Gene-Based Vaccines
Authors: Margaret A. Liu, Jeffrey B. Ulmer
Journal: Molecular Therapy (2000)

The inception of DNA vaccines is generally linked to the observation of Wolff et al. in 1990 that intramuscular injection of a plasmid DNA containing a reporter gene resulted in expression of the transgene in situ (1Wolff J.A. Malone R.W. Williams P. Chong W. Acsadi G. Jani A. Felgner P.L. Direct gene transfer into mouse muscle in vivo.Science. 1990; 247: 1465-1468Crossref PubMed Scopus (3117) Google Scholar). In fact, there are at least 14 specific examples published prior to 1989 of in vivo biological activity—including protein expression—after direct inoculation of DNA (linear or plasmid) into tissues of animals (see Table 1). Many of these studies involved the transfer of DNA encoding infectious virus, thereby providing an amplification of gene transfer, but nevertheless demonstrated that direct inoculation of plasmid DNA into tissues of animals could result in transfection of host cells by the plasmid. In one instance, antibody responses were induced against hepatitis B surface antigen (HbsAg) after inoculation of plasmid DNA encoding the hepatits B virus (HBV) genome (6Seeger C. Gaben D. Varmus H.E. The cloned genome of ground squirrel hepatitis virus is infectious in the animal.Proc. Natl. Acad. Sci. USA. 1984; 81: 5849-5852Crossref Scopus (40) Google Scholar), thereby suggesting the potential of this approach for vaccine application. However, 8 more years passed before another example of induction of humoral immune responses using plasmid DNA was published (10Tang D.C. Devit M. Johnston S.A. Genetic immunization is a simple method for eliciting an immune response.Nature. 1992; 356: 152-154Crossref PubMed Scopus (1302) Google Scholar). The following year, priming of cytotoxic T lymphocytes (CTL) with DNA encoding influenza nucleoprotein and protection from a lethal viral challenge were shown (11Ulmer J.B. et al.Heterologous protection against influenza by injection of DNA encoding a viral protein.Science. 1993; 259: 1745-1749Crossref PubMed Scopus (2076) Google Scholar). These results were particularly interesting, given the specialized nature of antigen presentation required to generate CTL. They were also important as they demonstrated proof-of-concept for DNA vaccines in an animal model of infectious disease. Since then, DNA vaccines encoding a multitude of antigens from viruses, bacteria, parasites, fungi, allergens, and tumors have been used successfully to induce immune responses in animal species ranging from mice to humans.TABLE 1ReferenceDNASpeciesRoute of inoculationDemonstration of expression in vivo2Israel M.A. Chan H.W. Hourihan S.L. Rowe W.P. Martin M.A. Biological activity of polyoma viral DNA in mice and hamsters.J. Virol. 1979; 29: 990-996PubMed Google ScholarPolyoma virus genomic DNAHamstersscTumor formation3Will H. Cattaneo R. Koch H.G. Daral G. Schaller H. Cloned HBV DNA causes hepatitis in chimpanzees.Nature. 1982; 299: 740-742Crossref PubMed Scopus (100) Google ScholarHBV-14 genomic DNAChimpanzeesiv, im, ihAntigen expression, viral DNA, hepatitis3Will H. Cattaneo R. Koch H.G. Daral G. Schaller H. Cloned HBV DNA causes hepatitis in chimpanzees.Nature. 1982; 299: 740-742Crossref PubMed Scopus (100) Google ScholarHBV-2 genomic DNAChimpanzeesiv, im, ihAntigen expression, viral DNA, hepatitis3Will H. Cattaneo R. Koch H.G. Daral G. Schaller H. Cloned HBV DNA causes hepatitis in chimpanzees.Nature. 1982; 299: 740-742Crossref PubMed Scopus (100) Google ScholarHBV-6 genomic DNAChimpanzeesiv, im, ihAntigen expression, viral DNA, hepatitis4Fung Y.K. Crittendon L.B.T. Fadly A.M. Kung H.J. Tumor induction by direct injection of cloned v-src DNA into chickens.Proc. Natl. Acad. Sci. USA. 1983; 80: 353-357Crossref Scopus (49) Google Scholarv-src geneChickensscTumor formation, src RNA, src DNA4Fung Y.K. Crittendon L.B.T. Fadly A.M. Kung H.J. Tumor induction by direct injection of cloned v-src DNA into chickens.Proc. Natl. Acad. Sci. USA. 1983; 80: 353-357Crossref Scopus (49) Google ScholarRSV genomic DNAChickensscTumor formation, src RNA, src DNA5Nicolau C. LePape A. Soriano P. Fargette F. Juhel M.F. In vivo expression of rat insulin after intravenous administration of the liposome-entrapped gene for rat insulin I.Proc. Natl. Acad. Sci. USA. 1983; 80: 1068-1072Crossref PubMed Scopus (135) Google ScholarInsulinRatsivProtein expression6Seeger C. Gaben D. Varmus H.E. The cloned genome of ground squirrel hepatitis virus is infectious in the animal.Proc. Natl. Acad. Sci. USA. 1984; 81: 5849-5852Crossref Scopus (40) Google ScholarGSHV genomic DNASquirrelsihAntigen expression, viral DNA, antibody responses7Bouchard L. Gelinas C. Asselin C. Bastin M. Tumorigenic activity of polyoma virus and SV40 DNAs in newborn rodents.Virology. 