Plasmid DNA Vaccines

INTRODUCTION

Conventional active vaccines are made of a killed or attenuated form of the infectious agent, a modified product of the infectious agent (toxoid) or a constituent of an infectious agent (such as the capsule). Relatively high and repeated doses are administered when a non-viable (killed organism, toxoid, capsule) vaccine is used and the protective immunity obtained is not long lasting. Moreover, usually a humoral but not a cell-mediated immune response is generated. On the other hand, low doses are administered when a viable attenuated vaccine is used, both humoral and cell-mediated immune responses are generated and immunity is usually long lasting. Successful vaccines for some infectious agents, in particular intracellular parasites, and tumors are yet to be developed. What is required in such cases, but not available, are antigens that are safe to use, that can be processed by the endogenous pathway and eventually activating cytotoxic T-lymphocytes (CTL). The activated CTL generated would destroy the parasite-infected cell.

Attempts were made to prepare vaccines that mimicked the effectiveness of an attenuated vaccine for those intracellular parasites for which no effective vaccines were available. Furthermore, safety concerns, including the possibility of attenuated vaccines reverting to their virulent form, and their use being contraindicated in immunodeficiency diseases, lead vaccinologists to attempt to prepare replacements for attenuated vaccines that would be as effective and might be safer to use.

In 1965, Youmans and Youmans [1] reported on the immunogenicity of mycobacterial ribosomal and ribonucleic acid preparations and in 1972 Berry and Venneman [2] reported on the immunogenicity of pure RNA extracted from Salmonella typhimurium. Both groups suggested that the RNA preparations were inducing a cell-mediated immune response. The idea of using nucleic acids as vaccines was later abandoned because it was thought that RNA was behaving as an adjuvant for the antigen contaminants in the Ribosome/RNA vaccines [3]. This idea was revived when Wolff et al. [4] in 1990 reported that intramuscular injection of plasmid DNA encoding several different reporter genes (a gene that encodes a product that can be assayed) could induce protein expression in muscle cells. Later, Tang et al. [5] demonstrated the production of anti-human Growth Hormone (hGH) antibodies in mice injected with plasmid DNA encoding hGH and Ulmer et al. [6] showed that both antibody and CTL responses were elicited in mice injected with a plasmid DNA encoding an influenza virus protein. Moreover, the mice were protected against influenza infection. So, instead of a vaccine that contains the antigen, the gene that codes for the production of the antigen is administered, it is expressed in the host cells and the antigen produced will eventually induce humoral and cellular immune responses. Thus it appeared that plasmid DNA vaccines were good candidates for use in inducing immunity to certain infectious diseases where no effective vaccines are available, and as a possible replacement for attenuated vaccines.

DNA vaccines for a number of infectious agents have been prepared and reported to induce both humoral and cell mediated immune responses in animal models. Moreover, DNA vaccines for some tumors and some allergens have been prepared. Prior to reviewing the DNA vaccine studies, an overview of the mechanisms involved in antigen processing/presenting and the generation of the immune response will be presented.

ANTIGEN PROCESSING/PRESENTING AND THE GENERATION OF THE IMMUNE RESPONSE

Antigen Processing/Presenting cells (APC) degrades a protein antigen into smaller peptides and presents the peptide to T-lymphocytes, which eventually are activated. Professional APC are cells that possess all the antigen processing/presenting machinery and include macrophages, dendritic cells, Langerhans cells of the skin and B-lymphocytes. Major Histocompatability Complex (MHC) class Iand class II molecules play a major role in presenting a processed antigen to T-lymphocytes. MHC class I molecules are expressed on the surface of practically all nucleated cells [7, 8]. They consist of two polypeptide chains; an alpha chain and beta-2-microglobulin. Genes that determine the production of alpha chains are located on chromosome numbers 6 and 17 in humans and mice, respectively. The alpha chain consists of three external regions (alpha-1, alpha-2 and alpha-3), a transmembrane region and an intracyto-plasmic region. Beta-2-microglobulin is non-covalently linked to the alpha chain, it does not transverse the cell membrane and the gene that determines its production in humans is located on chromosome number 15.

Endogenously synthesized proteins (synthesized within the APC) such as viral, protozoal, bacterial and tumor proteins are degraded in the cytoplasm of APC into small peptides by proteases (encoded by LMP2/LMP7 genes) in the proteasomes located in the cell cytosol. Transporter proteins (TAP1/TAP2) carry the peptides to the endoplasmic reticulum which contain newly synthesized MHC class I molecules. The peptides then bind to MHC class I molecules at sites called grooves, located between the alpha-1 and alpha 2 regions. The tri-molecular complex (alpha chain, beta-2-microglobulin and peptide) is then transported to the cell outer membrane and presented to the CD8⁺CTL. These lymphocytes become fully activated following second signals that include cytokine-receptor (such as IL-2 and IL2R) and co-stimulatory molecule-receptor (such as B7 and CD28) interactions. The activated CD8⁺ CTL produce perforins that polymerize in the cell membrane of the target cell (e.g. cell infected with the virus) causing its death [7].

