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Vaccines: DNA

  1. Jeffrey B Ulmer1,
  2. John J Donnelly2

Published Online: 21 DEC 2007

DOI: 10.1002/9780470015902.a0000492.pub2

eLS

eLS

How to Cite

Ulmer, J. B. and Donnelly, J. J. 2007. Vaccines: DNA. eLS. .

Author Information

  1. 1

    Novartis Vaccines and Diagnostics, Inc., Emeryville, California, USA

  2. 2

    Novartis Vaccines and Diagnostics Srl, Siena, Italy

Publication History

  1. Published Online: 21 DEC 2007

Design of Plasmids

  1. Top of page
  2. Design of Plasmids
  3. DNA Vaccine Delivery
  4. The Immune Response Induced by DNA Vaccines (CD8 T Cells, CD4 T Cells, B Cells)
  5. How to Secrete Gene Products to Improve Stimulation of B Cells and CD4+ T Cells
  6. Coexpression of Cytokines and Costimulatory Molecules to Improve Immunogenicity
  7. Immunomodulatory DNA Motifs
  8. Results from Animal Experiments (Infections, Tumours, etc.)
  9. Tumours
  10. Clinical Trials of DNA Vaccines
  11. Safety Considerations (e.g. Chromosome Integration)
  12. Prospects for DNA Vaccines
  13. References
  14. Further Reading

DNA vaccines, in their simplest form, consist of Escherichia coli-derived plasmids containing the following elements: (1) a strong eukaryotic promoter, such as the immediate early promoter from cytomegalovirus, (2) a transcription terminator, such as that from bovine growth hormone, (3) a gene of interest encoding an antigenic target expressed by a pathogen, (4) an antibiotic resistance gene to facilitate selection of transformed bacteria carrying the plasmid and (5) a bacterial origin of replication to allow for expression of the plasmid in E. coli. This combination yields a eukaryotic expression vector that results in the production of the antigen of interest in cells of the vaccinated animal (Wolff et al., 1990). See also Bacterial Plasmids, and Plasmids

A reasonable assumption on the relative potency of deoxyribonucleic acid (DNA) vaccines is that more antigen produced in situ will lead to higher levels of immune responses. Hence, DNA vaccine vectors have been designed to yield high levels of antigen expression. Where tissue-specific expression is desired, nonviral promoters may be useful. As examples, the use of the albumin promoter targeted expression of DNA vaccines in hepatocytes and use of the immunoglobulin promoter and enhancer elements resulted in preferential expression in bursa-derived lymphocytes (B cells). Little work has been reported on inducible promoters for DNA vaccines, such as those responsive to tetracycline or RU486, but these may prove to be very important, particularly for regulated expression of immunologically active molecules such as cytokines (see later). All of the aforementioned promoters require that the expression vector be transported into the nucleus for transcription to take place. This requirement may be obviated by use of a bacteriophage T7 promoter system, where expression of the T7 ribonucleic acid (RNA) polymerase can drive expression of antigen controlled by the T7 promoter without the need for host cell transcription machinery. This approach may also be useful for expressing proteins that require the involvement of other gene products (e.g. rev-dependent expression of human immunodeficiency virus (HIV) env). See also Bacteriophages: Tailed

DNA Vaccine Delivery

  1. Top of page
  2. Design of Plasmids
  3. DNA Vaccine Delivery
  4. The Immune Response Induced by DNA Vaccines (CD8 T Cells, CD4 T Cells, B Cells)
  5. How to Secrete Gene Products to Improve Stimulation of B Cells and CD4+ T Cells
  6. Coexpression of Cytokines and Costimulatory Molecules to Improve Immunogenicity
  7. Immunomodulatory DNA Motifs
  8. Results from Animal Experiments (Infections, Tumours, etc.)
  9. Tumours
  10. Clinical Trials of DNA Vaccines
  11. Safety Considerations (e.g. Chromosome Integration)
  12. Prospects for DNA Vaccines
  13. References
  14. Further Reading

Route of inoculation

Administration of DNA by one of several different routes can result in the generation of immune responses (see Table 1). For example, syringe administration of DNA by intramuscular, intradermal, intravenous and intrasplenic injection; gene gun inoculation into the skin (Tang et al., 1992); and mucosal delivery via oral and intranasal routes have all resulted in immune responses and/or protection in animal models. This could be explained by the possibility that the expression of antigen by any type of somatic cell may suffice to induce immune responses by DNA vaccination, unlike DNA delivery for gene therapy where tissue-specific expression sometimes is desired. It may be that expression of antigen by any cell can provide a source of antigen for transfer to professional antigen-presenting cells (APCs). Alternatively, a ‘professional’ APC (e.g. dendritic cell) may become transfected regardless of route of immunization. See also Antigens, Antigen-presenting Cells, Human Gene Therapy, and Vaccines: Presentation

Table 1. Delivery of DNA vaccines by various routes
SiteMethodAntigenResult
  1. CSP, circumsporozoite protein; CTL, cytotoxic T lymphocyte; HBsAg, hepatitis B surface antigen; NALT, nasal-associated lymphoid tissue; PLGA, polylactide coglycolide; TH, T-helper.

IntramuscularNeedleVariousSerum antibodies, CTL, TH cells
 Jet injectorHBsAg, Plasmodium falciparum CSPSerum antibodies, CTL, TH cells
 ElectroporationVariousSerum antibodies, CTL, TH cells
SubcutaneousNeedleVariousNo response
IntradermalNeedleVariousSerum antibodies, CTL, TH cells
 Jet injectorP. falciparum CSPSerum antibodies
 Gene gunVariousAntibodies, CTL, TH cells
 TattooInfluenza NPCTL
OralPLGALuciferaseSerum IgG, IgA, secretory IgA antibodies
IntranasalCholera toxinInfluenza haemagglutininAntibody-forming cells in NALT
 LiposomesVariousSerum IgG, IgA antibodies, CTL
IntravaginalLiposomesLuciferaseSecretory IgA
 Phosphate-buffered salineHIV gp160Secretory IgA
 Gene gunInfluenza HASecretory IgA

Pathogens in general enter hosts via mucosal surfaces. While protective immunity can be conferred by systemic immune responses, even against mucosal pathogens such as influenza virus, it may be desirable in many cases to include a mucosal component to the host immune response. Oral vaccination can also be a successful strategy for inducing systemic immune responses. For these reasons, the potential of mucosal DNA vaccination has been investigated. Immune responses and protective efficacy have been reported after mucosal administration of DNA vaccines and the responses can be enhanced when the DNA is delivered with the mucosal adjuvant cholera toxin. Delivery of DNA via mucosal routes, particularly oral, poses some significant challenges with respect to uptake of DNA at inductive sites and to the harsh environment through which the DNA must be transported. In this regard, poly(dl-lactide-coglycolide) (PLG) microspheres and cationic lipid complexes have been used successfully for oral or intranasal delivery of DNA vaccines for the induction of systemic and mucosal immune responses. Therefore, the prospect for induction of broad-based immunity by mucosal administration of DNA vaccines appears promising. See also Immune Responses: Primary and Secondary, and Immune Responses at Mucosal Surfaces