1984; 135: 53-64Crossref PubMed Scopus (18) Google ScholarPolyoma virus genomic DNARatsscAntigen expression, tumor formation, viral DNA7Bouchard L. Gelinas C. Asselin C. Bastin M. Tumorigenic activity of polyoma virus and SV40 DNAs in newborn rodents.Virology. 1984; 135: 53-64Crossref PubMed Scopus (18) Google ScholarMiddle T geneRatsscAntigen expression, tumor formation, viral DNA7Bouchard L. Gelinas C. Asselin C. Bastin M. Tumorigenic activity of polyoma virus and SV40 DNAs in newborn rodents.Virology. 1984; 135: 53-64Crossref PubMed Scopus (18) Google ScholarSV40 genomic DNAHamstersscAntigen expression, tumor formation, viral DNA7Bouchard L. Gelinas C. Asselin C. Bastin M. Tumorigenic activity of polyoma virus and SV40 DNAs in newborn rodents.Virology. 1984; 135: 53-64Crossref PubMed Scopus (18) Google ScholarLarge T geneHamstersscAntigen expression, tumor formation, viral DNA8Dubensky T.W. Campbell B.A. Villarreal L.P. Direct transfection of viral and plasmid DNA into the liver or spleen of mice.Proc. Natl. Acad. Sci. USA. 1984; 81: 7529-7533Crossref PubMed Scopus (84) Google ScholarPolyoma virus genomic DNAMiceis, ihViral DNA9Benvenisty N. Reshef L. Direct introduction of genes into rats and expression of the genes.Proc. Natl. Acad. Sci. USA. 1986; 83: 9551-9555Crossref PubMed Scopus (70) Google ScholarCAT, insulin, growth hormoneRatsipProtein expression Open table in a new tab One of the initial attractions of using an expression plasmid DNA as vaccine was the possibility of synthesizing antigens within cells of the inoculated host, thereby allowing processing and presentation by MHC molecules. It was reasoned that this would lead to priming of T-cell responses. In fact, DNA vaccines have turned out to be potent inducers of T-cell responses, but are less strong at priming antibody responses when compared to certain other means of immunization (e.g., recombinant protein in adjuvant). This is true for mice, monkeys, and, in the few examples studied to date, humans. One possible explanation may lie in the amount of antigen that is produced. Based on estimates from reporter gene studies, only picogram to nanogram amounts of protein are expressed (1Wolff J.A. Malone R.W. Williams P. Chong W. Acsadi G. Jani A. Felgner P.L. Direct gene transfer into mouse muscle in vivo.Science. 1990; 247: 1465-1468Crossref PubMed Scopus (3117) Google Scholar, 12Manthorpe M. et al.Gene therapy by intramuscular injection of plasmid DNA: Studies on firefly luciferase gene expression in mice.Hum. Gene Ther. 1993; 4: 419-431Crossref PubMed Scopus (395) Google Scholar). This small amount of protein given as a subunit vaccine would not be sufficient to induce a vigorous immune response. However, it has been shown that very little antigen, when presented in the context of an antigen-presenting cell (APC), is sufficient to induce T-cell responses (13Fields R.C. Shimizu K. Mulé J.J. Murine dendritic cells pulsed with whole tumor lysates mediate potent antitumor immune responses in vitro and in vivo.Proc. Natl. Acad. Sci. USA. 1998; 95: 9482-9487Crossref PubMed Scopus (402) Google Scholar). The means by which such T cell responses are primed is still being elucidated. However, it appears that at least two factors are important: the type of cells involved and the potential effect of immunostimulatory motifs within bacterial plasmid DNA. After injection of DNA vaccines, most of the antigen is produced by non-APCs, such as myocytes and cells of the skin. However, these cells do not directly prime T-cell responses; professional APCs derived from the bone marrow are required (14Corr M. Lee D.J. Carson D.A. Tighe H. Gene vaccination with naked plasmid DNA: Mechanism of CTL priming.J. Exp. Med. 1996; 184: 1555-1560Crossref PubMed Scopus (420) Google Scholar, 15Fu T.M. et al.Priming of cytotoxic T lymphocytes by DNA vaccines: Requirement for professional antigen presenting cells and evidence for antigen transfer from myocytes.Mol. Med. 1997; 3: 362-371Crossref PubMed Google Scholar, 16Iwasaki A. Torres C.A. Ohashi P.S. Robinson H.L. Barber B.H. The dominant role of bone marrow-derived cells in CTL induction following plasmid DNA immunization at different sites.J. Immunol. 