vaccines have progressed from a theoretical concept to a clinical reality over the last few decades. Compared to conventional vaccination methods, these vaccines have a number of benefits, such as substantial potency, rapid growth, inexpensive production, and safe administration. Nevertheless, their usefulness was restricted up to now due to worries about the erratic and ineffective circulation of mRNA in vivo. Thankfully, these worries have largely been allayed by recent technological developments, which have led to the creation of multiple mRNA vaccination platforms for cancer and viral infections. The mRNA vaccines have been demonstrated as a powerful alternative to traditional conventional vaccines because of their high potency, safety and efficacy, capacity for rapid clinical development, and potential for rapid, low-cost manufacturing. The paper will examine the present status of mRNA vaccine technology and suggest future paths for the advancement and application of this exciting vaccine platform as a common therapeutic choice.     

Heat shock protein 70 / MAGE-1 tumor vaccine can enhance the potency of MAGE-1–specific cellular immune responses in vivo

The cancer-testis antigen encoded by the MAGE-1 gene is an attractive antigen in tumor immunotherapy because it can be processed as a foreign antigen by the immune system and generate tumor-specific cellular immune response in vivo. However, increase of the potency of MAGE-1 DNA vaccines is still needed. The high degree of sequence homology and intrinsic immunogenicity of heat shock protein 70 (HSP70) have prompted the suggestion that HSP70 might have immunotherapeutic potential, as HSP70 purified from malignant and virally infected cells can transfer and deliver antigenic peptides to antigen-presenting cells to elicit peptide-specific immunity. In this research, we evaluated the enhancement of linkage of Mycobacterium tuberculosis HSP70 to MAGE-1 gene of the potency of antigen-specific immunity elicited by naked DNA vaccines. We found that vaccines containing MAGE-1-HSP70 fusion genes enhanced the frequency of MAGE-1–specific cytotoxic T cells in contract to vaccines containing the MAGE-1 gene alone. More importantly, the fusion converted a less effective DNA vaccine into one with significant potency against established MAGE-1–expressing tumors. These results indicate that linkage of HSP70 to MAGE-1 gene may greatly enhance the potency of DNA vaccines, and generate specific antitumor immunity against MAGE-1–expressing tumors.     

Overcoming immunity to a viral vaccine by DNA priming before vector boosting

Replication-defective adenovirus (ADV) and poxvirus vectors have shown potential as vaccines for pathogens such as Ebola or human immunodeficiency virus in nonhuman primates, but prior immunity to the viral vector in humans may limit their clinical efficacy. To overcome this limitation, the effect of prior viral exposure on immune responses to Ebola virus glycoprotein (GP), shown previously to protect against lethal hemorrhagic fever in animals, was studied. Prior exposure to ADV substantially reduced the cellular and humoral immune responses to GP expressed by ADV, while exposure to vaccinia inhibited vaccine-induced cellular but not humoral responses to GP expressed by vaccinia. This inhibition was largely overcome by priming with a DNA expression vector before boosting with the viral vector. Though heterologous viral vectors for priming and boosting can also overcome this effect, the paucity of such clinical viral vectors may limit their use. In summary, it is possible to counteract prior viral immunity by priming with a nonviral, DNA vaccine.     

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.     

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.     

Preclinical and clinical progress of particle-mediated DNA vaccines for infectious diseases

This review provides an overview of studies employing particle-mediated epidermal delivery (PMED) or the gene gun to administer DNA vaccines for infectious diseases in preclinical studies employing large animal models and in human clinical trials. It reviews the immunogenicity and protective efficacy of PMED DNA vaccines in nonhuman primates and swine and studies that have directly compared the effectiveness of PMED in these large animal models to existing licensed vaccines and intramuscular or intradermal delivery of DNA vaccines with a needle. Various clinical trials employing PMED have been completed and an overview of the immunogenicity, safety, and tolerability of this approach in humans is described. Finally, efforts currently in progress for commercial development of particle-mediated DNA vaccines are discussed.     

Enhanced potency of plasmid DNA microparticle human immunodeficiency virus vaccines in rhesus macaques by using a priming-boosting regimen with recombinant proteins

DNA vaccines have been used widely in experimental primate models of human immunodeficiency virus (HIV), but their effectiveness has been limited. In this study, we evaluated three technologies for increasing the potency of DNA vaccines in rhesus macaques. These included DNA encoding Sindbis virus RNA replicons (pSINCP), cationic poly(lactide-co-glycolide) (PLG) microparticles for DNA delivery, and recombinant protein boosting. The DNA-based pSINCP replicon vaccines encoding HIV Gag and Env were approximately equal in potency to human cytomegalovirus (CMV) promoter-driven conventional DNA vaccines (pCMV). The PLG microparticle DNA delivery system was particularly effective at enhancing antibody responses induced by both pCMV and pSINCP vaccines and had less effect on T cells. Recombinant Gag and Env protein boosting elicited rapid and strong recall responses, in some cases to levels exceeding those seen after DNA or DNA/PLG priming. Of note, Env protein boosting induced serum-neutralizing antibodies and increased frequencies of gamma interferon-producing CD4 T cells severalfold. Thus, PLG microparticles are an effective means of delivering DNA vaccines in nonhuman primates, as demonstrated for two different types of DNA vaccines encoding two different antigens, and are compatible for use with DNA prime-protein boost regimens.