Polynucleotide Vaccines Research Articles

DNA Vaccine Encoding HPV16 Oncogenes E6 and E7 Induces Potent Cell-mediated and Humoral Immunity Which Protects in Tumor Challenge and Drives E7-expressing Skin Graft Rejection.

We have previously shown that a novel DNA vaccine technology of codon optimization and the addition of ubiquitin sequences enhanced immunogenicity of a herpes simplex virus 2 polynucleotide vaccine in mice, and induced cell-mediated immunity when administered in humans at relatively low doses of naked DNA. We here show that a new polynucleotide vaccine using the same technology and encoding a fusion protein of the E6 and E7 oncogenes of high-risk human papillomavirus type 16 (HPV16) is immunogenic in mice. This vaccine induces long-lasting humoral and cell-mediated immunity and protects mice from establishment of HPV16-E7-expressing tumors. In addition, it suppresses growth of readily established tumors and shows enhanced efficacy when combined with immune checkpoint blockade targeted at PD-L1. This vaccine also facilitates rejection of HPV16-E7-expressing skin grafts that demonstrate epidermal hyperplasia with characteristics of cervical and vulvar intraepithelial neoplasia. Clinical studies evaluating the efficacy of this vaccine in patients with HPV16 premalignancies are planned.

An escalating dose study to assess the safety, tolerability and immunogenicity of a Herpes Simplex Virus DNA vaccine, COR-1

ABSTRACTThis paper describes a single site, open-label Phase I clinical trial evaluating the safety, tolerability and immunogenicity in healthy volunteers of a herpes simplex polynucleotide vaccine that has previously been shown to enhance immunogenicity and protect against lethal herpes simplex virus type 2 (HSV-2) challenge in mice. Five escalating doses of the vaccine, COR-1, were given by intradermal injection to HSV-1 and 2 seronegative healthy individuals. COR-1 was found to be safe and well-tolerated; the only vaccine-related adverse events were mild. While vaccine-induced antibody responses were not detectable, cell-mediated immune responses to HSV-specific peptide groups were identified in 19 of the 20 subjects who completed the study, and local inflammation at the immunisation site was observed. This study indicates COR-1 has potential to be used as a therapeutic vaccine for HSV-2 infection.

Incomplete tumour control following DNA vaccination against rat gliomas expressing a model antigen

BackgroundVaccination against tumour-associated antigens is one approach to elicit anti-tumour responses. We investigated the effect of polynucleotide (DNA) vaccination using a model antigen (E. coli lacZ) in a syngeneic gliosarcoma model (9L).MethodsFisher 344 rats were vaccinated thrice by intramuscular injection of a lacZ-encoding or a control plasmid in weekly intervals. One week after the last vaccination, lacZ-expressing 9L cells were implanted into the striatum.ResultsAfter 3 weeks, in lacZ-vaccinated animals the tumours were significantly smaller than in control-vaccinated animals. In cytotoxic T cell assays lysis rates of >50 % could only be observed in a few of the lacZ-vaccinated animals. This response was directed against lacZ-expressing and parental 9L cells but not against syngeneic MADB 106 adenocarcinoma cells. In Elispot assays interferon-γ production was observed upon stimulation with 9LlacZ and 9L wild-type but not MADB 106 cells. This response was higher for lacZ-immunized animals. All animals revealed dense infiltrates with CD8+ lymphocytes and, to a lesser extent, with NK cells. CD25-staining indicated cells possibly associated with the maintenance of peripheral tolerance to self-antigens. All tumours were densely infiltrated by microglia consisting mostly of ramified cells. Only focal accumulation of macrophage-like cells expressing ED1, a marker for phagocytic activity, was observed.ConclusionProphylactic DNA vaccination resulted in effective but incomplete suppression of brain tumour formation. Mechanisms other than cytotoxic T cell responses as measured in the generally used in vitro assays appear to play a role in tumour suppression.

Antirabies DNA Immunization

Cited are literary data related to the development of DNA vaccines against rabies virus. Research results regarding gene vaccination of different models of laboratory animals and different ways of vaccine introduction are presented. Possibility to potentiate immunogenicity of DNA vaccines using adjuvants and cytokines is considered. Ways of improving of polynucleotide vaccines are discussed.

