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DNA vaccines: improving expression of antigens
Genetic Vaccines and Therapyvolume 1, Article number: 2 (2003)
DNA vaccination is a relatively recent development in vaccine methodology. It is now possible to undertake a rational step-by-step approach to DNA vaccine design. Strategies may include the incorporation of immunostimulatory sequences in the backbone of the plasmid, co-expression of stimulatory molecules, utilisation of localisation/secretory signals, and utilisation of the appropriate delivery system, for example. However, another important consideration is the utilisation of methods designed to optimise transgene expression. In this review we discuss the importance of regulatory elements, kozak sequences and codon optimisation in transgene expression.
In 1990, the direct gene transfer of plasmid DNA into mouse muscle in vivo without the need for a special delivery system was demonstrated . Furthermore, intramuscular inoculation with plasmid DNA encoding reporter genes induced protein expression within the muscle cells. This study provided evidence for the idea that naked DNA could be delivered in vivo to direct protein expression. Subsequently, a further study reported the gene expression a year or more after intramuscular injection of plasmid DNA . Since these initial studies, many more experiments have been carried out to evaluate different factors that determine the efficiency of gene transfer and immunogenicity of plasmid DNA. Furthermore, plasmid DNA has been used to immunise against a variety of diseases (known as DNA vaccination). Alternatively, plasmid DNA has been used to treat genetic diseases and similar factors may affect the efficacy of this gene therapy.
DNA vaccines usually consist of plasmid vectors (derived from bacteria) that contain heterologous genes (transgenes) inserted under the control of a eukaryotic promoter, allowing protein expression in mammalian cells . An important consideration when optimising the efficacy of DNA vaccines is the appropriate choice of plasmid vector. The basic requirements for the backbone of a plasmid DNA vector are a eukaryotic promoter, a cloning site, a polyadenylation sequence, a selectable marker and a bacterial origin of replication . A strong promoter may be required for optimal expression in mammalian cells. For this, some promoters derived from viruses such as cytomegalovirus (CMV) or simian virus 40 (SV40) have been used. A cloning site downstream of the promoter should be provided for insertion of heterologous genes, and inclusion of a polyadenylation (polyA) sequence such as the bovine growth hormone (BGH) or SV40 polyadenylation sequence provides stabilisation of mRNA transcripts. The most commonly used selectable markers are bacterial antibiotic resistance genes, such as the ampicillin resistance gene. However, since the ampicillin resistance gene is precluded for use in humans, a kanamycin resistance gene is often used. Finally, the Escherichia coli ColE1 origin of replication, which is found in plasmids such as those in the pUC series, is most often used in DNA vaccines because it provides high plasmid copy numbers in bacteria enabling high yields of plasmid DNA on purification. This review describes the utilisation of methods designed to optimise transgene expression.
Various reports have described the strength of promoter/enhancers or other transcriptional elements in DNA vaccines (see Table 1) [5–20]. In general, virally-derived promoters have provided greater gene expression in vivo than other eukaryotic promoters. In particular, the CMV immediate early enhancer-promoter (known as the CMV promoter) has often been shown to direct the highest level of transgene expression in eukaryotic tissues when compared with other promoters. For example, in one study a plasmid expressing human immunodeficiency virus type 1 (HIV-1) Gag/Env under the regulation of the CMV promoter/enhancer was compared to a comparable plasmid utilising the endogenous AKV murine leukemia long terminal repeat . Analysis of the immune responses in macaques injected with the plasmids showed that the CMV-containing plasmid elicited higher Gag- and Env-specific humoral and T-cell proliferative responses, reflecting the greater transcriptional activity of the CMV promoter. Furthermore, it has been demonstrated that inclusion of the CMV intron A improved the level of expression of transgenes expressed by the CMV promoter or other promoter/enhancers . It is thought that the beneficial effect of introns on expression is primarily due to an enhanced rate of polyadenylation and/or nuclear transport associated with RNA splicing . However, some widely used virally-derived promoters, such as the CMV promoter, may not be suitable for some gene therapy applications since treatment with interferon-γ or tumour necrosis factor-α may inhibit transgene expression from DNA vaccines containing these promoters [23, 24]. Thus, alternatives to the CMV promoter have been sought. For example, the desmin promoter/enhancer, which controls expression of the muscle-specific cytoskeletal protein desmin, was used effectively to drive expression of the hepatitis B surface antigen priming both humoral and cellular immunity against the antigen . These responses were shown to be of a comparable magnitude to those in mice immunised with comparable DNA vaccines containing the CMV promoter. Other tissue-specific promoters that have been studied include the creatine kinase promoter, also specific to muscle cells [5, 25], and the metallothionein and 1,24-vitaminD(3)(OH)(2) dehydroxylase promoters, both of which are specific to keratinocytes .