MHC class II molecules are expressed mainly on APC [8, 9]. Certain cytokines such as gamma-interferon may induce other cell types to express them. MHC class II molecules consist of two non-covalently linked polypeptides; the alpha and beta chains. Genes that determine their production are also located on chromosome number 6 in humans and number 17 in mice. Each of the two chains consists of two external regions (alpha-1, alpha-2 and beta-1, beta-2), a transmembrane region and an intracytoplasmic region.

APC take up exogenous proteins by phagocytosis/pinocytosis/endocytosis [8, 10, 11]. The protein is degraded by enzymes into smaller peptides in the endosome/lysozome compartment of the APC. MHC class II molecules, synthesized in the endoplasmic reticulum and associated with the invariant chain are transported to the endosome/lysozome. The products of DM genes stabilize and prevent the inactivation of MHC molecules in the acidic environment. Moreover, DM molecules act as catalysts for peptide loading of the grooves, which are located between the alpha-1 and beta-1 regions, by causing the release of the invariant chain (Class II associated invariant peptide, CLIP). The peptide-MHC II complex formed is transported to the cell surface and is presented to CD4⁺ T-lymphocytes (T-helpers). These lymphocytes become activated following second signals that include cytokine-receptor and co-stimulatory molecule-receptor interactions. There are at least 2 sub-populations of T-helpers (Th), Th1 and Th2. Activation of one of the two sub-populations predominates, depending among other yet undetermined factors, on the cytokines produced (interleukin (IL)-12 favors a Th1 response and IL-4 favors a Th2 response), the co-stimulatory molecules involved (B7.1-CD28 interaction favors a Th1 response; B7.2-CD28 interaction favors a Th2 response) and the route of entry of the antigen [12]. Cytokines produced by activated Th1 but not Th2 lymphocytes are IL-2 and gamma-interferon. Cytokines produced by activated Th2 but not Th1 lymphocytes are IL-4, IL-5, Il-6, IL-10 and IL-13. Cytokines produced in a Th1 response mediate Delayed Type Hypersensitivity, are involved in the activation of CTL, macrophages and NK-cells and help B-lymphocytes to eventually produce IgG2a and IgG3 class antibodies. On the other hand, cytokines produced in a Th2 response promote eosinophil proliferation and production of IgG1 and IgE class antibodies by B-lymphocytes. It is worth noting at this stage that a maximized Th1 and CTL response are needed for protection against intracellular parasites and tumors.

Some reports indicated that exogenous proteins may be processed, complexes to MHC class I molecules and presented to CTL [13-19]. By a yet not well-defined mechanism called cross priming, the antigen may be either shuttled directly into the cytosol of the APC, or it may be engulfed by phagocytosis and leaks out of the endosome/ lysozome compartment into the cytosol.

PLASMID DNA VACCINES

Molecular Aspects and Preparation

DNA vaccines are bacterial plasmids that are designed to express a gene (reporter gene) in the cells of a host. The reporter gene encodes a protein, which could be among others, an antigen of a disease organism, a tumor antigen, an allergen, a cytokine or a co-stimulatory molecule. One or more sequences known to encode reporter genes in the host can be inserted in the plasmid.

Naturally occurring bacterial plasmids suitable for use in the preparation of a plasmid DNA vaccine do not exist and so they have to be genetically engineered. The one that is usually used is derived from E. coli and is engineered to include the following components: 1- a strong eukaryotic promotor to drive transcription of the reporter gene. Most studies use viral promoters, in particular the human cytomegalovirus immediate-early promoter, 2- a LacZ gene which encodes the first 146 amino acids of the enzyme beta galactosidase, 3- a cloning site within the LacZ gene to insert the reporter gene (one or more genes can be inserted), 4- a 3’ polyadenylation termination sequence (3’ polyA tail) derived from either the simian virus 40 or the bovine growth hormone (this renders stability to transcribed messenger RNA), 5- a prokaryotic origin of replication for plasmid vector amplification in bacteria (E. coli competent cells are mainly used) and 6- a selectable marker such as an antibiotic resistant gene (usually ampicillin or kanamycin is used) to select the tranformed E. coli [20-22].