Translational Research on Vaccines: Influenza as an Example

Influenza vaccine development is reviewed as an example of ongoing translational research, which is moving fundamental advances in biology into useful products. The types of new influenza vaccines span the gamut of modern biology research and include new methods of vaccine production (tissue culture compared with egg-produced vaccine). New vaccines being studied include recombinant proteins, polynucleotide vaccines, conjugate vaccines, peptides, expression vectors, and live attenuated vaccines; new adjuvants are being explored to reduce the quantity of antigen needed. The benefits of using reverse genetics are explored. The limits of translational research for predicting clinical results after wide use of vaccines are discussed, including one example of "overinterpretation" of data; some questions should be left to phase IV experience to answer. The pipeline for new influenza vaccines is robust, and recent investment by government and industry in newer vaccines will bring several new products to clinical use.

DNA vaccines for cancer

DNA vaccines, also referred to as genetic, plasmid or polynucleotide vaccines, represent a relatively simple and economical method to exploit gene transfer for immunization against tumor associated antigens. This review discusses the potential advantages of DNA vaccines for cancer immunotherapy as compared to conventional protein vaccines and viral vectors. The proposed mechanisms responsible for induction of immune responses following DNA-based immunization are summarized. The preclinical development of DNA vaccines and the clinical experience with DNA immunization for cancer to date are reviewed. The low toxicity associated with DNA vaccines favors its further development, but additional strategies to improve the potency of this approach are needed if it is to be successfully integrated into the clinical setting.

Codon Modified Human Papillomavirus Type 16 E7 DNA Vaccine Enhances Cytotoxic T-Lymphocyte Induction and Anti-tumour Activity

Polynucleotide immunisation with the E7 gene of human papillomavirus (HPV) type 16 induces only moderate levels of immune response, which may in part be due to limitation in E7 gene expression influenced by biased HPV codon usage. Here we compare for expression and immunogenicity polynucleotide expression plasmids encoding wild-type (pWE7) or synthetic codon optimised (pHE7) HPV16 E7 DNA. Cos-1 cells transfected with pHE7 expressed higher levels of E7 protein than similar cells transfected with pW7. C57BL/6 mice and F1 (C57× FVB) E7 transgenic mice immunised intradermally with E7 plasmids produced high levels of anti-E7 antibody. pHE7 induced a significantly stronger E7-specific cytotoxic T-lymphocyte response than pWE7 and 100% tumour protection in C57BL/6 mice, but neither vaccine induced CTL in partially E7 tolerant K14E7 transgenic mice. The data indicate that immunogenicity of an E7 polynucleotide vaccine can be enhanced by codon modification. However, this may be insufficient for priming E7 responses in animals with split tolerance to E7 as a consequence of expression of E7 in somatic cells.

Polynucleotide viral vaccines: codon optimisation and ubiquitin conjugation enhances prophylactic and therapeutic efficacy

Papillomavirus infection is a major antecedent of anogenital malignancy. We have previously established that the L1 and L2 capsid genes of papillomavirus have suboptimal codon usage for expression in mammalian cells. We now show that the lack of immunogenicity of polynucleotide vaccines based on the L1 gene can be overcome with codon modified L1, which induces strong immune responses, including conformational virus neutralising antibody and delayed type hypersensitivity. Conjugation of a ubiquitin gene to a hybrid gene incorporating L1 and the E7 non-structural papillomavirus protein improved E7 specific CTL responses, and induced protection against an E7 expressing tumour, but induced little neutralising antibody. However, a mixture of ubiquitin conjugated and non-ubiquitin conjugated polynucleotides induced virus neutralising antibody and E7 specific CD8 T cells. An optimal combined prophylactic/therapeutic viral vaccine might therefore comprise ubiquitin conjugated and non-ubiquitinated genes, to induce prophylactic neutralising antibody and therapeutic cell mediated immune responses.

Immunization of livestock with DNA vaccines: current studies and future prospects

Early studies using DNA immunization suggest the potential benefits of this form of immunization including: long-lived immunity, a broad spectrum of immune responses (both cell-mediated immunity and humoral responses) and the simultaneous induction of immunity to a variety of pathogens through the use of multivalent vaccines. Using marine and cow models, we studied methods to enhance and direct the immune response to polynucleotide vaccines. We demonstrated the ability to modulate the magnitude and direction of the immune response by co-administration of plasmid encoded cytokines and antigen. Also, we clearly demonstrated that the cellular components (cytosolic, membrane-anchored, or extracellular) to which the expressed antigen is delivered determines the types of immune responses induced. Since induction of immunity at mucosal surfaces (route of entry for many pathogens) is critical to prevent infection, various methods of delivering polynucleotide vaccines to animals including mucosal surfaces have been attempted and are described as future prospects for improving immune responses by DNA vaccination.

