Gene Vaccines[dhelix]

  1. Indresh K. Srivastava, PhD; and
  2. Margaret A. Liu, MD
  1. From Chiron Corporation, Emeryville, California; and Transgene, Strasbourg, France.
     
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    Abstract

    Gene vaccines are a new approach to immunization and immunotherapy in which, rather than a live or inactivated organism (or a subunit thereof), one or more genes that encode proteins of the pathogen are delivered. The goal of this approach is to generate immunity against diseases for which traditional vaccines and treatments have not worked, to improve vaccines, and to treat chronic diseases. Gene vaccines make use of advances in immunology and molecular biology to more specifically tailor immune responses (cellular or humoral, or both) against selected antigens. They are still under development in research and clinical trials.

    The mechanisms for inducing cellular (as opposed to humoral) responses against a particular antigen have been elucidated. Gene vaccines provide a means to generate specific cellular responses while still generating antibodies, if desired. In addition, by delivering only the genes that encode the particular proteins against which a protective or therapeutic immune response is desired, the potential limitations and risks of certain other approaches can be avoided.

    This article describes the rationale for, immunologic mechanisms involved in, and design of gene vaccines under development. Preclinical and clinical studies of these vaccines are discussed for various clinical applications, focusing on infectious diseases.

    Recent developments in immunology and molecular biology have permitted development of DNA and other gene-based vaccines. Unlike previous vaccines, many of which were designed to induce antibodies as the mechanism of protection against clinical disease caused by microorganisms, gene-based vaccines are designed to generate both cytotoxic cellular and antibody immune responses. An additional goal is to make vaccines that can be used to treat chronic diseases. Targeted diseases have expanded from infectious diseases to include cancer, allergies, and autoimmune diseases.

    The immune response comprises both cellular and humoral (antibody) components. For certain infectious agents, the efficacy of a vaccine may be correlated with induction of a particular level of circulating antibody in a vaccinated person. Until recently, much less was known about how to induce or measure clinically effective cellular immunity. To date, no known type or level of cellular response has been shown to correlate with efficacy of a vaccine or therapy in humans. Moreover, whereas antibodies can directly attack microorganisms, T cells work by killing infected or cancerous cells. Thus, by definition, T cells cannot prevent infection; rather, they contain or eliminate it.

    Instead of directly attacking a pathogen, a cytolytic T cell is activated to kill when its receptor binds to certain molecules on another cell and recognizes foreign elements (from the pathogen or from novel cancer proteins). The receptor has dual specificity, meaning that it sees two molecules on the surface of the target cell. One molecule is the MHC class I antigen, which is specific to an individual person, and the other molecule is a peptide derived from proteins of the pathogen or cancer cell. Thus, the T cell recognizes and is activated by foreign or abnormal peptides when those peptides are associated with a person's own MHC class I molecules. In an infected or cancerous cell, some of the newly synthesized viral (or other) proteins are degraded into peptides and enter the endoplasmic reticulum of the cell, where they meet and bind to newly synthesized MHC class I molecules (1). When this MHC class I–antigenic peptide complex is expressed on the surface of an antigen-presenting cell (along with co-stimulatory molecules, to which the T cell must also bind), T cells with receptors that specifically recognize this complex are activated and can kill infected cells (Figure 1).

    Figure 1. Newly synthesized viral protein in the cytoplasm of an infected cell is degraded into peptides that are transported into the endoplasmic reticulum and then to the Golgi apparatus. Peptides bind to newly synthesized major histocompatibility complex class I molecules; the complex is then expressed on the surface of the cell where, in the presence of appropriate accessory molecules, binding to the T-cell receptor of CD8 cells can occur. This binding results in activation of the cytolytic T lymphocyte. Adapted from McDonnell and Askari , with permission from the Massachusetts Medical Society.
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      Figure 1. Newly synthesized viral protein in the cytoplasm of an infected cell is degraded into peptides that are transported into the endoplasmic reticulum and then to the Golgi apparatus. Peptides bind to newly synthesized major histocompatibility complex class I molecules; the complex is then expressed on the surface of the cell where, in the presence of appropriate accessory molecules, binding to the T-cell receptor of CD8 cells can occur. This binding results in activation of the cytolytic T lymphocyte. Adapted from McDonnell and Askari , with permission from the Massachusetts Medical Society. Activation of cytolytic T lymphocytes.+(2)

      Efforts to specifically induce cytolytic T cell responses have greatly increased so that this component of the immune system might be used for vaccination. The challenge has been that if a protein is provided to an antigen-presenting cell, in general that cell will take up the protein into the endolysosomal pathway and degrade it into peptides associated with MHC class II rather than class I proteins. The MHC class II proteins recycle from the cell surface through the endolysosomal pathway, where they encounter peptide epitopes. These MHC class II–peptide complexes stimulate helper T cells that produce cytokines that in turn help activate cytolytic T cells and assist B cells to generate antibodies.