DNA uptake

The processes involved in uptake of DNA by cells in the inoculated animal have not yet been elucidated. After intramuscular injection, uptake of DNA by muscle cells does not appear to be mediated by transient damage to the cells. Rather, a physiological mechanism such as endocytosis may be involved (see Figure 1). Once inside cells, DNA must be transported out of the endosome/lysosome into the cytoplasm to avoid digestion, then transported into the nucleus before transcription can occur. In contrast to intramuscular injection, the gene gun is a fundamentally different approach. This technology transfects cells using DNA-coated gold beads that are propelled directly into cells by virtue of the mass of the particle and the force of the propulsion. In this way, active uptake of DNA is not required. Both modes of inoculation are effective at inducing protective immune responses, but the gene gun requires less DNA than does an intramuscular injection, presumably due to the lability of extracellular DNA prior to cellular uptake after intramuscular injection. The rate-limiting step in this sequence of events is not known, since an in vitro model to facilitate the study of passive DNA uptake by and intracellular trafficking within cells has eluded development. Without a full understanding of the specific cells required and the processes involved in DNA uptake by those cells, rational approaches to DNA vaccine delivery are difficult to formulate. See also Transfection of DNA into Mammalian Cells in Culture

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Figure 1. Mechanism of action of DNA vaccines. After administration, cellular uptake of DNA vaccines possibly occurs by a phagocytic process (step 1). Within the phagosome, some plasmid DNA molecules are partially digested to release immunostimulatory oligonucleotides that stimulate the innate immune system via TLR9 (step 2). Other plasmid DNA molecules enter the cytoplasm and are internalized by the nucleus, resulting in transcription and production of antigen encoded by the DNA vaccine. When expressed by APCs, the antigens can then be processed and presented by MHC class I or II molecules directly to naïve T cells (step 3). Alternatively, the antigens released from DNA-transfected cells (e.g. by secretion or apoptosis) can interact with B cells to prime antigen-specific antibodies (step 4) or be internalized by bystander APCs such as DC for indirect presentation by MHC molecules (step 5). This concomitant production of antigen and innate immune activation can promote memory immune responses to provide protection against subsequent exposure to the pathogen from which the antigen was derived.

Improving the delivery of DNA

Cationic lipids are very effective at facilitating transfection of cells in vitro, and it was this property that led investigators to test cationic lipids as potential DNA delivery vehicles in vivo. In general, cationic lipids are formulated with a neutral colipid, such as dioleylphosphatidylethanolamine, facilitate not only cell entry but also escape from the endosome/lysosome and release of DNA from the lipid complex. The use of cationic lipids for DNA vaccine delivery has been limited, but intramuscular injection of a cationic lipid-complexed DNA vaccine has been shown to induce higher levels of humoral and cellular immune responses in mice than did naked DNA. Although the means by which cationic lipid:DNA complexes enhance immune responses is not understood, it could include increased transfection of cells in vivo leading to higher levels of antigen production, or an adjuvant effect of the complex itself.

Microparticulates include DNA adsorbed to or entrapped in biodegradable microparticles such as poly lactide-co-glycolide or chitosan, or complexed with nonionic block copolymers or polycations such as polyethyleneimine (O'Hagan et al., 2004). Among the classical adjuvants, aluminum phosphate is noteworthy for its effectiveness and simplicity of preparation. Microparticulates appear to improve delivery of DNA to APCs by facilitating trafficking to local lymphoid tissue via the afferent lymph and facilitating uptake by dendritic cells (DC) (Denis-Mize et al., 2003). Alum phosphate does not bind DNA, and in fact cationic alum formulations that do bind DNA generally are not immunogenic. Alum phosphate is thought to act by recruiting APCs to the site of the intramuscular injection, where a proportion of muscle cells would be expressing the antigen encoded by the DNA vaccine.

Tissue damage or irritation leading to regeneration of myocytes may be important in enhancing immune responses to DNA vaccines. Early studies suggested that agents that caused muscle necrosis, such as cardiotoxin or bupivicaine, increased immune responses to DNA vaccines administered while the muscle was regenerating. This was thought to be due to increased protein expression in regenerating myocytes, but recruitment of APCs by inflammatory responses also may play some role. Later, hydrostatic damage caused by injection of relatively larger volumes of fluid was implicated as a mechanism for the relatively high immunogenicity of plasmid DNA vaccines when given intramuscularly in mice compared with larger animals. The polymer and adjuvant formulations currently under evaluation also may work in part through a local inflammatory component. Most recently electroporation, which has the potential both to force DNA into cells and to create damage to adjacent muscle cells, has emerged as the most potent method for delivering DNA intramuscularly.

Bacterial and viral vectors

Several live bacterial and viral vectors have been used as vaccine vehicles. In the case of viral vectors, for example recombinant adenovirus, vaccinia virus and herpes virus, expression of antigen occurs in host cells in situ after infection. In contrast, bacterial vectors deliver either protein antigen, as a result of expression of antigen in the organism in situ (e.g. recombinant Mycobacterium bovis, Bacillus Calmette-Guérin (BCG) or Listeria monocytogenes), or plasmid DNA, as a consequence of release of plasmid intracellularly from the vector (e.g. recombinant Shigella). These vectors offer the potential advantages of more efficient DNA uptake and targeting of antigen expression. However, there are certain issues one must bear in mind when considering vectors consisting of live organisms rather than plasmid DNA, including: (1) increased vaccine complexity, (2) additional potential safety concerns and (3) immunogenicity of the vector itself, which can limit the effectiveness of the vaccine in individuals with preexisting antibodies as a consequence of prior infection with the organism or vaccination with the vector. See also Vaccinia Virus Expression System

DNA vaccine priming followed by boosting with other vaccines

Various approaches have been tested that sought to take advantage of combining the ability of DNA to prime antibody responses with the ability of recombinant proteins and viral vectors to boost them. DNA–protein prime-boost regimens have been studied extensively in HIV (Barnett et al., 2003), and also have been studied in anthrax, tuberculosis and in transmission blocking vaccines for both vivax and falciparium malaria (Kongkasuriyachai et al., 2004). Both malaria and HIV have been used to test immunization regimens comprising a DNA prime and a viral vector boost. Malaria is acquired at a young age, and for complete protection it likewise may be necessary to immunize at a very early age. It was shown experimentally that seven-day old mice with maternal antibodies could acquire CD8+ related protective immunity with a circumsporozoite protein DNA vaccine together with granulocyte–macrophage colony-stimulating factor (GM-CSF), followed by boosting with the same antigen in a poxvirus vector at one month of age. DNA primes with viral vector boosts also may enhance protective responses to HIV env, although in most such studies the anti-env responses induced by this approach did not include neutralizing antibodies because the viral vectors were more effective at inducing expanded populations of circulating effector CTL. DNA prime-vector boost approaches are currently being evaluated clinically (see later) and may prove useful.