1997; 159: 1-4Google Scholar). This presents an apparent paradox, but can be explained as follows. T cells can be primed through antigen presentation after production of antigen within the APC itself, such as after virus infection, as well as after acquisition of antigen synthesized by another cell (i.e., cross-priming). Both of these processes appear to play a role in the induction of T cell responses after DNA vaccination. Evidence now exists for both production of antigen by APCs (17Condon C. Watkins S.C. Celluzzi C.M. Thompson K. Falo Jr., L.D. DNA-based immunization by in vivo transfection of dendritic cells.Nat. Med. 1996; 2: 1122-1228Crossref PubMed Scopus (778) Google Scholar, 18Chattergoon M.A. Robinson T.M. Boyer J.D. Weiner D.B. Specific immune induction following DNA-based immunization through in vivo transfection and activation of macrophages/antigen-presenting cells.J. Immunol. 1998; 160: 5707-5718PubMed Google Scholar, 19Akbari O. et al.DNA vaccination: Transfection and activation of dendritic cells as key events for immunity.J. Exp. 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Immunol. 1999; 163: 4721-4727PubMed Google Scholar) after DNA vaccination. The relative importance of these two distinct pathways in T-cell priming by DNA vaccines is the subject of current debate, and it may be dependent upon the route of DNA administration. Recent work has suggested that direct transfection of APCs plays a predominant role after gene gun administration of DNA (24Porgador A. et al.Predominant role for directly transfected dendritic cells in antigen presentation to CD8+ T cells after gene gun immunization.J. Exp. Med. 1998; 188: 1075-1082Crossref PubMed Scopus (479) Google Scholar), but that cross-priming is most important after intramuscular DNA injection (23Corr M. vonDamm A. Lee D.J. Tighe H. In vivo priming by DNA injection occurs predominantly by antigen transfer.J. Immunol. 1999; 163: 4721-4727PubMed Google Scholar). Regardless of which pathway is most important, both are sufficient, neither is necessary, and it is likely that both are involved in the induction of T-cell responses by DNA vaccines. Regarding the second important factor, DNA vaccines appear to have a built-in adjuvant that contributes to the induction of immune responses in quantitative and qualitative ways. More than 15 years ago, Tokunaga and colleagues discovered that DNA from Mycobacterium bovis had antitumor activity (25Tokunaga T. et al.Antitumor activity of deoxyribonucleic acid fraction from Mycobacterium bovis BCG. I. Isolation, physicochemical characterization, and antitumor activity.J. Natl. Cancer Inst. 1984; 72: 955-962PubMed Google Scholar) and subsequently traced the effect to immunostimulatory properties of a particular GC-rich motif (26Yamamoto S. et al.Unique palindromic sequences in synthetic oligonucleotides are required to induce TNF and augment TNF-mediated natural killer activity.J. Immunol. 1992; 148: 4072-4076PubMed Google Scholar). These properties of DNA were shown to be effective in vivo (27Messina J.P. Gilkeson G.S. Pisetsky D.S. Stimulation of in vitro murine lymphocyte proliferation by bacterial DNA.J. Immunol. 1991; 147: 1759-1764PubMed Google Scholar) and to have wideranging effects on cells of the immune system (28Krieg A.M. et al.CpG motifs in bacterial DNA trigger direct B-cell activation.Nature. 