      For a protein to enter the proteolytic pathway that generates the peptides that will bind to MHC class I molecules, it must be introduced into or generated in the cytoplasm of a cell. Thus, finding a means of introducing antigenic proteins from pathogens or cancer cells into this pathway has become one of the prime challenges in vaccine development. Introduction of proteins directly into the cytoplasm from extracellular spaces has proven difficult; a more feasible method is to deliver a gene encoding the desired protein to the cell (3). If the cell then transcribes and translates the gene, the protein will be in the cytoplasm for proper processing and presentation.

      Thus, a method of delivering adequate amounts of the desired gene, resulting in adequate expression of the encoded protein, is needed. In addition, because T cells can be stimulated to respond only when the MHC–antigen complex is on the surface of professional antigen-presenting cells that have other cell surface proteins that interact with the T cell, it was thought that the genes would have to be delivered specifically to antigen-presenting cells rather than to other cell types (4, 5).

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      Viral and Bacterial Vectors

      Viruses have highly evolved structures that enable them to bind to cells and deliver genes into the cells they infect. It may be feasible to use live virus vaccines to induce cytolytic T lymphocytes and thus treat a disease in which cellular immunity is critical. However, in some diseases (such as AIDS), concerns about the safety of a live attenuated vaccine are too great to make this a practical approach (6). In addition, a stronger immune response and an improved safety profile may be obtained by using a vaccine that delivers only the genes that encode the desired antigens (that is, the specific antigen against which the immune response important for protection is directed) rather than all the genes for a given pathogen. Therefore, efforts have been made to use harmless viruses or ones that have been rendered incapable of reproducing for delivery of genes derived from a different pathogen. In other words, a vector is made in which all the genes or key genes of a virus are removed or the virus is nonpathogenic or nonreplicative in humans. Genes encoding a protein antigen from the pathogen (that is, from a different virus or a tumor antigen) are then put in the virus particle. The virus acts as a transporter, in the manner of a Trojan horse, to deliver the gene encoding the antigen.

      The viral vector is itself antigenic, and certain viruses cause inflammatory responses. These responses may benefit the desired immune response, depending on their type, location, and extent, but they might also be drawbacks. Preexisting immunity due to previous infection (for example, adenovirus) or vaccination (for example, smallpox) may limit the potency of the vaccine by clearing the viral vector before it can infect cells to deliver its payload of antigen genes. Similarly, if the vector is reused for a booster immunization, the immune response against the virus itself may eliminate too many of the vector particles before they can deliver the genes. Much effort has been directed to making vectors from other strains of viruses, vectors that are not as immunogenic, and vectors that do not induce inflammatory responses.

      Bacteria are also being developed as delivery systems for plasmid DNA vaccines and as heterologous expression systems in which the genes encoding antigens are incorporated into the genome of the bacteria (7, 8). One attractive feature of bacterial vectors is their potential for oral administration, which would make use easier and may generate mucosal immune responses. Shigella(9-11), Salmonella(12-14), and Listeria monocytogenes (15) have been used as experimental delivery systems. In all cases, the bacteria used for such proposed vaccines must be attenuated strains, and reversion to virulence, preexisting immunity, and reactogenicity remain major concerns.

      Concerns about the safety of live vector systems are heightened by the well-known reversion of polio vaccine, an attenuated virus, which can cause wild-type polio. An experimental example is the death of infant monkeys immunized with an attenuated simian immunodeficiency virus that had been safe and effective in adult monkeys (16). Theoretical and actual concerns about the use of viral and bacterial vectors have led to efforts to deliver the genes into cells without using a pathogen-derived vector. The DNA vaccines, described below, appear capable of inducing cellular immune responses (by delivering genes into cells) without the potential drawbacks of viral and bacterial vectors.