Expression library immunization for identification of protective antigens

A technique for antigen discovery has been developed that combines the technologies of expression cloning and DNA vaccines, termed expression library immunization (ELI), whereby mixtures of plasmids encoding either fragments of a genome or discrete genes are tested for protective efficacy (Barry et al., 1995). Remarkably, pools of many thousand discrete plasmids can confer protection in certain animal models, such as Mycoplasma pulmonis. By successive fractionation and testing of these mixtures it may be possible to identify a single gene or small number of genes encoding a protective antigen of the pathogen. If so, this technique will greatly facilitate the identification of vaccine antigens.

The Immune Response Induced by DNA Vaccines (CD8 T Cells, CD4 T Cells, B Cells)

  1. Top of page
  2. Design of Plasmids
  3. DNA Vaccine Delivery
  4. The Immune Response Induced by DNA Vaccines (CD8 T Cells, CD4 T Cells, B Cells)
  5. How to Secrete Gene Products to Improve Stimulation of B Cells and CD4+ T Cells
  6. Coexpression of Cytokines and Costimulatory Molecules to Improve Immunogenicity
  7. Immunomodulatory DNA Motifs
  8. Results from Animal Experiments (Infections, Tumours, etc.)
  9. Tumours
  10. Clinical Trials of DNA Vaccines
  11. Safety Considerations (e.g. Chromosome Integration)
  12. Prospects for DNA Vaccines
  13. References
  14. Further Reading

CD8+ T cells

CD8+ cytotoxic T lymphocytes (CTLs) recognize peptides 8–10 amino acids in length that are displayed on the cell surface bound to MHC class I molecules. In general the peptides are thought to be generated by proteolysis in the cytosol, and then transported to the endoplasmic reticulum where they bind to nascent major histocompatibility complex (MHC) class I molecules. CD8+ CTLs can be demonstrated readily when lymph node or spleen cells from mice that have been injected intramuscularly with plasmid DNA encoding viral antigens are restimulated in vitro with antigen, or with mitogen and interleukin 2 (IL-2), or, in the case of lymphocytic choriomeningitis virus (LCMV), restimulated in vivo by viral infection. Effector CTLs that recognized epitope peptides appropriate to the murine MHC H-2 restriction element have been demonstrated in mice immunized with DNA encoding the nucleoprotein (NP) from influenza A virus, hepatitis B surface antigen (HBsAg) and core antigen, and HIV env. CTLs that were capable of recognizing and killing virus-infected targets have been induced in mice by DNA vaccines against influenza virus, against HIV (demonstrated using targets infected with vaccinia-HIV gp160 recombinants), against vaccinia virus or adenovirus-rabies virus glycoprotein recombinants, against LCMV, against herpes simplex virus (HSV), and against measles virus. In studies of influenza NP in BALB/c mice, a single intramuscular injection of as little as 1 mg of NPDNA induced CTLs that recognized the 147–155 epitope peptide from influenza NP, with higher doses yielding CTL precursors at progressively increasing frequencies. In other studies, anti-NP CTLs were found to persist for more than two years after immunization with influenza NP DNA. Repeated intramuscular immunization and boosting with DNA vaccines increased cell-mediated immune responses to influenza NP and HIV env, and have been reported to drive immune responses towards a type 1-like helper (TH1) phenotype. In rhesus monkeys injected intramuscularly with plasmids encoding either HIV env or gag genes, MHC class I-restricted, gag- or env-specific CTLs developed following one or two vaccinations. Anti-env CTLs were detected at least 11 months (the longest time point tested) following a final injection, demonstrating that these responses can be long-lived. See also AIDS: Clinical Manifestations, Epitopes, Influenza Viruses: Molecular Virology, Major Histocompatibility Complex (MHC), Major Histocompatibility Complex: Human, and T Lymphocytes: Cytotoxic

Induction of CTL responses after gene gun immunization was first demonstrated directly using the gene for the MHC antigen H-2Kb administered intrasplenically and intramuscularly after surgical exposure of the target tissues. Mice immunized by this combination of routes developed allospecific CTLs. In mice, three epidermal gene gun immunizations of 4 μg each of a construct encoding gp120 and 1 μg each of a construct encoding rev, induced CTL responses that were detected after two immunizations but were suppressed by a third immunization, while antibody responses appeared after dose 3. This result may have been related to a switch in helper T-cell phenotypes from TH1 to TH2, as the loss of CTL responsiveness was blocked by administration of antibody to IL-4. Gene gun immunization of rhesus monkeys using plasmids encoding simian immunodeficiency virus (SIV) env and gag genes induced env-specific CTLs in 3/3 monkeys although gag-specific CTLs were not detected. Intramuscular and intravenous injections of the same plasmids combined with gene gun immunization yielded env-specific CTLs in 3/3 animals and gag-specific CTLs in 2/3. See also Mice as Experimental Organisms

Recently, interest has been focused on the mechanisms by which DNA vaccines are able to induce CTLs. The intramuscular injection of plasmid DNA predominantly transfects striated muscle cells, while epidermal immunization by gene gun may transfect epidermal keratinocytes and Langerhans cells, and mononuclear cells present in epidermal capillary beds. Transplantation of C2C12 myoblasts stably transfected with the gene for influenza NP yielded NP-specific CTL responses, indicating that direct transfection of APCs is not required for the induction of CTLs by DNA vaccines. However, studies in several laboratories have shown that bone marrow-derived APCs are required for the induction of CTLs by DNA vaccines administered intramuscularly, or epidermally by gene gun. Studies in our laboratory have shown that APCs are required even when the NP protein is delivered by transplantation of stably transfected myoblast lines. Thus the induction of CTL responses by DNA vaccines may occur, at least in part, by transfer of antigen from non-APCs to APCs (termed ‘cross-priming’). DNA vaccines consisting of plasmids encoding proteins targeted for intracellular degradation, or minigenes encoding minimal epitopes, are still capable of inducing CTL, and thus in the case of DNA vaccines cross-priming may occur by transfer of processed peptide as well as of the mature protein. Bone marrow-derived APCs, such as DC, are potent inducers of CTL, and thus direct transfection of APCs may also play an important role, e.g. in gene gun immunization. See also Antigen-presenting Cells, and Dendritic Cells (T-lymphocyte Stimulating)

CD4+ T cells

CD4+ ‘helper’ T cells may be grouped operationally into functional subsets characterized by the particular cytokines they produce. In mice, type 1-like helper (TH1) T cells predominantly produce the cytokines IL-2 and interferon γ and support the development of cellular immune responses, including delayed-type hypersensitivity (DTH) and CTL, and antibody responses of the IgG2a immunoglobulin isotype. Type 2-like helper (TH2) T cells may produce IL-4, IL-5, IL-6 and IL-10, and promote B-cell activation and immunoglobulin class switching. Antibody responses driven by TH2-type T cells are typified by a predominance of the IgG1 immunoglobulin isotype. Intramuscular immunization of mice with DNA encoding a variety of antigens, such as influenza haemagglutinin (HA) and NP, HIV env, gag and rev, and Mycobacterium tuberculosis antigen 85 (Ag85), generates memory T-lymphocyte responses exemplified by antigen-specific T-cell proliferation and cytokine secretion during in vitro culture of splenocytes from vaccinees. Supernatants from antigen-stimulated cultures contained high levels of IL-2 and interferon γ with little or no IL-4 or IL-5, indicating that intramuscular DNA vaccination elicited TH1-like cytokine responses. Selective removal of lymphocyte populations for the NP and env studies indicated that CD4+ T lymphocytes were the primary responding cells in these assays. In contrast, vaccination using a gene gun, which delivers DNA primarily to the epidermis, can lead to TH2-like responses. Although there are no conclusive data to account for these differences, it has been suggested that the adjuvant effects of plasmid DNA may preferentially drive TH1-type responses in a dose-dependent manner. Thus, modalities such as intramuscular immunization that introduce higher doses of DNA may tend to preferentially activate TH1 cells. See also Antibody Responses: Development, Cytokines, Hypersensitivity: T Lymphocyte-mediated (Type IV), Interleukins, and T Lymphocytes: Helpers