Separation and purification of plasmid mixtures by continuous elution electrophoresis.

Separation and purification of plasmid mixtures by continuous elution electrophoresis.

Intra-pinna anti-tumor vaccination with self-replicating infectious RNA or with DNA encoding a model tumor antigen and a cytokine.

To optimize polynucleotide vaccinations for protective antitumor immunity we used a self-replicating RNA vaccine in which Semliki Forest virus replicase drives RNA expression of the lacZ gene coding for beta-galactosidase as model tumor-associated antigen (TAA). This was compared with replicase-deficient control RNA and with lacZ DNA plasmids with respect to gene expression in vitro and in vivo and for vaccination using the mouse ear pinna as an optimal immunization site. In vitro, the highest expression was observed with self-replicating RNA. Gene expression following pinna inoculation of either non-replicating DNA plasmids or self-replicating RNA was similar, lasting for 2-3 weeks. Higher antibody responses were obtained with RNA than with DNA. beta-Gal peptide specific CTL memory responses to lacZ DNA or RNA lasted for more than 6 weeks while respective responses induced by lacZ-transfected tumor cells lasted for only 2 weeks. To achieve a protective response against lacZ tumor cells with self-replicating RNA about a 100-fold lower dose of polynucleotide was sufficient in comparison to DNA. The extent of protective antitumor immunity not only depended on the gene dose used for vaccination, but also on the aggressiveness of the lacZ-transfected tumor line used for challenge. In comparison to lacZ-transfected tumor cells as vaccines, polynucleotide vaccination also demonstrated superiority with regard to cross-protection. Protective antitumor immunity could be strongly increased upon co-inoculation of lacZ DNA with IL-2 DNA or IL-12 RNA. IL-2 DNA, but not IL-12 RNA, also augmented the CTL response while IL-12 RNA, but not IL-2 DNA, reduced the antibody response. These results demonstrate efficient protective antitumor immunity after intra-pinna lacZ TAA polynucleotide vaccination and show additional immunomodulatory effects by co-administration of cytokine polynucleotides.

Development of a polynucleotide vaccine from melanoma antigen recognized by T cells-1 and recombinant protein from melanoma antigen recognized by T cells-1 for melanoma vaccine clinical trials.

MART-1, a melanoma antigen recognized by T cells-1, is a melanocyte lineage-differentiation antigen expressed only in melanocytes and melanoma cells. This protein is recognized by many T-lymphocyte lines that are human leukocyte antigen (HLA)-A2 restricted and melanoma reactive. These observations have culminated in an array of clinical trials of MART-1 immunization using recombinant viruses or MART-1 immunodominant peptides. Polynucleotide immunization is a promising alternative to recombinant viral vaccines that allows delivery of the full-length cDNA encoding all potential peptide epitopes in a vector that is uncompromised by anti-viral immunity. In preparation for a phase I clinical trial of MART-1 polynucleotide immunization in patients with resected melanoma who were at significant risk for recurrence, the authors constructed a plasmid DNA encoding the MART-1 cDNA under transcriptional regulatory control of the cytomegalovirus immediate early promoter-enhancer and partially deleted intron A. This plasmid directs high-level MART-1 expression in transduced myoblasts and maturing myocytes diffusely throughout the cytoplasm. Immunization of mice with this construct by intramuscular injection elicited MART-1-specific immune responses in all animals. Previous trials of MART-1 immunization have been unable to examine the humoral immune response to MART-1 because of a lack of sufficient, highly purified protein. We have produced and purified Escherichia coli recombinant MART-1 protein using a glutathione-S-transferase fusion protein expression system. Protein staining of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a band of MART-1 protein at approximately 20 kD; and Western immunoblotting with an anti-MART-1 monoclonal antibody confirmed a doublet at approximately 20 kD. These findings are consistent with previous reports using different expression systems for recombinant MART-1. This protein preparation functioned well in enzyme-linked immunosorbent assays (ELISAs) to detect anti-MART-1 antibody responses in a mouse model; and a panel of healthy donor human sera showed minimal binding to ELISA plates coated with the protein, supporting its utility in monitoring human anti-MART-1 antibody responses. The glutathione-S-transferase fusion method yielded approximately 200 micrograms MART-1 per 2-L bacterial culture, enough to coat 100 ELISA plates.