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      DNA Vaccines

      Plasmid DNA is a simple ring of double-stranded DNA that contains a gene encoding the desired protein (an antigen, in the case of vaccines) and elements needed for the gene to be expressed in whatever cell has taken up the DNA (that is, a promoter at the beginning of the gene and a terminator at the end). It had long been assumed that simple plasmid DNA, called naked DNA because neither a chemical formulation nor a viral coat or envelope structure surrounds it, would not be taken up by cells or could not reach the nucleus for transcription of genes. In 1990, Wolff and colleagues provided compelling evidence that plasmid DNA can transfect cells in vivo (17). This finding opened new possibilities for delivering genes in vivo without the use of viral vectors. Plasmid DNA is considerably simpler to manipulate in terms of putting genes into the plasmid and producing and purifying large quantities of plasmid. Thus, plasmid DNA offers several potential advantages in delivering genes, whether as a vaccine or as a therapy.

      A major attribute of DNA vaccines is their ability to deliver genes into cells for generation of MHC class I–restricted cytolytic T lymphocyte responses. These responses require that the antigens reach the cytosol of specific antigen-presenting cells, and it was previously not clear whether professional antigen-presenting cells could take up plasmid DNA or whether the protein antigen encoded by the DNA (taken up by other cells) would reach the correct antigen-presenting cells and subcellular locations.

      Ulmer and colleagues demonstrated that immunization of mice by intramuscular injection of plasmid DNA encoding an influenza viral protein generated specific cytolytic T cells and protected the mice against subsequent challenge with live influenza virus (18). Morbidity and mortality from infection with a strain of virus that differed from the strain from which the vaccine was made were decreased (Figure 2). Because cytolytic T cells recognize epitopes derived from proteins, including conserved (often internal) proteins, a vaccine that generates cytolytic T cells may protect against different strains. Although antibodies can also be directed against conserved proteins, often the surface or envelope proteins of a virus vary; this variation is often the basis for the categorization of viruses (such as influenza) into different strains (Figure 2, bottom). Viruses escape immune responses by mutating the regions against which an immune response is generated. Although this can occur for both cytolytic T cell and antibody epitopes, the great variability of the envelope of HIV and the ease with which viruses such as influenza mutate their outer proteins to escape antibody responses pose a challenge for making antibody-based vaccines. In contrast, internal proteins (against which antibody responses are often ineffective owing to their intraviral location) still provide cytolytic T-cell epitopes and are conserved to a greater degree. Thus, much effort has been devoted to making vaccines that generate cytolytic T-cell responses against epitopes derived from conserved proteins (which are often internal or functional). Protection was obtained against a strain of influenza virus that was not only a different subtype from the strain from which the gene for the DNA vaccine was derived but also had arisen 34 years later (Figure 2, bottom). This demonstrated in vivo that DNA vaccines could result in production of the encoded protein, which could then enter the appropriate cellular processing pathway and stimulate production of protective cytolytic T cells. As a result, this approach to making new vaccines has now been extended to a variety of disease models.

      Figure 2. . Survival rates among mice immunized against influenza by using a DNA vaccine encoding the nucleoprotein of influenza from the 1934 H1N1 strain ( ). When infected with a lethal dose of influenza from a strain different from the strain from which the vaccine was made, immunized mice had a higher survival rate than did control mice immunized with a control ( ). This protection was mediated by cytolytic T lymphocytes that recognized portions of the nucleoprotein. . The strains from which the DNA vaccine was made ( ) and used to infect ( ) the mice. The influenza nucleoprotein was more highly conserved than the surface proteins. Reproduced with permission from the British Society for Immunology. Donnelly JJ, Ulmer JB, Liu MA. Immunization with polynucleotides. Immunology. 1994; 2:20-6.
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        Figure 2. . Survival rates among mice immunized against influenza by using a DNA vaccine encoding the nucleoprotein of influenza from the 1934 H1N1 strain ( ). When infected with a lethal dose of influenza from a strain different from the strain from which the vaccine was made, immunized mice had a higher survival rate than did control mice immunized with a control ( ). This protection was mediated by cytolytic T lymphocytes that recognized portions of the nucleoprotein. . The strains from which the DNA vaccine was made ( ) and used to infect ( ) the mice. The influenza nucleoprotein was more highly conserved than the surface proteins. Reproduced with permission from the British Society for Immunology. Donnelly JJ, Ulmer JB, Liu MA. Immunization with polynucleotides. Immunology. 1994; 2:20-6. Heterosubtypic protection.TopsquarescirclesBottomleftright