B cells

Administration of plasmid DNA has proved to be an effective means of generating humoral immune responses against proteins of diverse origins. Antibody responses induced by DNA vaccination were first demonstrated in mice against human growth hormone and human α-1-antitrypsin after particle bombardment of gold beads coated with DNA encoding these proteins. Antibodies against viral proteins were demonstrated after intramuscular injection of DNA vaccines encoding influenza NP and HA, as well as other antigens of infectious agents such as HIV envelope protein, bovine herpes virus glycoprotein and hepatitis B surface antigen. Later, specific antibodies were induced in preclinical animal models by DNA vaccines encoding idiotypic antibody of a B-cell lymphoma, carcinoembryonic antigen (CEA), human Ig V region, MHC class I molecules, rabies virus glycoprotein, herpes simplex glycoproteins B and D, HIV rev, papillomavirus L1, hepatitis C virus nucleocapsid, hepatitis B core protein, duck hepatitis B virus surface antigen, hepatitis D and E virus structural proteins, human T-lymphotropic virus type 1 (HTLV-1) envelope protein, St Louis and Russian encephalitis virus prM/E, Japanese encephalitis virus NS1, dengue-2 envelope protein, cytomegalovirus tegument pp65, bovine respiratory syncytial virus G protein, foot and mouth disease virus, Schistosoma japonicum paramyosin, Plasmodium yoelii circumsporozoite protein, Leishmania major gp63, Bacillus thuringensis δ-endotoxin, fragment C of tetanus toxin, Brucella abortus ribosomal genes, My. pulmonis and M. tuberculosis Ag85 and heat shock protein 65 (hsp65). Introduction of plasmid DNA to mucosal surfaces by feeding of plasmid encapsulated in microspheres, or by direct application of plasmid with mucosal adjuvants or cationic lipids, has been reported to yield secretory immune responses in mice. Therefore, DNA vaccination is an effective way of obtaining antigen expression in situ, leading to humoral immune responses against various viral, bacterial, parasitic, tumour and eukaryotic proteins. Because DNA vaccination results in the expression of antigen in host cells in situ, proper antigenicity of proteins of eukaryotic origin in general, and of viral proteins in particular, may be achieved more readily than with other types of vaccines, for example inactivated viruses or recombinant or purified protein subunits. See also B Lymphocytes, Immunity: Experimental Transfer, Plasmids, and Vaccination of Animals

How to Secrete Gene Products to Improve Stimulation of B Cells and CD4+ T Cells

  1. Top of page
  2. Design of Plasmids
  3. DNA Vaccine Delivery
  4. The Immune Response Induced by DNA Vaccines (CD8 T Cells, CD4 T Cells, B Cells)
  5. How to Secrete Gene Products to Improve Stimulation of B Cells and CD4+ T Cells
  6. Coexpression of Cytokines and Costimulatory Molecules to Improve Immunogenicity
  7. Immunomodulatory DNA Motifs
  8. Results from Animal Experiments (Infections, Tumours, etc.)
  9. Tumours
  10. Clinical Trials of DNA Vaccines
  11. Safety Considerations (e.g. Chromosome Integration)
  12. Prospects for DNA Vaccines
  13. References
  14. Further Reading

Early studies of antibody responses induced by DNA vaccines focused on viral envelope proteins, such as influenza HA, gD of HSV, rabies virus glycoprotein, HBsAg and HIV env, all of which naturally are expressed in membrane-anchored form. In the cases of influenza HA, HBsAg and HIV env, in addition to the membrane-anchored form, a soluble form of the mature protein also may be generated naturally by enzymatic cleavage or by shedding of virus-like particles. Robust antibody responses, including conformationally specific virus-neutralizing antibodies, were readily demonstrated using these DNA vaccines. Later studies of DNA vaccines that encoded proteins that were not normally targeted for secretion, such as L1 of cottontail rabbit papillomavirus, showed that conformationally specific, virus-neutralizing antibodies could be induced even though the protein product lacked secretion signal sequences. DNA vaccines that encode influenza NP also induce strong antibody responses, although the protein product lacks a conventional secretion signal sequence and contains a nuclear localization signal. DNA vaccines that encode secreted forms of soluble proteins, such as human growth hormone, immunoglobulin single-chain variable region fragments (Fvs), or an N-terminal fragment of HSV gB, have also been shown to be capable of eliciting antibody responses. In contrast, DNA vaccines that encode proteins that are degraded intracellularly, e.g. by ubiquitin-mediated proteolysis, produce strong cellular immunity but generally are ineffective or of much reduced effectiveness in inducing antibody responses. Thus the ability of a DNA vaccine to produce antibody responses may depend more on its ability to produce mature protein in an appropriate conformation than on whether the protein is membrane-anchored or soluble, and whether it is targeted for secretion by conventional mechanisms. The effects of protein trafficking on CD4+ T-cell responses may be quite protein-specific and thus difficult to predict. DNA vaccines encoding influenza NP are able to induce robust CTL and IgG antibody responses in mice after intramuscular injection at quite low doses (<1 μg), while CD4+ T-cell proliferative responses are barely detected unless much higher doses (>30 μg) are given. In contrast, HIV gag induces CD4+ T-cell responses effectively. Thus a reasonable approach to developing DNA vaccines to an unknown protein that is not normally secreted would be to express the protein both with and without an exogenous secretion signal sequence. For transmembrane proteins, expression of both full-length membrane-anchored and truncated soluble forms would be appropriate. This empirical approach is necessitated by our present limited ability to predict the disposition in vivo of proteins encoded by DNA vaccines. See also T Lymphocytes: Helpers, T-Lymphocyte Responses: Development, and Viral Capsids and Envelopes: Structure and Function

Coexpression of Cytokines and Costimulatory Molecules to Improve Immunogenicity

  1. Top of page
  2. Design of Plasmids
  3. DNA Vaccine Delivery
  4. The Immune Response Induced by DNA Vaccines (CD8 T Cells, CD4 T Cells, B Cells)
  5. How to Secrete Gene Products to Improve Stimulation of B Cells and CD4+ T Cells
  6. Coexpression of Cytokines and Costimulatory Molecules to Improve Immunogenicity
  7. Immunomodulatory DNA Motifs
  8. Results from Animal Experiments (Infections, Tumours, etc.)
  9. Tumours
  10. Clinical Trials of DNA Vaccines
  11. Safety Considerations (e.g. Chromosome Integration)
  12. Prospects for DNA Vaccines
  13. References
  14. Further Reading

