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Anatomy of plasmid vectors used in genetic immunization Brazilian Journal of Medical and Biological Research (1999) 32: 147-153 Main features of DNA-based
immunization vectors

Departamentos de 1Biologia Geral, 2Microbiologia, and3 Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil4Instituto de Investigaciones en Ingenieria Genetica y Biologia Molecular(INGEBI-CONICET-UBA), Buenos Aires, Argentina Abstract
Correspondence
DNA-based immunization has initiated a new era of vaccine research.
Key words
One of the main goals of gene vaccine development is the control of the levels of expression in vivo for efficient immunization. Modifying the vector to modulate expression or immunogenicity is of critical importance for the improvement of DNA vaccines. The most fre- quently used vectors for genetic immunization are plasmids. In this article, we review some of the main elements relevant to their design such as strong promoter/enhancer region, introns, genes encoding antigens of interest from the pathogen (how to choose and modify them), polyadenylation termination sequence, origin of replication for plasmid production in Escherichia coli, antibiotic resistance gene as selectable marker, convenient cloning sites, and the presence of immunostimulatory sequences (ISS) that can be added to the plasmid to enhance adjuvanticity and to activate the immune system. In this review, the specific modifications that can increase overall expression as well as the potential of DNA-based vaccination are also discussed.
Introduction
therefore they are of limited interest for the purpose of immunization. In contrast, DNA plasmids encoding antigen(s) are more fre- quently used because they do not have the involves transfer of a gene encoding an anti- inconvenience of classical vaccines: they are genic protein cloned in expression vectors to safe, inexpensive, easy to produce, heat stable a host, leading to the induction of an immune response. During the last two decades, dif- this review, we will deal with some of the main elements relevant to the design of vec- have been developed as well as new methods tors used for genetic vaccination. These vec- for direct gene transfer. Several reviews on tors are Escherichia coli-derived plasmids the subject have been published (1-3). Direct capable of expressing foreign genes in eu- gene transfer may be undertaken using either karyotic cells. Conceptually, their struc- viral vectors or recombinant plasmid DNA.
ture can be divided into two distinct units: i) Viral vectors have the disadvantages of be- a transcription complex unit that drives anti- ing derived from pathogens like traditional gen synthesis and that contains a promoter/ vaccines based on attenuated viruses, and enhancer region, introns with functional splic- ing donor and acceptor sites, sequences en- coding an antigenic protein, and signals re- quired for efficient polyadenylation of the transcript. ii) Prokaryotic elements such as lian promoters, have also been used. These replication origin, multiple cloning sites and promoter elements are particularly interest- a selectable marker to facilitate the construc- ing for vaccine development since they may tion, propagation and amplification of re- combinant vectors in bacteria. This plasmid pathogenic or tumor-causing viruses for ap- backbone can also carry immunostimulatory plications involving humans or animals.
sequences (ISS) with adjuvant activity (Fig- Gurunathan et al. (14) reported that the bo- vine promoter/enhancer from the major his- Basic structure of plasmid DNA
gene gave significantly better protection than vaccines
that obtained with hCMV-derived plasmids.
The transcription complex unit
including those from the beta-actin, muscle- specific heavy chain of myosin and muscle Enhancer/promoter regions. Almost all creatine kinase genes (mck) (7,15,16).