Since the rate of transcriptional initiation is generally increased by the use of strong promoter/enhancers, the rate of transcriptional termination may become rate-limiting . In addition, the efficiency of primary RNA transcript processing and polyadenylation is known to vary between the polyadenylation sequences of different genes. Thus, the polyadenylation sequence used within a DNA vaccine may also have significant effects on transgene expression. For example, it was demonstrated that the commonly used SV40 polyadenylation sequence was less efficient than the minimal rabbit β-globin and bovine growth hormone polyadenylation sequences in mouse liver, although addition of a second SV40 enhancer downstream of the SV40 polyadenylation signal did increase expression to a level comparable to the other signals . Therefore, it is possible that the strategy of inserting a second SV40 enhancer downstream of a SV40 polyadenylation sequence may be utilised in the construction of more efficient vectors.
Sequences flanking the AUG initiator codon within mRNA influence its recognition by eukaryotic ribosomes. As a result of studying the conditions required for optimal translational efficiency of expressed mammalian genes, the 'Kozak' consensus sequence has been shown to be important [28, 29]. It has been proposed that this defined translational inititiating sequence (-6 GCCA/GCCAUGG +4) should be included in vertebrate mRNAs located around the initiator codon . It has also been suggested that efficient translation is obtained when the -3 position contains a purine base or, in the absence of a purine base, a guanine is positioned at +4 . Prokaryotic genes and some eukaryotic genes do not possess Kozak sequences. Therefore, the expression level of these genes might be increased by the insertion of a Kozak sequence.
Codon bias is observed in all species, and the use of selective codons in genes often correlates with gene expression efficiency . In general, taxonomically-close organisms, such as E. coli and Salmonella enterica serovar Typhimurium, for example, use similar codons for their protein synthesis whereas taxonomically-distant organisms, such as E. coli and Saccharomyces cerevisiae, utilise very different codons . Mammalian codon usage is also different from that of microorganisms . Nagata et al.  studied the effect of codon optimisation for mammalian cells of cytotoxic T-lymphocyte (CTL) epitopes derived from the intracellular bacterium, Listeria monocytogenes, and the parasite Plasmodium yoelii, and reported that the codon optimisation level of the genes correlated well with translational efficiency in mammalian cells.
The greatest deviation from random codon usage in an organism occurs in the most highly expressed genes as a result of selection for codons that maximise translational efficiency . Minor tRNA species are avoided in highly expressed genes. Thus, differences between codon usage in a heterologous gene and the host organism may affect expression. To improve expression of human immunodeficiency virus type 1 gp120 from a DNA vaccine vector, André et al. generated a synthetic gp120 sequence in which most of the wild-type codons were replaced with codons from highly expressed human genes. The resulting construct showed increased in vitro expression of gp120 compared to the wild-type sequence. In addition, significantly increased antibody titres and CTL reactivity were observed following administration of the vector containing the synthetic sequence. Similarly, a DNA vaccine vector encoding a synthetic epitope of listeriolysin O with mammalian codon usage showed higher translation efficiency than a vector containing the wild-type sequence in murine cells . Furthermore, the first DNA vaccine was capable of inducing specific CD8+ T cells able to confer partial protection against challenge with L. monocytogenes where the second DNA vaccine could not. A number of other studies have reported that increased immune responses may be obtained by DNA vaccination with a transgene sequence with optimised codon usage. [36–40].