        Figure 3 shows the mechanism of antigen presentation for generation of cytolytic T-cell responses after DNA vaccination. Whereas non–antigen-presenting cells, such as myocytes, can take up plasmid and synthesize the antigen, only antigen-presenting cells can prime cytolytic T cells. Thus, if a non–antigen-presenting cell takes up the DNA vaccine and produces the protein antigen, it must deliver the antigen in some form to a professional antigen-presenting cell by a process called cross-priming, in order for cytolytic T cells to be induced (4).

        Figure 3. Studies in bone marrow chimeric mice demonstrated that after immunization with plasmid DNA encoding an antigen, cytolytic T lymphocytes were activated by antigen-presenting cells ( ) that either had been directly transfected by the DNA or had received antigen via cross-priming, in which a non–antigen-presenting cell initially produces the protein encoded by the DNA vaccine, then transfers the antigen in some form to a professional antigen-presenting cell for generation of MHC class I restricted cytolytic T cells. Production of the protein antigen in non–antigen-presenting cells, such as myocytes, cannot result in direct stimulation of cytolytic T cells .
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          Figure 3. Studies in bone marrow chimeric mice demonstrated that after immunization with plasmid DNA encoding an antigen, cytolytic T lymphocytes were activated by antigen-presenting cells ( ) that either had been directly transfected by the DNA or had received antigen via cross-priming, in which a non–antigen-presenting cell initially produces the protein encoded by the DNA vaccine, then transfers the antigen in some form to a professional antigen-presenting cell for generation of MHC class I restricted cytolytic T cells. Production of the protein antigen in non–antigen-presenting cells, such as myocytes, cannot result in direct stimulation of cytolytic T cells . Mechanism of antigen presentation for generation of cytolytic T cells after DNA vaccination.APC(3)

          Another means of immunizing with a DNA vaccine is to put the plasmid DNA onto gold beads and propel the beads into the skin by using a gene gun. This approach was first shown to induce antibodies against the protein encoded by the plasmid (19). Thus, both the intramuscular and epidermal routes of DNA delivery are effective for generating immune responses, but the responses differed slightly depending on the route of immunization (20-22).

          Compared with other types of vaccine technologies, DNA vaccines potentially have more widespread applications. Once a manufacturing and purification process for the plasmid DNA has been established, a similar process can be applied to a different DNA vaccine, since only the inserted gene will be different. In contrast, each attenuated, inactivated, or recombinant protein vaccine requires a unique manufacturing and purification process. In addition, plasmid DNA is relatively more stable than other existing vaccines, meaning that DNA vaccines may be more suitable for worldwide distribution. Thus, DNA vaccines are considered to be a new platform for making future vaccines, one that can generate cytolytic T lymphocytes in addition to antibodies.

          Other reasons for using a DNA vaccine rather than a live virus vaccine include potential safety advantages. Although attenuated live virus vaccines can stimulate T-cell responses, for certain diseases (such as AIDS) an attenuated live virus vaccine is considered to be too risky; DNA provides a potentially safer alternative. Moreover, some viral proteins have undesired effects (for example, they down-regulate immune responses) or act as decoys for the immune system (for example, regions of HIV Env that generate nonfunctional or highly strain-specific immune responses). Thus, by providing only the genes that encode antigens against which an immune response is desired, a DNA vaccine can induce immune responses in a more specific manner than can a live virus vaccine.

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          Cytolytic T-Cell Responses

          The initial interest in DNA vaccines arose from their ability to induce potent cellular responses mediated by MHC class I–restricted CD8+ cytolytic T cells in animal models. Effector cytolytic T cells that recognized epitope peptides appropriate to the MHC restriction element have been demonstrated in mice immunized with DNA encoding the nucleoprotein from influenza A virus (18), hepatitis B surface antigen and core antigen (23, 24), and HIV Env and Gag antigens (25-29). In addition, cytolytic T cells that could recognize and kill virus-infected targets were demonstrated in models of influenza virus (18), lymphocytic choriomeningitis virus (30, 31), and herpes simplex virus (32).