1995; 374: 546-549Crossref PubMed Scopus (3052) Google Scholar). These effects include upregulation of expression of several different cytokines, such as IL-12, IL-6, and interferon-γ in B cells and macrophages (29Klinman D.M. Yi A.K. Beaucage S.L. Conover J. Krieg A.M. CpG motifs present in bacteria DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon gamma.Proc. Natl. Acad. Sci. USA. 1996; 93: 2879-2883Crossref PubMed Scopus (1349) Google Scholar), as well as upregulation of activation markers in dendritic cells (30Jakob T. Walker P.S. Krieg A.M. Udey M.C. Vogel J.C. Activation of cutaneous dendritic cells by CpG-containing oligodeoxynucleotides: A role for dendritic cells in the augmentation of Th1 responses by immunostimulatory DNA.J. Immunol. 1998; 161: 3042-3049PubMed Google Scholar). The rapidity with which these responses are manifest (hours) suggests that CpG motifs stimulate innate immune responses. How does this relate to DNA vaccines? It is known that plasmid DNA is rapidly degraded by extracellular nucleases in the injected tissue (31Barry M.E. et al.Role of endogenous endonucleases and tissue site in transfection and CpG-mediated immune activation after naked DNA injection.Hum. Gene Ther. 1999; 10: 2461-2680Crossref PubMed Scopus (149) Google Scholar). This likely liberates oligonucleotides, some of which may contain CpG motifs, and these may serve to “ready” the immune system for priming of an adaptive immune response against the antigen expressed by the DNA vaccine. Moreover, the Th1 bias of CpG motifs may explain why DNA vaccines preferentially induce strong cellular immune responses of the Th1 type. That CpG motifs within DNA vaccines play a role in their potency was demonstrated by the conversion of a low-potency DNA vaccine into a high-potency one merely by cloning in a known active CpG motif (32Sato Y. et al.Immunostimulatory DNA sequences necessary for effective intradermal gene immunization.Science. 1996; 273: 352-354Crossref PubMed Scopus (950) Google Scholar). However, it is now apparent that several factors may determine the CpG effect within DNA vaccines, including species-specific sequences, regions of the plasmid flanking the CpG motif, and other sequences within the plasmid that interfere with the immunostimulatory effects of active motifs (33Krieg A.M. et al.Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs.Proc. Natl. Acad. Sci. USA. 1998; 95: 12631-12636Crossref PubMed Scopus (354) Google Scholar). The general applicability of DNA vaccines at inducing broad-based immune responses in small animal models is now well established [for review, see (34Donnelly J.J. Ulmer J.B. Shiver J.W. Liu M.A. DNA vaccines.Annu. Rev. Immunol. 1997; 15: 617-648Crossref PubMed Scopus (1132) Google Scholar)]. However, the magnitude of immune responses induced in primates is generally lower than that in small animals. Furthermore, the amount of DNA required for effective immunization of primates is much higher than for small animals and often requires multiple immunizations involving prime-boost scenarios with recombinant protein or viral vectors. In addition, data from phase I human clinical studies have been published with modest (or no) immune responses reported (see Table 2). Other phase I clinical trials have been conducted involving influenza, hepatitis B, and herpes simplex genes, but no results have been reported from those studies. Therefore, it is generally regarded that naked DNA vaccines are not sufficiently potent in primates (including humans) for successful application and that an enabling technology is required.TABLE 2ReferencesGene insertRoute of inoculationImmune responses35MacGregor R. et al.First human trial of a DNA-based vaccine for treatment of human immunodeficiency virus type 1 infection: Safety and host response.J. Infect. Dis. 1998; 178: 92-100Crossref PubMed Scopus (531) Google ScholarHIV-1 env/revimVery modest increase in antibodies, CTL in HIV+ volunteers36Wang R. et al.In duction of antigen-specific cytotoxic T lymphocytes in humans by a malaria DNA vaccine.Science. 1998; 282: 476-480Crossref PubMed Scopus (677) Google ScholarMalaria circumsporozoiteimCTL, no antibodies37Calarota S. et al.Cellular cytotoxic response induced by DNA vaccination in HIV-1-infected patients.Lancet. 1998; 351: 1320-1325Abstract Full Text Full Text PDF PubMed Scopus (364) Google ScholarHIV-I nef, rev, tatimTransient, CTL in HIV+ volunteers38Calarota S.A. et al.Immune responses in asymptomatic HIV-1-infected patients after HIV-DNA immunization followed by highly active antiretroviral treatment.J. Immunol. 