In this review the methodologies by which antigen expression has been optimised to date, i.e. optimisation of vector and transgene sequences, have been discussed. It is clear that transgene expression may be increased through the use of optimised promoters and polyA sequences. However, in some circumstances it may be necessary to optimise DNA vaccines to produce reduced transgene expression. For example, the weaker SV40 promoter has been used rather than the CMV promoter to drive expression of antigens that induce cell death upon overexpression . Tissue-specificity is also considered important. Such tissue-specific expression systems may be able to produce stable expression by reducing the probability of inducing an immune response to the transgene. It may be possible to design vectors for gene therapeutic purposes that avoid inducing unwanted immune responses against the encoded antigen by using tissue-specific promoters . Restricting the site of expression of genes should minimise the risks related to aberrant expression of a gene product. Furthermore, it should be possible to develop expression systems where gene products are only expressed in the critical cell types for DNA vaccination or gene therapy, for example, dendritic cells (DCs). As a better understanding of the proteins whose expression is limited to DCs is obtained, novel expression systems will be generated. Finally, through increased knowledge of the regulation of expression of antigens, it is now possible to produce multivalent systems whereby multiple antigens may be expressed from a single DNA vaccine vector .
It is clear that the optimisation of antigen expression is an important consideration in DNA vaccine vector design. However, it is important to recognise that other aspects of vector design may influence the efficacy of the vaccine/gene therapy. A rational approach to improve the efficacy of DNA vaccination or gene therapy would optimise the: (i) vector backbone DNA sequence; (ii) transgene sequence; (iii) co-expression of stimulatory sequences; (iv) delivery system used for the vector; (v) targeting of the vector for appropriate immune stimulation.
The backbone of a DNA vaccine vector could be further modified to enhance immunogenicity via the manipulation of the DNA to include certain sequences, so that the DNA itself will have an adjuvantising effect. DNA vaccine vectors contain many CpG motifs (consisting of unmethylated CpG dinucleotides flanked by two 5' purines and two 3' pyrimidines) that, overall, induce a Th1-like pattern of cytokine production , and are thought to account for strong CTL responses frequently seen following DNA vaccination . It is possible to augment responses to DNA vaccine vectors by incorporating CpG motifs into the DNA backbone of the plasmid . Alternatively, immune responses may be modulated or enhanced by the co-expression of stimulatory molecules or cytokines [46, 4] or through the use of localisation or secretory signals [47–49], or ligand fusions [50–54] to direct antigens to sites appropriate for immune modulation. Finally, a variety of routes of administration of DNA vaccines have been studied, including intramuscular, intradermal, subcutaneous, intravenous, intraperitoneal, oral, vaginal, intranasal and, more recently, non-invasive delivery to the skin (reviewed by Gurunathan et al. ).
The approaches outlined above will together allow for the rational and optimised design for DNA vaccines and gene therapy vectors. The ability to improve antigen expression through the use of optimisation of regulatory elements, kozak sequences and codon usage is highlighted in this review, as part of this rational approach.
Wolff JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A, Felgner PL: Direct gene transfer into mouse muscle in vivo. Science. 1990, 247: 1465-1468.
Wolff JA, Ludtke JJ, Acsadi G, Williams P, Jani A: Long-term persistence of plasmid DNA and foreign gene expression in mouse muscle. Hum. Mol. Gen. 1992, 1: 363-369.
Davis HL: Plasmid DNA expression systems for the purpose of immunisation. Curr. Opin. Biotechnol. 1997, 8: 635-640. 10.1016/S0958-1669(97)80041-9.
Gurunathan S, Klinman DM, Seder RA: DNA vaccines: immunology, application, and optimization. Ann. Rev. Immunol. 2000, 18: 927-974. 10.1146/annurev.immunol.18.1.927.
Bartlett RJ, Secore SL, Singer JT, Bodo M, Sharma K, Ricordi C: Long-term expression of a fluorescent reporter gene via direct injection of plasmid vector into mouse skeletal muscle: comparison of human creatine kinase and CMV promoter expression levels in vivo. Cell Transplant. 1996, 5: 411-419. 10.1016/0963-6897(95)02026-8.
Hannas-Djebbara Z, Didier-Bazs M, Sacchettoni S, Prod'hon C, Jouvet M, Belin MF, Jacquemont B: Transgene expression of plasmid DNAs directed by viral or neural promoters in the rat brain. Brain Res. Mol. Brain Res. 1997, 46: 91-99. 10.1016/S0169-328X(96)00276-8.