The potential use of immunoregulatory molecules (e.g. cytokines, chemokines or costimulatory molecules) to enhance or modulate the immune response to antigens encoded by DNA vaccines has been effected by coadministration of recombinant cytokines with DNA or coexpression of cytokines in situ, through the use of DNA plasmids encoding such molecules (Xiang and Ertl, 1995). With respect to the latter, DNA expression plasmids encoding the following cytokines have been demonstrated to enhance the potency of coadministered DNA vaccines: (1) GM-CSF, a pluripotent molecule that stimulates growth and differentiation of various progenitor cells; (2) IL-2, a TH1-type cytokine that stimulates growth and differentiation of T cells and immunoglobulin production from B cells; (3) IL-12, which promotes cellular immune responses through the differentiation of TH1 cells; and (4) IL-1b, which has a wide variety of effects on immune and inflammatory responses. In addition, a DNA construct encoding the β-chemokine TCA-3, a chemoattractant for inflammatory cells, was shown to increase the immunogenicity of a DNA vaccine. A different approach has been to use DNA constructs encoding costimulatory molecules (B7-1/B7-2 or CD80/CD86) that are known to be important for providing a signal to T cells during engagement of T cells with APCs, and CD40 ligand (CD154), which is expressed transiently on T cells. Interaction of CD154 with APCs bearing CD40 promotes antigen presentation to T cells. A potential safety issue posed by this approach is the consequence of expressing immunologically active molecules in an unregulated manner, and it may be necessary to use an inducible promoter system to regulate more tightly the timing and magnitude of cytokine expression. See also Chemokines and Chemokine Receptors, Cytokines, Immunoregulation, and Interleukins

Immunomodulatory DNA Motifs

  1. Top of page
  2. Design of Plasmids
  3. DNA Vaccine Delivery
  4. The Immune Response Induced by DNA Vaccines (CD8 T Cells, CD4 T Cells, B Cells)
  5. How to Secrete Gene Products to Improve Stimulation of B Cells and CD4+ T Cells
  6. Coexpression of Cytokines and Costimulatory Molecules to Improve Immunogenicity
  7. Immunomodulatory DNA Motifs
  8. Results from Animal Experiments (Infections, Tumours, etc.)
  9. Tumours
  10. Clinical Trials of DNA Vaccines
  11. Safety Considerations (e.g. Chromosome Integration)
  12. Prospects for DNA Vaccines
  13. References
  14. Further Reading

The immunostimulatory effects of bacterial DNA have been known for several years (Yamamoto et al., 1992), but have only recently been studied in detail as potential vaccine adjuvants. A specific nucleotide sequence motif, consisting of -purine-purine-C-G-pyrimidine-pyrimidine-, is a potent inducer of lymphocyte proliferation in vitro resulting in the secretion of cytokines such as IL-6, IL-12 and interferon γ (Krieg et al., 1995). Coadministration of such oligonucleotides with protein vaccines can enhance antigen-specific immune responses and modulate the response to include CTLs, suggesting that the presence of CpG motifs in DNA vaccines may account for the bias towards the induction of TH1-type responses. In fact, bacterial-derived plasmid DNA can have both immunomodulatory effects, as demonstrated by the observation that coinoculation of noncoding plasmid DNA with a protein antigen can modulate the immunoglobulin isotype profile of antibodies against the protein, and enhancing effects, as demonstrated by the observation that antibody responses induced by a DNA vaccine can be enhanced by the inclusion of additional noncoding plasmid. The potential role of CpG motifs in the effectiveness of DNA vaccines has been further suggested by apparent improvements in potency of some DNA vaccines by inclusion of the specific motif AACGTT in the vector backbone. The mechanism of action of these motifs is believed to be, at least in part, due to their dependence on signalling the innate immune system via Toll-like receptor 9 (TLR9) (Tudor et al., 2005). Recent advances on the importance of the innate immune system in modulating and enhancing adaptive immune responses has provided insight into the potential adjuvant effects of these immunostimulatory motifs.

As with all vaccine adjuvants, safety issues must also be considered, such as the potential of inducing deleterious inflammatory or autoimmune responses by nonspecifc activation of immune cells. However, DNA vaccine modifications to include CpG motifs in the vector or to coinject plasmid with oligonucleotides are desirable means of increasing vaccine potency, in that they do not significantly increase the complexity of the vaccine.

Results from Animal Experiments (Infections, Tumours, etc.)

  1. Top of page
  2. Design of Plasmids
  3. DNA Vaccine Delivery
  4. The Immune Response Induced by DNA Vaccines (CD8 T Cells, CD4 T Cells, B Cells)
  5. How to Secrete Gene Products to Improve Stimulation of B Cells and CD4+ T Cells
  6. Coexpression of Cytokines and Costimulatory Molecules to Improve Immunogenicity
  7. Immunomodulatory DNA Motifs
  8. Results from Animal Experiments (Infections, Tumours, etc.)
  9. Tumours
  10. Clinical Trials of DNA Vaccines
  11. Safety Considerations (e.g. Chromosome Integration)
  12. Prospects for DNA Vaccines
  13. References
  14. Further Reading

Viral diseases

Influenza was the first disease for which protective immunity induced by DNA vaccines was demonstrated in animal models. Cross-strain protection, in which mice immunized against one influenza subtype were rendered resistant to challenge by a different subtype, was induced by immunization with DNA encoding NP (Ulmer et al., 1993); protective responses against homologous influenza strains were demonstrated in mice, chickens (Fynan et al., 1993) and ferrets using DNA encoding HA (Table 2). In ferrets, immunization with a combination of DNA plasmids encoding HA and two internal conserved proteins, NP and matrix, provided protection from an antigenically distinct isolate of influenza, as measured by a decrease in nasal viral shedding that was equivalent to protection provided by DNA encoding the homologous HA (Donnelly et al., 1995). Protective immune responses have been demonstrated in a number of other preclinical animal models including human immunodeficiency virus (Letvin et al., 1997), bovine herpes virus, a murine and a mucosal guinea-pig model of human HSV, rabies virus, LCMV, cottontail rabbit papilloma virus, hepatitis B virus, bovine respiratory syncytial virus, Japanese encephalitis virus, Russian spring/summer encephalitis virus, Central European encephalitis virus and foot and mouth disease virus. While in most of the models, immunity was dependent upon the generation of protective antibody responses, for LCMV and for the influenza studies with NP DNA the protection seen was based upon cellular responses. See also Avian Influenza Viruses, and Influenza Viruses: Molecular Virology

Table 2. Effector responses to DNA vaccines
Disease agentEffector typeChallenge outcome
  1. CTL, cytotoxic T lymphocyte; MUC-1, epithelial cell that is glycosylated abnormally in some breast carcinomas; TH, T-helper.