carry the immediate early promoter/enhancer exogenous enhancer elements of aB crystal- from pathogenic viruses. Although these pro- line gene (cryB) or mck can be incorporated moter elements are from pathogenic viruses, upstream in relation to the enhancer/pro- moter region. Hartikka et al. (17) and Dai et therapy and genetic immunization thanks to al. (18) reported that these entities prolong their high transcription initiation ability in and increase expression by the CMV promo- most mammalian tissues (4). The most com- tor/enhancer. In fact, the main function of monly used promoter is the one from human these elements is to control the gene initia- tion transcription rate. In spite of the exist- strong and constitutive expression in a vari- ence of various gene delivery systems, the ety of cell types (5-7). The use of alternative main question to be addressed is if the initia- tion of the transferred gene transcription is cussed, including Rous sarcoma virus (RSV), efficient and how long it lasts. The modula- tion of this initiation rate can be essential to obtain a positive genetic immunization ef- Figure 1 - Plasmid DNA patternfor genetic immunization. i) The repeat (LTR) promoter (8-10). Among them, It is sometimes advisable to introduce an the hCMV promoter gave the highest levels inducible promoter to control in vivo expres- of expression of the reported genes tested sion. This was done by Dhawan et al. (19) and Liang et al. (20) who modified plasmids to make tetracycline (Tc)-dependent tran- scriptions. When tetracycline is administered to transfected mice it can act in a repressive or activating way depending on the position of the tetracycline-operator (tetO) control sequence within these Tc-controlled plas- livers adjuvant and mitogenicactivity via immunostimulatory mids. The inducible systems have the advan- tage of overcoming potential immunological Anatomy of plasmid vectors used in genetic immunization tolerance that may exist in genetic immuni- cording to the different transcriptional gene terminators. Moreover, as the rate of tran- Introns. Intervening sequences (or in- scriptional initiation is increased by the use trons) have a beneficial effect on antigen of strong promoter/enhancer, the process of expression. This can be attributed to an en- transcriptional termination may become rate- hanced rate of RNA polyadenylation and/or limiting (18). Although transcriptional ter- minators are not widely recognized as gene (21), but can also indicate the presence of regulatory elements, Hartikka et al. (17) transcriptional enhancers within the introns showed that modifications in transcriptional terminator sequences such as their replace- ment by other types of terminator sequences nation also include intron A from hCMV.
or the construction of chimeric termination Indeed, some studies have revealed that ex- sequences with more efficient polyadenyla- pression and stimulation of an immune re- tion signal or the lack of 3' untranslated sponse were enhanced by the addition of an region (UTR) sequences led to increases in intron sequence upstream of the coding re- expression which can reflect the differences gion (9,23). This location prevents the utili- in transcriptional termination efficiency.
zation of possible cryptic 5' donor splicing sites within DNA sequences (21). These cryp- carried by DNA vaccine constructs usually tic sites hamper expression due to aberrant sites of different genes, and the branchpoint site can be optimized to match the consensus late SV40-derived sequence terminators. The sequence leading to an increased expression efficient and increases the steady-state level of RNA approximately 5-fold more than the improved transcriptional efficiency 10- to early SV40 polyadenylation signal (27).
served little effect on gene expression when multiple cloning sites. This location facili- compared to cells transfected in culture.
tates efficient processing of cloned genes Various interpretations are possible but the which may not have an efficient polyadeny- most plausible explanation is that introns contain DNA sequences that are recognized Sequences encoding an antigenic pro- tein. Recombinant DNA technology has en- not required after transfection, in established cell lines. In conclusion, the studies that with an optimized structure for genetic im- examine the role of introns in expression munization. To be optimized, synthetic genes plasmid vectors show that the increase in in require elimination of large hairpin struc- vivo expression depends not only on the tures in the 5'-end UTR mRNAs. It has been presence or absence of introns, but also on shown that these structures reduce the level the position of introns within the transcrip- of in vitro and in vivo translation in higher eukaryotes (28,29). To initiate translation, Polyadenylation signal. Polyadenylation an ATG must be present in the inserted gene.