Cutaneous vaccination: the skin as an immunologically active tissue and the challenge of antigen delivery

Vaccination is one of the major achievements of modern medicine. As a result of vaccination, diseases such as polio and measles have been controlled and small pox has been eradicated. However, despite these successes there are still many microbial diseases that cause tremendous suffering because there is no vaccine or the vaccines available are inadequate. In addition, even if vaccines were available for all infectious diseases there is no guarantee that people would use them routinely. One of the major impediments to ensuring vaccine efficacy and compliance is that of delivery. Presently most vaccines are given by intramuscular administration. Unfortunately this is often traumatic, especially in infants. Thus, if it was possible to replace intramuscular immunization by mucosal (oral/intranasal) or transdermal delivery it may be possible to both enhance mucosal immunity as well as improve overall compliance rates. The transdermal route has been used by the pharmaceutical industry for the delivery of various low molecular weight drugs. Some of the approaches used for smaller compounds may also have potential for delivery of either protein or polynucleotide vaccines. However, there is a greater challenge to delivering large molecular weight molecules through the skin due to size, charge and other physicochemical properties. This review will describe the recent advances that have been made in dermal and topical delivery as related to vaccines.

Immunization of animals: from DNA to the dinner plate

Recently, there has been a great deal of interest in polynucleotide vaccination also referred to as DNA vaccines or genetic immunization for inducing long-term immunity in various animals and humans. The main attraction of this technology is the possibility to induce a broad range of immune responses without the use of conventional adjuvants. To date, most of the studies (>500 reports) have focused on DNA vaccination in mice. The present report summarizes the limited number of trials that have used target animal species to not only test the immune responses but also correlate them to protection.

Polynucleotide vaccines: potential for inducing immunity in animals

Polynucleotide immunization has been described as the Third Revolution in Vaccinology. Early studies suggest the potential benefits of this form of immunization including: long-lived immunity, a broad-spectrum of immune responses (both cell mediated immunity, and humoral responses) and the simultaneous induction of immunity to a variety of pathogens through the use of multivalent vaccines. Using a murine model, we studied methods to enhance and direct the immune response to polynucleotide vaccines. We demonstrated the ability to modulate the magnitude and direction of the immune response by co-administration of plasmid encoded cytokines and antigen. Also, we clearly demonstrated that the cellular components (cytosolic, membrane-anchored, or extracellular) to which the expressed antigen is delivered determines the types of immune responses induced. Since induction of immunity at mucosal surfaces (route of entry for many pathogens) is critical to prevent infection, various methods of delivering polynucleotide vaccines to mucosal surfaces have been attempted and are described. Expansion of studies in various species, using natural models, should be extremely helpful in demonstrating the universality of this approach to immunization and more importantly, accurately identify parameters that are critical for the development of protective immunity.

Developing dendritic cell polynucleotide vaccination for prostate cancer immunotherapy

Immunotherapy has been successfully used to treat some human malignancies, principally melanoma and renal cell carcinoma. Genetic-based cancer immunotherapies were proposed which prime T lymphocyte recognition of unique neo-antigens arising from specific mutations. Genetic immunization (polynucleotide vaccination, DNA vaccines) is a process whereby gene therapy methods are used to create vaccines and immunotherapies. Recent findings indicate that genetic immunization works indirectly via a bone marrow derived cell, probably a type of dendritic antigen presenting cell (APC). Direct targeting of genetic vaccines to these cells may provide an efficient method for stimulating cellular and humoral immune responses to infectious agents and tumor antigens. Initial studies have provided monocytic-derived dendritic cell (DC) isolation and culture techniques, simple methods for delivering genes into these cells, and have also uncovered potential obstacles to effective cancer immunotherapy which may restrict the utility of this paradigm to a subset of patients.

Vaccines for Ovarian Carcinoma.

BACKGROUND: Metastasis to the abdominal cavity is the primary cause of morbidity and mortality in patients with ovarian cancer. Beyond surgery and chemotherapy combinations, strategies that target tumor cells in vivo are being investigated, such as the use of recombinant cytokines to up-regulate or modulate the cell-mediated or humoral immune response. METHODS: The authors report on their experience with tumor vaccines, including first-generation vaccines, peptide vaccines, and polynucleotide vaccines, in the treatment of ovarian cancer. RESULTS: Cytokines may stimulate proliferation or activation of effector cells that mediate either major histocompatibility restricted cytotoxicity (adaptive immunity) or natural (innate) immunity. Cytokines are often pleiotropic, and their effects may depend on concentration, scheduling, and responsiveness of the cell populations to which they are directed. They also have been used to enhance the efficacy of tumor vaccines that have reached a higher level of sophistication. Recently designed tumor vaccines are capable of stimulating antitumor immune responses that recognize tumor cell epitopes or that have the potential to act synergistically with cytokines such as interleukin-2 and interleukin-12. CONCLUSIONS: Enthusiasm for antitumor vaccine strategies is supported by accumulating clinical reports of responses following treatments using a variety of vaccines. Additional research is needed to determine optimum vaccine approaches for the treatment or prevention of ovarian cancer.