          Induction of antigen-specific cytolytic T-cell responses by DNA vaccines has likewise been observed in nonhuman primates (26, 33-36) and, more recently, in human clinical trials (37). In several independent experiments, certain regimens of immunization with plasmid DNA encoding HIV or simian immunodeficiency virus env or gag genes induced MHC class I–restricted cytolytic T cells directed against Env and Gag epitopes.

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          Humoral Responses

          DNA vaccines have been shown to elicit antibodies against various viral, bacterial, parasitic, tumor, and eukaryotic proteins, including proteins from influenza (18, 38), HIV (39-43), hepatitis B surface antigen (44), rabiesvirus glycoprotein (45, 46), herpes simplex glycoproteins B and D (47-50), papillomavirus L1 (51, 52), hepatitis C nucleocapsid protein (53, 54), hepatitis B core protein (55), Mycoplasma pulmonis(56), Mycobacterium tuberculosis antigen 85 and heat-shock proteins, (57-59), plasmodial antigens (60), and Leishmania(61) and Schistosoma(62) antigens.

          In a DNA vaccine, the antigen is synthesized in situ (by the immunized host); therefore, it has the potential advantage over a recombinant protein that the resulting antigen would more likely have the conformation and post-translational modifications made by that host during infection by the pathogen. For example, the glycosylation pattern of proteins produced recombinantly in bacterial or yeast cells might differ from the glycosylation pattern of proteins made in mammalian cells. In addition, a transmembrane protein (such as the gp160 Env protein of HIV) cannot be readily produced and purified in a recombinant manner, whereas the protein could be produced in situ by the immunized host after DNA immunization.

          Many infectious diseases enter the host through mucosal surfaces. Thus, it would be advantageous to generate mucosal antibody responses in addition to systemic responses. To date, intramuscular injection and gene gun delivery of naked plasmid DNA have had limited ability to induce secretory mucosal IgA responses. However, formulation of plasmid DNA with cationic lipids (63), monophosphoryl lipid A, or QS-21 (64), encapsulated in poly(lactide-coglycolide) microparticles (65), macroaggregated polyethyleneimine–albumin conjugates (66), or biodegradable alginate microspheres (67) that are delivered intramuscularly, orally, or intranasally, induce significant secretory IgA responses at the mucosal sites. Recently, Wang and colleagues reported induction of strong mucosal immunity against the simian immunodeficiency virus in primates by using DNA encoding intact noninfectious virions (68). The levels of secretory IgA in the rectal secretions of the immunized primates were higher than the levels achieved through natural infection (68).

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          Protection by DNA Vaccines in Preclinical Disease Models

          In addition to the initial demonstration of the efficacy of DNA vaccines to protect against infectious challenge in a mouse model of influenza virus, DNA vaccines have been shown to protect against influenza in mice, ferrets, and primates (69-71); lymphocytic choriomeningitis virus (30, 31); herpes simplex virus in guinea pigs and mice (47-49); rabies virus (45, 46); cottontail rabbit papillomavirus (51); hepatitis B virus (72); malaria (Plasmodium falciparum) (60, 73-75); and HIV in nonhuman primates (25, 26, 35, 43, 76-79).

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          Mixed-Modality Vaccines

          Recent studies have focused on using DNA vaccines in combination with other types of vaccines. If a DNA vaccine encoding an antigen is given as a prime, followed by a different type of vaccine as a boost (for example, a viral vector encoding the same protein), the immune responses or protection observed may be enhanced compared with use of either vaccine alone. For example, when monkeys were first given plasmid DNA encoding HIV Env, then were boosted with recombinant Env protein, they were protected against subsequent infection with a chimeric simian–human immunodeficiency virus (80). Mice that were immunized with DNA encoding malarial antigens and then boosted with recombinant vaccinia (a strain known as modified vaccinia virus Ankara) expressing the same antigens had 100% survival against a subsequent challenge with the parasite. In contrast, the challenge caused substantial mortality among animals given the DNA vaccine alone, the modified vaccinia virus Ankara vector vaccine alone, or the combination of those two vaccines in the reverse order (81). In the malaria model, Sedegah and colleagues showed the increased protective efficacy of DNA vaccines when used in combination with modified vaccinia virus Ankara (74) or another poxvirus (75) vectors. Similarly, in rhesus macaques primed with plasmid DNA encoding multiple proteins of simian immunodeficiency virus (Gag, Pol, Vif, and Vpr) and HIV (Env, Tat, and Rev) and boosted with modified vaccinia Ankara encoding simian immunodeficiency virus proteins Gag and Pol and the HIV protein Env, potent cytolytic T-cell and weak antibody responses were observed. Furthermore, these animals were protected from AIDS when they were mucosally challenged with a highly pathogenic simian–human immunodeficiency virus (35).