Mukhtar M, Duan L, Bagasra O, Pomerantz RJ: Evaluation of relative promoter strengths of the HIV-1-LTR and a chimeric RSV-LTR in T lymphocyte cells and peripheral blood mononuclear cells: promoters for anti-HIV-1 gene therapies. Gene Ther. 1996, 3: 725-730.
Lee AH, Suh YS, Sung JH, Yang SH, Sung YC: Comparison of various expression plasmids for the induction of immune response by DNA immunization. Mol Cells. 1997, 7: 495-501.
Tucker C, Endo M, Hirono I, Aoki T: Assessment of DNA vaccine potential for juvenile Japanese flounder Paralichthys olivaceus, through the introduction of reporter genes by particle bombardment and histopathology. Vaccine. 2000, 19: 801-809. 10.1016/S0264-410X(00)00233-4.
Xu Z-L, Mizuguchi H, Ishii-Watabe A, Uchida E, Mayumi T, Hayakawa T: Optimization of transcriptional regulatory elements for constructing plasmid vectors. Gene. 2001, 272: 149-156. 10.1016/S0378-1119(01)00550-9.
Kwissa M, von Kampen J, Zurbriggen R, Gluck R, Reimann J, Schirmbeck R: Efficient vaccination by intradermal or intramuscular inoculation of plasmid DNA expressing hepatitis B surface antigen under desmin promoter/enhancer control. Vaccine. 2000, 18: 2337-2344. 10.1016/S0264-410X(00)00030-X.
Loriat D, Li Z, Mancini M, Tiollais P, Paulin D, Michel ML: Muscle-specific expression of hepatitis B antigen: no effect on DNA-raised immune responses. Virology. 1999, 260: 74-83. 10.1006/viro.1999.9795.
Xiang ZQ, Spitalnik SL, Cheng J, Erikson J, Wojczyk B, Ertl HC: Immune responses to nucleic acid vaccines to rabies virus. Virology. 1995, 209: 569-579. 10.1006/viro.1995.1289.
Kodihalli S, Goto H, Kobasa DL, Krauss S, Kawaoka Y, Webster RG: DNA vaccine encoding haemagglutinin provides protective immunity against H5N1 influenza virus in mice. J. Virol. 1999, 73: 2094-2098.
Saurez DL, Schultz-Cherry S: The effect of eukaryotic expression vectors and adjuvants on DNA vaccines in chickens using an avian influenza model. Avian Dis. 2000, 44: 861-868.
van Drunen Littel-van den Hurk S, Braun RP, Lewis PJ, Karvonen BC, Baca-Estrada ME, Snider M, McCartney D, Watts D, Babiuk LA: Intradermal immunization with a bovine herpesvirus-1 DNA vaccine induces protective immunity in cattle. J. Gen. Virol. 1998, 79: 831-839.
Galvin TA, Muller J, Khan AS: Effect of different promoters on immune responses elicited by HIV-1 gag/env multigenic DNA vaccine in Macaca mulatta and Macaca nemestrina. Vaccine. 2000, 18: 2566-2583. 10.1016/S0264-410X(99)00569-1.
Watts AM, Bright RK, Kennedy RC: DNA cancer vaccination strategies target SV40 large tumour antigen in a murine experimental metastasis model. 2000
Garapin A-C, Ma L, Pescher P, Lagranderie M, Marchal G: Mixed immune response induced in rodents by two naked DNA genes coding for mycobacterial glycosylated proteins. Vaccine. 2001, 19: 2830-2841. 10.1016/S0264-410X(01)00012-3.
Gill DR, Smyth SE, Goddard CA, Pringle IA, Higgins CF, Colledge WH, Hyde SC: Increased persistence of lung gene expression using plasmids containing the ubiquitin C or elongation factor 1-alpha promoter. Gene Ther. 2001, 8: 1539-1546. 10.1038/sj.gt.3301561.
Chapman BS, Thayer RM, Vincent KA, Haigwood NL: Effect of intron A from human cytomegalovirus (Towne) immediate-early gene on heterologous expression in mammalian cells. Nucleic Acids Res. 1991, 19: 3979-3986.
Huang MT, Gorman CM: Intervening sequences increase efficiency of RNA 3' processing and accumulation of cytoplasmic RNA. Nucleic Acids Res. 1990, 18: 937-947.