Viruses
Bovine herpes virusAntibodiesProtection
Bovine respiratory syncytial virusAntibodiesProtection
Canine parvovirusAntibodiesProtection
Central European encephalitis virusAntibodiesProtection
Cottontail rabbit papilloma virusAntibodiesProtection
Foot and mouth disease virusAntibodiesProtection
Hepatitis B virusAntibodiesProtection
HIVAntibodies, CTLsProtection
Infuenza virusAntibodies, CTLsProtection
Japanese encephalitis virusAntibodiesProtection
Lymphocytic choriomeningitis virusCTLsProtection
Rabies virusAntibodiesProtection
Russian spring/summer encephalitis virusAntibodiesProtection
   
Bacteria
Brucella abortusAntibodiesNo protection
Clostridium tetani (fragment C of toxin)AntibodiesProtection
Mycobacterium tuberculosisTH cells, CTLsProtection
Mycoplasma pulmonisUnknownProtection
Salmonella typhiAntibodiesNo challenge
   
Metazoa
Schistosoma spp.TH cells, antibodiesProtection
   
Protozoa
Leishmania majorTH cellsProtection
Plasmodium yoeliiCTLsProtection
   
Tumours
Carcinoembryonic antigenAntibodiesProtection
MUC-1CTLs, TH cellsProtection

Bacterial diseases

A potential advantage of applying DNA vaccination to viral diseases is that viral proteins expressed in situ after DNA vaccination may attain proper folding, posttranslational modification and intracellular transport, since viral proteins are normally expressed by eukaryotic cells of the host. In contrast, bacterial proteins are expressed not by the host cell protein synthesis machinery but by the invading organism. Thus, bacterial proteins expressed by eukaryotic cells may receive different types of posttranslational modifications and may attain nonnative conformations. For example, M. tuberculosis Ag85 is modified by N-linked oligosaccharides during expression in eukaryotic cells. Nevertheless, injection of plasmid DNA can be effective for expression of bacterial proteins in situ (both reporter proteins and antigens), and DNA vaccines can be effective in animal models. As revealed by the My. pulmonis experiments referred to above, a DNA vaccine consisting of several thousand distinct plasmids encoding proteins of the lung pathogen My. pulmonis, used in combination in a technique termed expression library immunization, conferred protection. In this study the specific component(s) of the mixture responsible for protection were not identified, but these results indicate that at least one of the plasmids encoded a protective immunogen of My. pulmonis. See also Bacterial Plasmids, and Proteins: Postsynthetic Modification – Function and Physical Analysis

DNA vaccines encoding M. tuberculosis antigens have also been protective in animal models. The efficacy of DNA vaccines encoding Ag85 and hsp65 was shown in mice using aerosol and systemic M. tuberculosis challenge models, respectively. The magnitude of reductions in the titre of mycobacteria in the lungs induced by these DNA vaccines encoding single M. tuberculosis was comparable to that induced by the M. bovis BCG vaccine itself. A DNA vaccine encoding the tetanus toxin C fragment provided protection in an animal challenge model. Antibody responses also have been raised against the OmpC porin protein of Salmonella typhi. A DNA vaccine encoding a ribosomal antigen from B. abortus induced an immune response but was not protective for mice. Therefore, DNA vaccines for bacterial diseases may be feasible in selected experimental systems. See also Immunity to Bacteria, and Tuberculosis

Parasitic diseases

DNA vaccines have also been used to raise protection against parasites. Antibodies have been raised against proteins from Schistosoma mansoni, S. japonicum, L. major and P. yoelii, by intramuscular injection of plasmid DNA. Immunized mice were protected against challenge with L. major and P. yoelii most likely by a cell-mediated mechanism. BALB/c mice are normally susceptible to L. major infection due to an inability to mount a TH1 response, unless given exogenous IL-12, suggesting that DNA vaccination may provide a means to overcome this deficit. Protection against P. yoelii may require CD8+ CTL, interferon γ or nitric oxide. Determinant selection by MHC molecules limits the ability of plasmids encoding individual P. yoelii proteins to protect different strains of inbred mice; combining together several constructs encoding different malaria proteins has been used to provide complementary epitopes. The method of administration of the DNA has been found to affect the immune responses generated against different malaria antigens. No route was conclusively favoured over the other in all instances and different anatomic sites appeared to provide optimal immunogenicity for different malaria proteins. See also Immunity to Protozoa, Leishmania, Malaria, Plasmodium, and Schistosomiasis and Other Trematode Infections

Tumours

  1. Top of page
  2. Design of Plasmids
  3. DNA Vaccine Delivery
  4. The Immune Response Induced by DNA Vaccines (CD8 T Cells, CD4 T Cells, B Cells)
  5. How to Secrete Gene Products to Improve Stimulation of B Cells and CD4+ T Cells
  6. Coexpression of Cytokines and Costimulatory Molecules to Improve Immunogenicity
  7. Immunomodulatory DNA Motifs
  8. Results from Animal Experiments (Infections, Tumours, etc.)
  9. Tumours
  10. Clinical Trials of DNA Vaccines
  11. Safety Considerations (e.g. Chromosome Integration)
  12. Prospects for DNA Vaccines
  13. References
  14. Further Reading

The identification of tumour-associated antigens as well as reports that local delivery of appropriate cytokines may have therapeutic effects in animal models have increased interest in developing antitumour vaccines. Recently, several laboratories have described antitumour DNA vaccines. DNA vaccines encoding variable regions of immunoglobulins, alone or coinjected with plasmids encoding the IL-2 or interferon γ genes, have been used to elicit anti-idiotype antibody responses. This approach may provide a useful approach to therapy against lymphomas expressing surface immunoglobulins. Vaccination of mice with DNA encoding the human CEA, which is expressed at high levels in human colon, breast and nonsmall-cell lung cancer, elicited CEA-specific immune responses and protected mice from subsequent challenge with syngeneic tumours expressing CEA. Similarly, a DNA vaccine containing the MUC-1 gene, which encodes the polymorphic epithelial mucin (PEM) associated with breast, pancreatic and colon cancers, protected mice from challenge with syngeneic, PEM-expressing tumour cells. Therapeutic effects of DNA plasmids encoding cytokines have been described in a number of laboratories. Thus DNA vaccines may have some potential applications beyond infectious diseases. See also Tumours: Immunotherapy, and Vaccination

Clinical Trials of DNA Vaccines

  1. Top of page
  2. Design of Plasmids
  3. DNA Vaccine Delivery
  4. The Immune Response Induced by DNA Vaccines (CD8 T Cells, CD4 T Cells, B Cells)
  5. How to Secrete Gene Products to Improve Stimulation of B Cells and CD4+ T Cells
  6. Coexpression of Cytokines and Costimulatory Molecules to Improve Immunogenicity
  7. Immunomodulatory DNA Motifs
  8. Results from Animal Experiments (Infections, Tumours, etc.)
  9. Tumours
  10. Clinical Trials of DNA Vaccines
  11. Safety Considerations (e.g. Chromosome Integration)
  12. Prospects for DNA Vaccines
  13. References
  14. Further Reading

DNA encoding HIV antigens were among the earliest of DNA vaccines to be tested in humans (first as therapeutic vaccines in infected individuals, then later as preventive vaccines in uninfected people). Owing to the induction of modest immune responses in these early clinical trials, recent efforts have focused on the use of DNA as a prime followed by a heterologous booster vaccine, such as with a viral vector. This prime-boost approach has been substantially more potent than either DNA or vector alone in nonhuman primate models and is being evaluated in clinical trials of HIV and other pathogens in uninfected humans. See also Vaccination of Humans

Therapeutic HIV DNA vaccines have the potential to reduce or clear the levels of circulating virus in infected individuals. Recent trials involving repeated immunization with DNA encoding rev/env and gag/pol genes in the presence of highly active antiretroviral treatment have been evaluated. This combined treatment has led to better stability of CTL responses than in placebo-immunized individuals, suggesting that DNA-based immunotherapy could be beneficial. Other HIV vaccines evaluated in clinical trials include DNA formulated with PLG microparticles, as well as combinations of DNA plasmids encoding genes derived from several clades of virus, followed by boosts of adenovirus or modified vaccinia Ankara (MVA) vectors (Hanke et al., 2007).

