DNA vaccines are usually circular plasmids that include a gene encoding the target antigen (or antigens) under the transcriptional control of a promoter region active in human cells. The coding region of the inserted gene is followed by transcription termination and polyadenylation sequences. To permit selection of plasmid-containing bacteria during the production process, the plasmid also contains an antibiotic resistance gene with a bacterial origin of replication. DNA is generally less costly to produce than peptide or protein vaccines, and is chemically stable under a variety of conditions. DNA vaccines are generally administered intramuscularly, using either a needle and syringe or a needle-free injector. DNA vaccines were first tested in human beings with HIV infection,(1) and subsequently in uninfected people as preventive vaccines against HIV(2,3) and malaria.(4) While immune responses to DNA alone have been relatively weak in humans, combination with adjuvants or with recombinant viral vectors in prime-boost approaches have resulted in appreciable HIV-specific CD8 responses(5) and have induced protective responses in primate models (see below).
Safety Issues
The following issues have been raised with regard to DNA vaccines
1) Integration into cellular DNA
Available evidence suggests that DNA vaccines currently being tested rarely integrate into cellular DNA. However, as vectors are modified or adjuvanted with the goal of increasing immunogenicity, the likelihood of integration may increase. The concern is that an integrated vaccine may result in insertional mutagenesis through the activation of oncogenes or inactivation of tumor suppressor genes. In addition, an integrated plasmid DNA vaccine may in theory result in chromosomal instability through the induction of chromosomal breaks or rearrangements. FDA continues to recommend integration studies for new DNA products.(6) These studies are conducted for each plasmid vaccine product using accepted assays in animals prior to the initiation of human trials. Typically, if integration is detected at all, it is found to occur at rates that are orders of magnitude below the spontaneous mutation frequency.(7)
2) Development of autoimmunity
Studies in mice have shown that systemic autoimmunity is unlikely to result from DNA vaccination,(6) and early human studies did not detect increases in antinuclear or anti-DNA antibodies.(1,4) Participants in human trials of DNA vaccines are followed for possible signs and symptoms of autoimmunity, and laboratory markers of autoimmunity are sometimes monitored as well. To date there has been no convincing evidence of DNA vaccine-associated autoimmunity.
3) Antibiotic resistance
Part of the production process of DNA plasmids involves selection of bacterial cells carrying the plasmid. This selection is accomplished by culturing the cells in the presence of an antibiotic to which resistance is conferred by a gene in the plasmid. Concern has been raised that resistance to the same antibiotic might be introduced in participants when the plasmid is used in clinical trials. Two precautions make this outcome unlikely. First, the antibiotic resistance genes contained by vaccine plasmids are driven by a bacterial origin of replication sequence (not a mammalian one) and are therefore expressed only in bacteria, not in host cells. Second, the antibiotic resistance employed does not involve antibiotics commonly used to treat human infections.
The FDA has developed specific advice on safety testing of DNA vaccines,(8) as has the European Union.(9)
Immunogenicity
The immune response to DNA vaccines results from uptake of plasmids into cells (including dendritic and muscle cells), where expression of the target antigen gene (or genes) occurs. The resulting proteins undergo processing as intracytoplasmic antigens, producing peptides that bind to Class I MHC molecules. The presentation of these MHC-bound peptides on the cell surface stimulates CD8 T-lymphocyte responses. Antibody responses to plasmid-encoded proteins are also observed, suggesting that plasmid-encoded protein antigens reach and stimulate B lymphocytes as well. DNA vaccines thus mimic viral infection by inducing both cellular and humoral immune responses.
The magnitude of these responses is generally modest when DNA is used alone. Primate studies(10,11,12-16) and preliminary results of human trials(5,17) suggest that more potent specific immune responses may be induced by combining DNA with adjuvants, by boosting with a recombinant viral vector or protein, or by both adjuvanting and boosting.
Challenge Studies in Primates
In primates, immunization regimens including a DNA vaccine have been found to provide protection (prevention of infection, or decreased viral load with improved clinical outcomes) following challenge with an immunodeficiency virus. The following studies provide evidence of such protection from DNA vaccine-containing regimens analogous to those currently being developed for human HIV vaccine trials:
DNA plasmid prime + recombinant modified vaccinia Ankara (MVA) boost(10)
DNA plasmid with cytokine (interleukin-2) adjuvant(11)
DNA plasmid with nonionic blocked copolymer adjuvant, with or without recombinant adenovirus boost(12)
DNA plasmid prime + recombinant fowlpox virus boost(13,16)
DNA plasmid prime with cytokine (interleukin-12) adjuvant + recombinant gp140 protein boost(18)
Of note, other studies have failed to find protection in primates challenged with SIV or SHIV viruses following DNA vaccine-containing regimens,(19,20) or have found protection of limited duration.(21)