1999; 162: 2330-2338Google ScholarHIV-I nef, rev, tatimCMI, low antibodies in HIV+ volunteers39Boyer J.D. et al.Enhancement of cellular immune responses in HIV-1 seropositive individuals: A DNA-based trial.Clin. Immunol. 1999; 90: 100-107Crossref PubMed Scopus (84) Google Scholar, 40Boyer J.D. et al.Vaccination of seronegative volunteers with a human immunodeficiency virus type I env/rev DNA vaccine induces antigen-specific proliferation and lymphocyte production of ?-chemokines.J. Infect. Dis. 2000; 181: 476-483Crossref PubMed Scopus (149) Google ScholarHIV-I env/revimCMI at high dose group41Tacket C.O. et al.Phase I safety and immune response studies of a DNA vaccine encoding hepatitis B surface antigen delivered by a gene delivery device.Vaccine. 1999; 17: 2826-2829Crossref PubMed Scopus (190) Google ScholarHBsAgGene gunNone Open table in a new tab Although DNA vaccines have generated significant interest based on their simplicity and surprising robustness in preclinical models, as with many new technologies they will benefit from, or perhaps be critically dependent upon, both a more complete understanding of their mechanisms of action (e.g., the role of cross-priming versus direct transfection of APCs, role of CpG motifs) and improvements in potency. Approaches that may enable successful human application include stronger expression vectors, facilitated delivery systems for DNA, adjuvants, and combinations thereof. Promising second-generation DNA vaccines (for example, see Fig. 1) are now being evaluated preclinically utilizing formulated plasmids (42Singh M. Briones M. Ott G. O'Hagan D. Cationic microparticles: A potent delivery system for DNA vaccines.Proc. Natl. Acad. Sci. USA. 2000; 97: 811-816Crossref PubMed Scopus (455) Google Scholar), in vivo electroporation (43Widera G. et al.In creased DNA vaccine delivery and immunogenicity by electroporation in vivo.J. Immunol. 2000; 164: 4635-4640Crossref PubMed Scopus (449) Google Scholar), and self-amplifying systems (44Dubensky T.W. et al.Sindbis virus DNA-based expression vectors: Utility for in vitro and in vivo gene transfer.J. Virol. 1996; 70: 508-514PubMed Google Scholar). If one considers each of the potential steps from the administration of the plasmid through cellular uptake, to nuclear localization, to expression, it is clear that a number of processes can be targeted for improvement. Additionally, adjuvantation of the immune response, possibly via the sequence of the DNA itself, via traditional forms of adjuvants, or via cytokines, may be useful. Some of these developments move DNA vaccines away from their original simple concept of an inert gene delivery system toward entities that have some of the characteristics of viral or bacterial vectors. The benefits of these improvements in terms of increasing potency, safety, or delivery will need to be weighed against the consequences of increased complexity to manufacture, inherent immunogenicity of the vector, and any resulting effect upon potential safety perception. Moreover, while DNA vaccines have been evaluated by somewhat different regulatory/evaluative groups than other gene therapy approaches, little if any distinction exists in terms of both clinical applications and the actual technology employed in the clinic. For example, plasmids encoding angiogenic proteins differ only in the protein encoded and the muscle injected compared to vaccine plasmids. Similarly, while cancer applications are largely therapeutic versus infectious disease vaccine applications, which so far have mostly been prophylactic, the actual constructs focus upon antigens and cytokines in both cases. Hence, the technologies developed and safety information gained from preclinical and clinical studies for vaccines may be useful for other gene therapy applications. Gene-based vaccines thus could be considered a subset of gene therapy extending the clinical applications from therapy to prophylaxis of disease. By better understanding how to utilize these gene delivery systems to induce specific immune responses, such information can be exploited both for immune therapies of noninfectious diseases and to learn how to suppress immune responses for applications of delivery of therapeutic proteins.