Genes encoding malaria proteins have previously demonstrated safety and immunogenicity in several human clinical trials. Recently, the DNA prime, MVA boost approach has been evaluated in humans, where T-cell responses were elicited against all five malaria antigen-encoding plasmids in approximately half of the immunized individuals. To assess efficacy, challenge studies were conducted with live Plasmodium falciparum. A reasonable correlation between T-cell responses and delay of onset of parasitaemia was observed. However, certain individuals with high levels of T-cell responses were not protected, suggesting that other immune parameters are involved (Dunachie et al., 2006).

Clinical trials utilizing gene-based vectors for cancer have included DNA alone, as well as the prime-boost approach. Key barriers to success for cancer vaccines targeting tumour antigens (versus preventive vaccines against infectious diseases) are overcoming immunologic tolerance and weakened immune systems. For these reasons, few successes have been reported. However, in chronic carriers of hepatitis B, a DNA vaccine was able to induce or expand antigen-specific T-cell responses. Furthermore, serum viral DNA levels diminished in half of the vaccinated individuals with complete clearance in one. Strategies to improve DNA vaccines for cancer have included the use of DNA encoding immunologically active proteins (such as cytokines), as well as facilitated DNA delivery technologies. Results from a clinical trial of a DNA vaccine encoding prostate-specific antigen (PSA) along with recombinant GM-CSF and IL-2 demonstrated that cellular and humoral immune responses against PSA could be induced and the vaccine appeared safe. Facilitated delivery of DNA vaccines using electroporation in vivo has shown substantial effectiveness in preclinical trials and is currently being evaluated for PSA DNA (Miller et al., 2005).

Licensed DNA-based vaccines

Two DNA vaccines were licensed in 2005, targeting West Nile virus for horses and infectious haematopoietic necrosis virus for salmon. In addition, a therapeutic DNA vaccine to treat melanoma in dogs received conditional approval from the US Department of Agriculture in 2007. These approvals represent important steps in the maturation of the DNA vaccine technology. First, they illustrate that DNA vaccines can be manufactured at commercial scale and relatively low cost (e.g. for fish). Second, they demonstrate that large animals can be successfully immunized with DNA vaccines (e.g. for horses). Previously, the low potency of DNA vaccines in humans compared to the robust responses in preclinical models was thought to be due, in part, to the large size difference between humans and the various preclinical small animal models. These recent successes should reinvigorate the search for enabling technologies for human DNA vaccines.

Safety Considerations (e.g. Chromosome Integration)

  1. Top of page
  2. Design of Plasmids
  3. DNA Vaccine Delivery
  4. The Immune Response Induced by DNA Vaccines (CD8 T Cells, CD4 T Cells, B Cells)
  5. How to Secrete Gene Products to Improve Stimulation of B Cells and CD4+ T Cells
  6. Coexpression of Cytokines and Costimulatory Molecules to Improve Immunogenicity
  7. Immunomodulatory DNA Motifs
  8. Results from Animal Experiments (Infections, Tumours, etc.)
  9. Tumours
  10. Clinical Trials of DNA Vaccines
  11. Safety Considerations (e.g. Chromosome Integration)
  12. Prospects for DNA Vaccines
  13. References
  14. Further Reading

Regulatory agencies charged with reviewing applications of DNA vaccines in humans have identified the following as key areas to consider: the potential for integration of the plasmid DNA into the genome of host cells; the potential for induction of immune tolerance or of autoimmunity; and the potential for induction of antibodies to the injected plasmid DNA. See also Autoimmune Disease: Pathogenesis, and Immunological Tolerance: Mechanisms

Potential for integration

The integration of injected plasmid DNA into the genome of host cells in a living animal may be silent; potentially could be mutagenic, if the integration event disrupted a cellular gene; or potentially could be carcinogenic, if the integration event inactivated a regulatory gene for cell division or activated an oncogene. In the case of DNA vaccination, the plasmid does not contain an origin of replication that is functional in eukaryotic cells, the cells thought to be transfected are for the most part nondividing, and the plasmids used contain only very limited sequence homology with mammalian DNA. Direct studies of integration in mice injected intramuscularly with plasmid DNA encoding influenza NP with sensitive polymerase chain reaction (PCR) methods (1 copy/150 000 nuclei, which has been calculated to be three orders of magnitude less than the spontaneous mutation rate) have not detected integration of the injected plasmid thus far. See also Mutagenesis, and Oncogenes

Potential for induction of immunological tolerance and autoimmunity

Since the amount of antigen produced after DNA immunization is thought to be small, the formal possibility exists that antigen-specific unresponsiveness might be induced by DNA vaccines rather than protective immunity. However, the studies described above have demonstrated the ability of DNA vaccination to induce functional immune responses. Even in neonatal mice, injection of DNA vaccines may induce priming rather than unresponsiveness, depending upon the experimental system used. It is conceivable that the ability of bacterial DNA to activate innate mechanisms of immunity, described above in the section on ‘Immunomodulatory DNA motifs’, might be essential to the immunogenicity of DNA vaccines. Current theories suggest that professional APCs must be activated when antigen is presented for immunogenic signals to be delivered. Such activation probably could occur as a result of cytokine secretion induced by bacterial DNA. This might account for the relative paucity of examples in which DNA vaccines have induced tolerance, even in neonates, in comparison to the many examples of robust immune responses to DNA vaccines. Autoimmune responses might occur as a result of immune-mediated destruction of cells expressing the antigen genes. However, the destruction of tissue cells occurs also in the course of viral and bacterial infections as well as in normal processes of tissue remodelling. So far, there is little evidence that DNA vaccines would pose a greater risk in this regard than conventional viral or bacterial vaccines. See also Antigen-presenting Cells, Autoimmune Disease: Pathogenesis, and Immunological Tolerance: Mechanisms