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          Clinical Trials

          The DNA vaccines have entered the clinic for initial safety and immunogenicity testing in humans. To date, the potency of the immune responses has been disappointing; nevertheless, humoral and cellular (T helper and cytolytic T cell) responses have been seen (82, 83) (Table 1). Moreover, patients who did not have cellular responses against epitopes derived from the HIV protein Nef nevertheless responded to a DNA vaccine by making the desired cytolytic T-cell responses. Clinical trials of DNA vaccines have been performed or are under way for various diseases, including cancer, influenza, hepatitis B, HIV, and malaria. Phase I clinical trials were initiated to evaluate the safety and immunogenicity of HIV-1 env/rev DNA constructs in infected and uninfected persons (84-87). The uninfected persons who received the highest dose of DNA vaccine had antigen-specific lymphoproliferative responses and antigen-specific production of interferon-γ and β-chemokines (87), but these responses were weak and did not persist. In the infected persons, an HIV-1 env/rev DNA vaccine construct boosted the env-specific antibodies; however, no consistent effect was observed on cellular responses to HIV. Another phase I clinical trial evaluated HIV regulatory genes, such as rev, nef, and tat. Immunization of infected persons with these genes enhanced cellular responses but produced no consistent changes in lymphocytes subsets or viral load (82, 84-86). The DNA vaccines were well tolerated in doses from 20 µg up to 2500 µg; no significant local or systemic reactions were observed, and no participant dropped out of the study (85, 88).

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          Table 1. Clinical Findings of DNA Vaccines

          In a phase I clinical trial of malaria DNA vaccines, three intramuscular injections of Plasmodium falciparum circumsporozoite construct induced antigen-specific, CD8+ T cell–dependent cytolytic T lymphocytes. The cellular responses were directed against multiple epitopes and were restricted by six HLA class I alleles (37). In the same study, despite induction of the excellent cytolytic T cell responses, DNA vaccination failed to induce detectable antigen-specific antibodies in any participant (88).

          A phase I trial of an experimental HIV vaccine that includes an HIV A subtype gag gene and more than 40 bits of DNA encoding regions of HIV proteins is in progress in Kenya. This is the first component of a prime–boost vaccination strategy, and will be followed by a second vaccine using modified vaccinia virus as a vector (89). Another phase I clinical trial was recently started in infected and uninfected persons to directly compare vaccines in which the gene is delivered as naked DNA or by attenuated adenovirus (90).

          A DNA vaccine against hepatitis B virus was evaluated for safety and immunogenicity in a phase I clinical trial involving naive healthy volunteers. A gene gun was used to propel the DNA into the skin. The hepatitis B DNA vaccine was found to be safe, well tolerated, and immunogenic (83). All of the volunteers developed protective antibody responses of at least 10 mIU/mL. Furthermore, this vaccine induced antigen-specific CD8+ T-cell responses in the volunteers who were positive for HLA class 1 A2 allele. These antigen-specific CD8+ T cells bound HLA-A2–hepatitis B surface antigen 335 to 343 tetramers, secreted interferon-γ, and lysed target cells presenting a hepatitis B surface antigen epitope.

          Current phase I and II trials are studying DNA vaccines as potential immunotherapies for various cancers, including colon cancer, human follicular lymphoma, and cutaneous T-cell lymphoma. These studies should soon yield detailed information on the ability of DNA vaccination to induce potent immune responses in humans.

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          Second-Generation DNA Vaccines

          As simple plasmids, DNA vaccines effectively induce immune responses and protection in various animal models of disease, but they have induced only modest immune responses in clinical trials. Thus, different strategies have been used to increase the potency of the DNA vaccines by targeting various points along the pathway for its in vivo delivery into cells for production of the encoded antigen. For example, the plasmids themselves have been altered to increase expression of the encoded proteins (91, 92). Direct modification of the plasmid to increase the immune response (Figure 4) or coadministration of plasmid DNA encoding chemokines, cytokines, and other molecules, such as immunostimulatory sequences (Figure 4), has in some cases increased immunogenicity (64, 75, 79, 93-101).