This ATG must also be the first one in the 5' and translation (26) which in turn vary ac- region and in the translational start site. Out- of-frame ATGs can reduce the rate of gene produces a transcription unit capable of mod- translation (29). Kozak (29) has proposed a ulating genetic expression. The exact modu- lation of the expression level elicits the de- located around the start site (-9 GCCGCCA/ GCCAUGG+4). She also pointed out that an
Discovery of new antigenic proteins.
efficient translation is obtained in the -3 position containing a purine base. In the opportunity to quickly discover new anti- absence of a purine base, the efficiency of gens and to handle the antigenicity of the translation can be maintained with a guanine protein at the sequence level with the advan- tage of not requiring protein production and organisms and some eukaryotic genes do not purification. Since it is easy and rapid to have this consensus sequence. Its insertion clone and modify genes in plasmid expres- into the 5'-region of these genes might in- produced and tested in a short period of time In contrast to prokaryotic sequences, limi- such as entire expression libraries which can tations of size and availability often dictate be cloned and injected in a shot-gun fashion that the eukaryotic sequences to be expressed very interesting application of DNA vacci- struct a synthetic gene it may be necessary to nation has been developed to identify new protective antigens. This strategy (termed excise them during processing of the pri- mary transcript. The presence of introns dur- ing processing can sometimes increase the levels of cytoplasmic messenger RNA (30).
of the pathogen. It was initially developed by Barry et al. (34) who demonstrated that im- sequences by addition or deletion of secre- munization with partial expression libraries tory control signals have also been evalu- made from genomic DNA of Mycoplasma ated, and surprisingly they either had no pulmonis protects mice challenged with the pathogen. Similarly, Alberti et al. (35) in- (23,31). However, Hoffman et al. (32) re- jected mice with an expression genomic li- ported that in-frame gene fusions with the brary of Trypanosoma cruzi. Although the sequence encoding the leader peptide of hu- protection was not assayed, it was possible to detect expression of T. cruzi antigens in the muscle and the specific IgG antibodies to a DNA vaccine, Boyle et al. (33) directed The identification of antigens, their com- the antigen to sites of immune-response in- binations and forms is the most effective way to raise protective responses and is one antigen-ligand fusion proteins. These two ligands bind to receptors that are present on endothelial venule cells of lymph nodes or Plasmid backbone unit
that both the humoral and cellular immune Multiple cloning region, replication ori- enhanced using either antigen-targeting strat- gin and prokaryotic selectable marker. The egy. The construction of optimized synthetic backbone of plasmid DNA vectors carries a multiple cloning site (MCS) that can be con- Anatomy of plasmid vectors used in genetic immunization sidered to be additional elements of the tran- cassette is possible but does not ease public scription unit. This MCS is especially de- concern over its potential clinical use. The signed to avoid formation of hairpin struc- use of auxotrophic markers can also be pos- tures in the 5' end of the transcribed RNA sible but there is a high contamination risk since hairpin structures can interfere with with the essential component that is not nec- host. It should be remembered that applica- tion of direct gene transfer for genetic immu- multicopy plasmid, ColE1. It is the best char- nization requires the availability of plasmid acterized copy number and incompatibility DNA that is free of all contaminants, partic- system and allows the maintenance at steady ularly toxic or immunogenic substances. This level of more than 20 copies per E. coli cell, resulting in a high DNA plasmid yield, im- purification technology combined with se- portant in gene vaccine production. The pres- lected E. coli strains and growth optimiza- ervation of a high number of copies depends tion is used for each plasmid construction on the mechanisms which initiate replica- tion and is controlled by the ColE1 origin Sequences as immunostimulatory ele- (36). DNA vectors can also contain a viral ments. It has been shown that certain DNA origin of replication. In this regard, SV40 sequences can induce cytokine secretion and parently, the inclusion or deletion of such stimulatory, whereas similar sequences from other species are not. These observations levels of the foreign peptides (37). These suggest that manipulation of DNA vaccines findings, together with the necessity of satis- to contain or to avoid these sequences may fying concerns related to their potential clini- affect the immunogenicity of the antigens cal use, indicate that replication sequences expressed by the vector. In this regard, Sato of viral origin should be deleted from DNA containing a bacterial ampR produced a stron- ger immune response than a similar expres- select for growth only in those cells which sion vector containing the kanamycin resis- contain a vector. Such markers are of two tance gene (kanR). In vitro transfection ex- types: drug resistant and auxotrophic. Drug periments revealed that the ability to stimu- resistant markers enable cells to detoxify an late the immune response was not due to the different amounts of antigen expressed by wise kill the cell. Auxotrophic markers al- low cells to synthesize an essential compo- ISS was identified within the kanR gene.