Potential for induction of anti-DNA antibodies

A third safety consideration concerns the potential for induction of immune responses against the plasmid DNA itself. Although pathogenic anti-DNA antibodies are generally believed to be a hallmark of systemic lupus erythematosus (SLE), it is likely that several factors play a role in these responses, including genetic susceptibility and underlying immune dysfunction. It is not clear whether exposure to DNA can induce or exacerbate SLE, but inoculation of spontaneously autoimmune mice with bacterial DNA can in some instances ameliorate disease. Therefore, the likelihood of induction of pathogenic anti-DNA antibodies by vaccination with plasmid DNA needs to be considered carefully. However, studies in laboratory animals have shown that purified double-stranded DNA (dsDNA) does not readily induce anti-DNA antibodies. Anti-dsDNA antibodies can be induced in normal mice by inoculation with DNA that has been denatured, complexed with methylated bovine serum albumin (mBSA) and coadministered with complete Freund's adjuvant (CFA). Vaccination of mice with dsDNA regardless of origin is not effective in inducing anti-DNA antibodies as measured by enzyme-linked immunosorbent assay (ELISA), immunoblot or radioimmunoassay. Therefore, while it is not yet known whether DNA vaccines will induce anti-DNA antibodies in humans, preclinical animal studies conducted so far have not given cause for concern. See also Enzyme-linked Immunosorbent Assay, Mice as Experimental Organisms, Systemic Lupus Erythematosus, and Vaccination of Animals

Prospects for DNA Vaccines

  1. Top of page
  2. Design of Plasmids
  3. DNA Vaccine Delivery
  4. The Immune Response Induced by DNA Vaccines (CD8 T Cells, CD4 T Cells, B Cells)
  5. How to Secrete Gene Products to Improve Stimulation of B Cells and CD4+ T Cells
  6. Coexpression of Cytokines and Costimulatory Molecules to Improve Immunogenicity
  7. Immunomodulatory DNA Motifs
  8. Results from Animal Experiments (Infections, Tumours, etc.)
  9. Tumours
  10. Clinical Trials of DNA Vaccines
  11. Safety Considerations (e.g. Chromosome Integration)
  12. Prospects for DNA Vaccines
  13. References
  14. Further Reading

DNA vaccines have proven to be very effective in many animal models of infectious and noninfectious diseases, yet have shown only modest immunogenicity in several human clinical trials. This has spawned much effort towards discovering and developing enabling technologies for DNA vaccines, so far with only limited success. However, the recent licensure of DNA vaccines for animal health applications has perhaps changed the outlook for this technology. Ultimately, success for human vaccines will depend on building upon the knowledge gained from both the limited immune responses in humans and the recent successes in fish, horses and dogs.

References

  1. Top of page
  2. Design of Plasmids
  3. DNA Vaccine Delivery
  4. The Immune Response Induced by DNA Vaccines (CD8 T Cells, CD4 T Cells, B Cells)
  5. How to Secrete Gene Products to Improve Stimulation of B Cells and CD4+ T Cells
  6. Coexpression of Cytokines and Costimulatory Molecules to Improve Immunogenicity
  7. Immunomodulatory DNA Motifs
  8. Results from Animal Experiments (Infections, Tumours, etc.)
  9. Tumours
  10. Clinical Trials of DNA Vaccines
  11. Safety Considerations (e.g. Chromosome Integration)
  12. Prospects for DNA Vaccines
  13. References
  14. Further Reading
  • Barnett S, Otten G, Srivastava I et al. (2003) Enhanced DNA prime-protein boost vaccines induce potent immune responses against HIV-1. In: Retroviruses of Human AIDS and Related Animal Diseases, XIII Cent Gardes Symposium, pp. 145–153. Amsterdam, The Netherlands: Elsevier.
  • Barry MA, Lai WC and Johnston SA (1995) Protection against mycoplasma-infection using expression-library immunization. Nature 377: 632635.
  • Denis-Mize K, Dupuis M, Singh M et al. (2003) Mechanisms of increased immunogenicity for DNA-based vaccines absorbed onto cationic microparticles. Cellular Immunology 225: 1220.
  • Donnelly JJ, Friedman A, Martinez D et al. (1995) Preclinical efficacy of a prototype DNA vaccine: enhanced protection against antigenic drift in influenza virus. Nature Medicine 1: 583587.
  • Dunachie SJ, Walther M, Epstein JE et al. (2006) A DNA prime-modified vaccinia virus Ankara boost vaccine encoding thrombospondin-related adhesion protein but not circumsporozoite protein partially protects healthy malaria-naive adults against Plasmodium falciparum sporozoite challenge. Infection and Immunity 74: 59335942.
  • Fynan EF, Webster RG, Fuller DH et al. (1993) DNA vaccines – protective immunizations by parenteral, mucosal, and gene-gun inoculations. Proceedings of the National Academy of Sciences of the USA 90: 1147811482.
  • Hanke T, McMichael AJ and Dorrell L (2007) Clinical experience with plasmid DNA – and modified vaccinia virus Ankara-vectored human immunodeficiency virus type 1 clade A vaccine focusing on T-cell induction. Journal of General Virology 88: 112.
  • Kongkasuriyachai D, Bartels-Andrews L, Stowers A et al. (2004) Potent immunogenicity of DNA vaccines encoding Plasmodium vivax transmission-blocking vaccine candidates Pvs25 and Pvs28-evaluation of homologous and heterologous antigen-delivery prime-boost strategy. Vaccine 22: 32053213.
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  • Letvin NL, Montefiori DC, Yasutomi Y et al. (1997) Potent protective anti-HIV immune responses generated by bimodal HIV envelope DNA plus protein vaccination. Proceedings of the National Academy of Sciences of the USA 94: 93789383.
  • Miller AM, Ozenci V, Kiessling R and Pisa P (2005) Immune monitoring in a phase 1 trial of a PSA DNA vaccine in patients with hormone-refractory prostate cancer. Journal of Immunotherapy 28: 389395.
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  • Tang DC, De Vit M and Johnston SA (1992) Genetic immunization is a simple method for eliciting an immune response. Nature 356: 152154.
  • Tudor D, Dubuquoy C, Gaboriau V et al. (2005) TLR9 pathway is involved in adjuvant effects of plasmid DNA-based vaccines. Vaccine 23: 12581264.
  • Ulmer JB, Donnelly JJ, Parker SE et al. (1993) Heterologous protection against influenza by injection of DNA encoding a viral protein. Science 259: 17451749.
  • Wolff JA, Malone RW, Williams P et al. (1990) Direct gene transfer into mouse muscle in vivo. Science 247: 14651468.
  • Xiang Z and Ertl HC (1995) Manipulation of the immune response to a plasmid-encoded viral antigen by coinoculation with plasmids expressing cytokines. Immunity 2: 129135.
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Further Reading

  1. Top of page
  2. Design of Plasmids
  3. DNA Vaccine Delivery
  4. The Immune Response Induced by DNA Vaccines (CD8 T Cells, CD4 T Cells, B Cells)
  5. How to Secrete Gene Products to Improve Stimulation of B Cells and CD4+ T Cells
  6. Coexpression of Cytokines and Costimulatory Molecules to Improve Immunogenicity
  7. Immunomodulatory DNA Motifs
  8. Results from Animal Experiments (Infections, Tumours, etc.)
  9. Tumours
  10. Clinical Trials of DNA Vaccines
  11. Safety Considerations (e.g. Chromosome Integration)
  12. Prospects for DNA Vaccines
  13. References
  14. Further Reading