Marked enhancement of direct respiratory tissue transfection by aurintricarboxylic acid.

Simple, nontoxic, and pharmaceutically defined methods for genetic modification of respiratory tissues may enable development of a variety of molecular medicines. Clinical applications for such medicines include treatment of inborn errors of metabolism, interventions for asthma and iatrogenic pulmonary fibrosis, and disease prophylaxis via mucosal polynucleotide vaccination. "Free," "direct," or "naked" plasmid administration is a simple, apparently safe, and pharmaceutically defined gene delivery method. Murine, macaque, and clinical human studies have demonstrated transfection of respiratory tissues after direct application of free plasmid. The aim of this study was to develop a simple and safe alternative to respiratory tissue transduction, and specifically to provide a theoretical framework for developing a category of adjuvants, nuclease inhibitors, that augment the transfection activity of free plasmid. Plasmid employing the human CMV IE promoter/enhancer to drive expression of the Photinus pyralis luciferase reporter protein was administered intratracheally into mouse lung with or without the nuclease inhibitor aurintricarboxylic acid (ATA). Lavage samples and tissue extracts were used to demonstrate inhibition of lung nuclease activity. ATA dose escalation studies were performed using lung homogenate assays to characterize transfection. Potential toxicity was assessed histologically. The data indicate that nucleases present in respiratory fluids accelerate clearance of biologically active plasmid from lung, that intratracheal coadministration of ATA together with plasmid reduces extracellular DNA clearance, and that this treatment results in marked enhancement of reporter protein expression. The effective dose for ATA enhancement of direct lung transfection was 0.5 microg/g mouse weight, and the LD50 was approximately 6 microg/g. These findings provide a theoretical and practical foundation for further development of an alternative gene delivery system: free plasmid-based respiratory transfection technology.

Targeting the skin for genetic immunization.

One of the most promising applications of recent advances in gene therapy is the development of immunization strategies based on the delivery of antigen-encoding DNA. DNA-based vaccination, also referred to as genetic vaccination or polynucleotide vaccination, offers considerable promise for improvement over existing immunization strategies, and the skin offers unique potential as a target tissue for genetic vaccines. The expression of genetically introduced antigens in a cutaneous microenvironment rich in both professional antigen-presenting cells and accessory cells, which are capable of producing immunostimulatory cytokines, has the potential to overcome the historical limitations of vaccinology and immunotherapy. Though the precise molecular mechanisms of genetic immunization remain unclear, a general working model of the events through which antigen-encoding plasmids introduced into the skin initiate an immune response can be constructed. The finding that Langerhans cells can be transfected in vivo raises the exciting possibility that these migrating professional antigen-presenting cells can be genetically engineered in vivo. By designing strategies to codeliver genes encoding antigens with genes encoding immunoregulatory molecules to the same antigen-presenting cell, it may be possible to either induce or suppress antigen-specific immune responses in the host. Though many aspects of the biology of cutaneous DNA immunization remain unknown, the skin appears to offer unique potential for the application of advances in gene therapy to vaccination and genetic engineering of the immune response.

Broadening the approaches to developing more effective vaccines

Although vaccination has had a dramatic impact on reducing economic losses due to infectious diseases, vaccination technology has not changed dramatically over the last 200 years. However, with the advent of biotechnology and our understanding of virulence factors of infectious agents combined with our knowledge of the host immune response, we are now witnessing a revolution in the number of new agents which may potentially be controlled by vaccination, as well as the approaches being used to develop vaccines. These approaches include subunit vaccines, genetically modified live vaccines and most recently, polynucleotide vaccines. Pathogens involved in bovine respiratory disease are used as models to describe recent advances in developing new vaccines that have the potential to be safer, more economical and more efficacious. Emphasis will be placed on identification of specific proteins involved in inducing protective immunity and producing these in a mammalian expression system as subunit vaccines formulated with adjuvants. To increase the duration of immunity, the genes encoding these antigens have been introduced directly into animals as polynucleotide vaccines. The benefits and short-comings, as well as the practical problems associated (both scientific and regulatory) with eventual acceptance of these vaccines, are discussed.