prokaryotic selectable marker is the beta- Klinman et al. (41) demonstrated that the lactamase ampicillin resistance gene (ampR) elimination of CpG motifs from the plasmid which confers resistance to penicillin-based antibiotics. Penicillin can induce anaphylac- immunogenicity, and that this effect could tic shock in sensitized individuals. Trace be reversed by co-administering exogenous amounts of this antibiotic may be present as beta-lactamase gene with another antibiotic equaled the effect of incorporating these to test various regulatory modules, to clone ISS, to remove unwanted or safetywise un- findings indicate that the backbone of the acceptable sequences, to identify and modify immunoprotective epitopes, are already avail- genic activity via ISS, suggesting that the able. In addition, obtaining high-level gene expression is not a difficult task. However, important consideration in designing a DNA there is some controversy over the role of high gene expression levels in genetic im- munization. The key to the problem depends vaccines induces B-cell proliferation, im- on the antigen used as well as the type of immune response expected, i.e., cellular, cretion, and promotes the generation of a humoral, or both. The exact modulation of the expression level is necessary for each newly tested antigen to elicit the desired immune responses without modifying or shut- used in DNA vaccines. Optimizing the num- ber and the exact sequences of ISS can greatly negative effects similar to those of tradi- enhance the potency of DNA-based vaccina- have been identified, efforts must be con- Concluding remarks
centrated on testing their different combina- tions and evaluating their immunogenic po- tential. The study of potential biosafety risks of DNA vaccines such as chromosomal inte- deal of modularity. It is therefore possible to efficient combinations of promoter, enhancer, Acknowledgments
introns, gene from a pathogen and signal of polyadenylation to undertake specific tasks in the genetic immunization protocol. The adjuvanticity of CpG motifs turns them into Cultura Inglesa, Belo Horizonte, Brazil for helpful comments and for reviewing the En- References
on-g inhibits transgene expression driven Hasler K, Fleckenstein B & Schaffner W (1985). A very strong enhancer is located tor), Molecular and Cell Biology of Human Genetic Therapeutics. Chapman & Hall, MHC I promoter. Human Gene Therapy, human cytomegalovirus. Cell, 41: 521-530.
systems for the purpose of immunization.
Current Opinion in Biotechnology, 8: 635- transgenic mice. Molecular and Cellular of human cytomegalovirus. Proceedings Donnelly JJ, Ulmer JB & Liu MA (1997).
of the National Academy of Sciences, DNA vaccines. Life Sciences, 60: 163-172.
Dang N, Miyazaki J, Bothwell M & Martin Harms JS & Splitter GA (1995). Interfer- GM (1994). Activity assays of nine hetero- Anatomy of plasmid vectors used in genetic immunization 18. Dai Y, Roman M, Naviaux RK & Verma IM cultured cells. In Vitro Cellular and Devel- 30. Whalen RG (1995). Promoters, enhancers opmental Biology Animal, 30A: 300-305.
blasts: long-term expression of factor IX and inducible elements for gene therapy.
9. Lee AH, Suh YS, Sung JH, Yang SH & protein following transplantation in vivo. In: Wolff J (Editor), Gene Therapeutics.
Proceedings of the National Academy of Sciences, USA, 89: 10892-10895.
31. Xiang ZQ, Spitalnik SL, Cheng J, Erikson immune response by DNA immunization.
19. Dhawan J, Rando TA, Elson SL, Bujard H J, Wojczyk B & Ertl HC (1995). Immune Molecules and Cells, 7: 495-501.
& Blau HM (1995). Tetracycline-regulated responses to nucleic acid vaccines to ra- 10. Miyoshi H, Blomer U, Takahashi M, Gage bies virus. Virology, 209: 569-579.