Qin L, Ding Y, Pahud DR, Chang E, Imperiale MJ, Bromberg JS: Promoter attenuation in gene therapy: interferon-gamma and tumor necrosis factor-alpha inhibit transgene expression. Hum. Gene Ther. 1997, 8: 2019-2029.
Harms JS, Oliveira SC, Splitter GA: Regulation of transgene expression in genetic immunization. Braz. J. Med. Biol. Res. 1999, 32: 155-162.
Gebhard JR, Ahu J, Cao X, Minnick J, Araneo BA: DNA immunization utilizing a herpes simplex virus type 2 myogenic DNA vaccine protects mice from mortality and prevents genital herpes. Vaccine. 2000, 18: 1837-1846. 10.1016/S0264-410X(99)00418-1.
Itai K, Sawamura D, Meng X, Hashimoto I: Keratinocyte gene therapy: inducible promoters and in vivo control of transgene expression. Clin Exp Dermatol. 2001, 26: 531-535. 10.1046/j.1365-2230.2001.00883.x.
Proudfoot NJ: How RNA polymerase II terminates transcription in higher eukaryotes. Trends Biochem. Sci. 1989, 14: 105-110. 10.1016/0968-0004(89)90132-1.
Kozak M: At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 1987, 196: 947-950.
Kozak M: Recognition of AUG and alternative initiator codons is augmented by G in position +4 but is not generally affected by the nucleotides in positins +5 and +6. EMBO J. 1997, 16: 2482-2492. 10.1093/emboj/16.9.2482.
Makoff AJ, Oxer MD, Romanos MA, Fairweather NF, Ballantine S: Expression of tetanus toxin fragment C in E. coli: high level expression by removing rare codons. Nucleic Acids Res. 1989, 17: 10191-10202.
Ikemura T: Correlation between the abundance of yeast transfer RNAs and the occurrence of the retrospective codons in protein genes. Differences in synonymous codon choice patterns of yeast and Escherichia coli wth reference to the abundance of isoaccepting transfer RNAs. J. Mol. Biol. 1982, 158: 573-597.
Ikemura T: Codon usage and tRNA content in unicellular and multicellular organisms. Mol. Biol. Evol. 1985, 2: 13-34.
Nagata T, Uchijima M, Yoshida A, Kawashima M, Koide Y: Codon optimization effect on translational efficiency of DNA vaccine in mammalian cells: analysis of plasmid DNA encoding a CTL epitope derived from microorganisms. Biochem. Biophys. Res. Comm. 1999, 261: 445-451. 10.1006/bbrc.1999.1050.
Grosjean H, Fliers W: Preferential codon usage in prokaryotic genes: the optimal codon-anticodon interaction energy and the selective codon usage in efficiently expressed genes. Gene. 1982, 18: 299-209. 10.1016/0378-1119(82)90157-3.
Andre S, Seed B, Eberly J, Schraut W, Haas J: Increased immune response elicited by DNA vaccination with a synthetic gp120 sequence with optimized codon usage.J. Virol. 1998, 72: 1497-1503.
Uchijima M, Yoshida A, Nagata T, Koide Y: Optimization of codon usage of plasmid DNA vaccine is required for the effective MHC class-I restricted T cell responses against an intracellular bacterium. J. Immunol. 1998, 161: 5594-5599.
Deml L, Bojak A, Steck S, Graf M, Wild J, Schirmbeck R, Wolf H, Wagner R: Multiple effects of codon usage optimization on expression and immunogenicity of DNA vaccine candidate vaccines encoding the human immunodeficiency virus type 1 gag protein. J. Virol. 2001, 75: 10991-11001. 10.1128/JVI.75.22.10991-11001.2001.
Narum DL, Kumar S, Rogers WO, Fuhrmann SR, Liang H, Oakley M, Taye A, Sim BKL, Hoffman SL: Codon optimization of gene fragments encoding Plasmodium falciparum merzoite proteins enhances DNA vaccine protein expression and immunogenicity in mice. Infect. Immun. 2001, 69: 7250-7253. 10.1128/IAI.69.12.7250-7253.2001.
Stratford R, Douce G, Zhang-Barber L, Fairweather N, Eskola J, Dougan G: Influence of codon usage on the immunogenicity of a DNA vaccine against tetanus. Vaccine. 2001, 19: 810-815. 10.1016/S0264-410X(00)00246-2.