          Figure 4. Sites within a DNA plasmid can be altered to potentially increase the potency of or alter the type of immune responses induced by the DNA vaccine. BGH = bovine growth hormone; CMVintA = cytomegalovirus promoter with the intron A sequence; mRNA = messenger RNA.
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            Figure 4. Sites within a DNA plasmid can be altered to potentially increase the potency of or alter the type of immune responses induced by the DNA vaccine. BGH = bovine growth hormone; CMVintA = cytomegalovirus promoter with the intron A sequence; mRNA = messenger RNA. Designer gene vaccines.

            An alternative physical delivery system is a device that generates an electric current, in vivo electroporation, to create temporary holes in the cellular membrane. These holes allow the plasmid DNA to diffuse into the cells more effectively, resulting in increased expression and potency of the DNA vaccine (102, 103). Certain formulations or manipulations, such as surface absorption of the DNA onto particles, have also increased the immunogenicity of DNA vaccines, presumably by increasing their transfection efficiency in vivo (104, 105). Of note, plasmid DNA is not simply an inert gene template but has immunostimulatory activity (106-109). Because it is made in bacteria, plasmid DNA has sequences known as CpG motifs that are recognized by the human immune system as foreign (106, 107). Hence, plasmid itself stimulates nonspecific production of cytokines that increases the specific immune response directed against the encoded antigen (108-110).

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            Summary

            In conclusion, recent developments in immunology and molecular biology have permitted development of DNA vaccines, which have wide-ranging applications (Table 2). Vaccines for such diseases as HIV infection, malaria, and tuberculosis are being developed by using plasmid DNA or viral or bacterial vectors to deliver the genes encoding antigens from pathogens to the host. As with the live attenuated virus vaccines that have been made for decades, antigenic proteins will be made in situ by the host, engendering cellular and humoral immune responses. But unlike live attenuated vaccines, gene-based vaccines are being designed to deliver only the genes encoding the antigens for the vaccine. The vectors themselves cannot replicate or revert to pathogenicity because they are rings of DNA (plasmid) or are viral or bacterial vectors designed at a molecular level to carry only the desired genetic sequences.

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            Table 2. Applications of DNA Vaccines

            These DNA and other gene-based vaccines should enable generation of specific types of immune responses (cytolytic T cell, antibodies, and the desired type of T-helper cell). The ability to generate cellular as well as humoral responses may be crucial in making effective vaccines against diseases caused by viruses (such as HIV), intracellular bacteria (such as tuberculosis), and parasites (such as malaria), as well as cancer. Similarly, the ability to generate certain forms of an immunogen, such as a protein with a particular structure that can be formed only by mammalian cells in situ, may be a critical feature of gene vaccines. It is thought that gene vaccines will be amenable to production and distribution on a global scale to provide them to the groups most in need of prevention.

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            Article and Author Information

            • Acknowledgment: The authors thank Nelle Cronen for editorial assistance.

            • Potential Financial Conflicts of Interest:Employment: I. Srivistava (Chiron Corp.), M.A. Liu (Bill & Melinda Gates Foundation, Transgene, Chiron Corp., Merck & Co., Inc.); Consultancies: M.A. Liu; Honoraria: M.A. Liu (Bill & Melinda Gates Foundation); Stock ownership or options (other than mutual funds): I. Srivistava (Chiron Corp.), M.A. Liu (Merck & Co., Inc., Chiron Corp.); Grants received: I. Srivistava (Chiron Corp.); Patents received and pending: M.A. Liu (Merck & Co., Inc., Chiron Corp.).

            • Requests for Single Reprints: Margaret A. Liu, MD, Transgene, 11 rue de Molsheim, 67082 Strasbourg, France; e-mail, liu{at}transgene.fr.

            • Current Author Addresses: Dr. Srivastava: Chiron Corporation, 4560 Horton Street, MS 4.3, Emeryville, CA 94608.

            • Dr. Liu: Transgene, 11 rue de Molsheim, 67082 Strasbourg, France.

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            Summary for Patients

            • Summaries for Patients:Gene Vaccines Offer a Precise and Powerful Approach to Treating Serious Infections Ann Intern Med April 1, 2003 138:I-48
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            1. Ann Intern Med April 1, 2003 vol. 138 no. 7 550-559
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