FH & Verma IM (1998). Development of a into mouse skeletal muscle. Somatic Cell self-inactivating lentivirus vector. Journal and Molecular Genetics, 21: 233-240.
20. Liang X, Hartikka J, Sukhu L, Manthorpe 11. Ulmer JB, Donnelly JJ, Parker SE, Rhodes M & Hobart P (1996). Novel, high express- Margalith M & Hedstrom RC (1997). Strat- ing and antibiotic-controlled plasmid vec- egy for development of a pre-erythrocytic tors designed for use in gene therapy.
Plasmodium falciparum DNA vaccine for human use. Vaccine, 15: 842-845.
21. Huang MTF & Gorman CM (1990). Inter- 33. Boyle JS, Brady JL & Lew AM (1998).
Montgomery DL & Liu MA (1993). Heter- encoding a fusion antigen that is directed injection of DNA encoding a viral protein.
cytoplasmic RNA. Nucleic Acids Re- to sites of immune induction. Nature, 392: 12. Pande H, Campo K, Tanamachi B, Forman 22. Chapman BS, Thayer RM, Vincent KA & 34. Barry MA, Lai WC & Johnston SA (1995).
SJ & Zaia JA (1995). Direct DNA immuni- using expression-library immunization.
pression in mammalian cells. Nucleic Ac- levels of circulating antibody to the en- coded protein. Scandinavian Journal of In- Izquierdo L, Infante JF, Finlay CM & Sierra fectious Diseases, 99 (Suppl): 117-120.
Felgner P & Wheeler C (1997). Develop- mouth disease virus capsid proteins. Jour- of Trypanosoma cruzi. Vaccine, 16: 608- nal of Virology, 72: 4454-4457.
plications. Vaccine, 15: 801-803.
24. Senapathy P, Shapiro MB & Harris NL 36. Lewin B (1994). The replicon: unit of repli- cation. In: Genes V. Oxford University Reiner SL, Charest H, Glaichenhaus N & identification, and applications to genome project. Methods of Enzymology, 183: Felgner P & Wheeler C (1997). Develop- parasite antigen confers protective immu- nity to mice infected with Leishmania major. Journal of Experimental Medicine, Gelinas RE & Palmiter RD (1988). Introns increase transcriptional efficiency in trans- 38. Davis HL, Schleef M, Moritz P, Mancini 15. Skarli M, Kiri A, Vrbova G, Lee CA & genic mice. Proceedings of the National Goldspink G (1998). Myosin regulatory el- Academy of Sciences, USA, 85: 836-840.
26. Jackson RJ & Standart N (1990). Do the intramuscular injection. Gene Therapy, 5: poly(A) tail and 3' untranslated region con- immunization. BioTechniques, 21: 92-99.
trol mRNA translation? Cell, 62: 15-24.
39. Krieg AM, Yi AK, Matson S, Waldschmidt 16. Bartlett RJ, Secore SL, Singer JT, Bodo 27. Carswell S & Alwine JC (1989). Efficiency M, Sharma K & Ricordi C (1996). Long- of utilization of the simian virus 40 late & Klinman DM (1995). CpG motifs in bac- term expression of a fluorescent reporter polyadenylation site: effects of upstream terial DNA trigger direct B-cell activation.
gene via direct injection of plasmid vector sequences. Molecular and Cellular Biol- 40. Sato Y, Roman M, Tighe H, Lee D, Corr motor expression levels in vivo. Cell Carson DA & Raz E (1996). Immunostimu- (1992). Post-transcriptional regulation of 17. Hartikka J, Sawdey M, Cornefert-Jensen fective intradermal gene immunization.
beta 1 gene. Journal of Biological Chem- 41. Klinman DM, Barnhart KM & Conover J (1998). CpG motifs as immune adjuvants.
for direct injection into skeletal muscle.
Human Gene Therapy, 7: 1205-1217.
mRNAs. Molecular and Cellular Biology,

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