Vinner L, Nielsen HV, Bryder K, Corbet SL, Nielsen C, Fomsgaard A: Gene gun DNA vaccination with Rev-independant synthetic HIV-1 gp160 envelope gene using mammalian codons. Vaccine. 1999, 17: 2166-2175. 10.1016/S0264-410X(98)00474-5.
Weeratna RD, Wu T, Efler SM, Zhang L, Davis HL: Designing gene therapy vectors: avoiding immune responses by using tissue-specific promoters. Gene Ther. 2001, 8: 1872-1878. 10.1038/sj.gt.3301602.
Mumper RJ, Ledebur HC, Rolland AP, Tomlinson E: Controlled plasmid delivery and gene expression. DNA Vaccines: methods and protocols. Edited by: Lowrie DB and Whalen RG. 2000, Towata, New Jersey, Humana Press, 267-286.
Klinman DM, Yamshchikov G, Ishigatsubo Y: Contribution of CpG motifs to the immunogenicity of DNA vaccines. J. Immunol. 1997, 158: 3635-3639.
Krieg AM, Yi A-K, Schorr J, Davis HL: The role of CpG dinucleotides in DNA vaccines. Trends Microbiol. 1998, 6: 23-27. 10.1016/S0966-842X(97)01145-1.
Weeratna R, Millan CLB, Krieg AM, Davis HL: Reduction of antigen expression from DNA vaccines by coadministered oligodeoxynucleotides. Antisense Nucleic Acid Drug Devel. 1998, 8: 351-356.
Haddad D, Ramprakash J, Sedegah M, Charoenvit Y, Baumgartner R, Kumar S, Hoffman SL, Weiss WR: Plasmid vaccine expressing granulocyte-macrophage colony-stimulating factor attracts infiltrates inducing immature dendritic cells into injected muscles. J. Immunol. 2000, 165: 3772-3781.
Boyle JS, Koniaras C, Lew AM: Influence of cellular location of expressed antigen on the efficacy of DNA vaccination: cytotoxic T lymphocytes and antibody responses are suboptimal when antigen is cytoplasmic after intramuscular DNA immunization. Int. Immunol. 1997, 9: 1897-1906. 10.1093/intimm/9.12.1897.
Lewis PJ, Cox GJM, van Drunen Littel-van den Hurk S, Babiuk LA: Polynucleotide vaccines in animals: enhancing and modulating responses. Vaccine. 1997, 15: 861-864. 10.1016/S0264-410X(96)00279-4.
Rice J, King CA, Spellerberg MB, Fairweather N, Stevenson FK: Manipulation of the pathogen-derived genes to influence antigen presentation via DNA vaccines. Vaccine. 1999, 17: 3030-3038. 10.1016/S0264-410X(99)00171-1.
Aris A, Feliu JX, Knight A, Coutelle C, Villaverde A: Exploiting viral cell-targeting abilities in a single polypeptide, non-infectious, recombinant vehicle for integrin-mediated DNA delivery and gene expression. Biotechnol. Bioeng. 2000, 68: 688-696.
Deliyannis G, Boyle JS, Brady JL, Brown LE, Lew AM: A fusion DNA vaccine that targets antigen-presenting cells increases protection from viral challenge. Proc. Natl. Acad. Sci. USA. 2000, 97: 6676-6680. 10.1073/pnas.120162497.
Hung C-F, Cheng W-F, Chai C-Y, Hsu K-F, He L, Ling M, Wu T-C: Improving vaccine potency through intracellular spreading and enhanced MHC class I presentation of antigen. The Journal of Immunology. 2001, 166: 5733-5740.
Wu Y, Wang X, Csencsits KL, Haddad A, Walters N, Pascual DW: M cell-targeted DNA vaccination. Proc. Natl. Acad. Sci. USA. 2001, 98: 9318-9323. 10.1073/pnas.161204098.
You Z, Huang X, Hester J, Toh HC, Chen S-Y: Targeting dendritic cells to enhance DNA vaccine potency. Cancer Res. 2001, 61: 3704-3711.
HSG, KAB and RWT produced the manuscript together. All authors read and approved the final manuscript.