A plasmid-reporter gene construct is prepared by treating the plasmid and the DNA containing the reporter gene with the same restriction endonuclease, mixing the two preparations and adding ligase. E. coli possessing the chromosomal gene that codes for a segment of beta-galactosidase is cultured on medium containing the recombinant preparation, isopropylthiogalactoside (IPTG, inducer of the lactose operon), 5-bromo-4-chloroindolyl-beta-galactoside (X-gal) and an antibiotic (ampicillin or kanamycin). Non-transformed cells will not grow in the presence of the antibiotic. To distinguish between the E. coli containing the plasmid-reporter gene construct and those containing reporter gene free plasmid (both of which would grow in the presence of the antibiotic because they now possess the antibiotic resistant gene), a blue/white screening protocol was designed based on the activity of the beta- galactosidase enzyme. This is achieved because the cloning site is in the middle of the LacZ gene. As mentioned, this gene codes for the first 146 amino acids of the beta-galactosidase enzyme, and a chromosomal gene of E. coli encodes the other segment of the enzyme. A process known as alpha-complementation to form an enzymatically active protein associates both segments. IPTG present in the medium will induce the LacZ gene (which is under control of a promoter sequence) and the colonies formed by E. coli containing the reporter gene-free plasmids will appear blue due to hydrolysis of X-Gal, a chromogenic substrate for the active beta-galactosidase. On the other hand, when the reporter gene is successfully inserted into the cloning site located in LacZ gene, inactive beta-galactosidase is produced. X-Gal is not hydrolyzed and the E. coli colonies will appear white [23, 24].

The plasmid-reporter gene construct is isolated from white colonies by lysis of the bacteria and purifying the plasmid construct using gel chromatography and/or ultracentrifugation on cesium chloride gradients.

Nowadays, commercially available mammalian expression systems are available, which facilitate the preparation of the plasmid DNA construct. One such a system is the pTargeT^*TM Mammalian Expression Vector System (Promega, Madison, WI). PCR amplified reporter gene will directly ligate to the thymidine overhangs of the plasmid. This plasmid vector system contains a version of the alpha peptide of beta-galactosidase that allows recombinants to be screened using blue/white colony selection. Moreover, purification of the plasmid can be accomplished using commercial kits such as FlexiPrep^R(Pharmacia Biotech, Uppsula, Sweden). This method includes alkaline cell lysis, RNase treatment and isopropanol precipitation. Plasmid DNA is purified using Sephaglas^TM FP, a commercial glass matrix that selectively binds to DNA. A chaotropic salt required for this binding denatures and removes remnants of proteins. The plasmid DNA is eluted in a final step using a buffer of low ionic strength.

Routes of Administration and Dose

Several routes of plasmid DNA inoculation have been undertaken in animal models. These include, intra-muscular, subcutaneous, intra-peritoneal, intra-dermal, subcutaneous, intravenous, oral, rectal, intra-bursal, intra-orbital, intra-tracheal, intra-nasal, and vaginal routes [21, 22, 25-28]. In the case of a plasmid DNA vaccine for a tumor, it can be injected into the tumor site [21]. The most common routes of administration are by injecting the plasmid DNA dissolved in saline intra-muscularly or intra-dermally using a hypodermic needle or by bombarding plasmid DNA coated onto colloidal gold micro-particles in the dermis or muscle using a gene gun. The gene gun accelerates the particles into the target tissue by a controlled discharge through a shock wave created by a chemical propellant, expansion of a compressed gas or an electric spark.

The dose used in mice depends on the method of administration. Usually an immune response is generated when 10-100ug of plasmid DNA is injected and 0.1-1ug when it is administered with a gene gun [25]. The immune response is increased when 1 or 2 boosters are given [29-35]. However, time intervals between boosters appear to be critical. In two studies it has been shown that an increase in the time interval between immunizing doses resulted in an increased immune response [36, 37].

The Generation of an Immune Response to Reporter Gene Encoded Product

If the reporter gene of the administered plasmid DNA vaccine codes for an antigenic portion of a pathogen or a tumor cell, then this will result in the in situ expression of the antigen and the induction of an antigen specific immune response [22,38]. The plasmid DNA enters the cell and makes it’s way to the nucleus where it is transcribed. It is thought that the plasmid DNA is retained in the host cell nucleus in an extra-chromosomal location where it remains in an expression competent and non replicating form for long periods of time [27, 39-42]. The transcribed messenger RNA enters the cytoplasm and is translated on the ribosomes. The expressed antigen is then processed/presented and subsequently a humoral and cell mediated immune response is generated.

Antigen Processing/Presenting Cells

A number of studies aimed at determining the APC that take up the plasmid DNA, process the product of the reporter gene by the endogenous pathway and eventually activate CTL. Initially it was thought that myocytes and keratinocytes were APC’s. Results of studies by Wolff et al. [4] and Ulmer et al. [6, 43] suggested that myocytes behaved as APC’s when the plasmid DNA was administered by the intra-muscular route. However, Myocytes express MHC class I molecules, but they do not express co-stimulatory molecules that are needed to optimize CTL activation. Agadjanyan et al. [44] showed that antigen-specific CTL responses could be induced by muscle cells only when the mice were vaccinated with plasmid DNA possessing an antigen and a B7 (co-stimulatory molecule) reporter genes. Moreover, Torres et al. [45] showed that removal of the muscle injection site within 10 minutes following immunization did not alter the generation of the immune response suggesting that the plasmid DNA might have quickly left muscle tissue and gained access to immunocompetent cell rich sites. On the other hand, they showed that when the skin administration site of plasmid DNA was removed within minutes following immunization, no immune response was generated. Furthermore, Klinman et al. [46] were able to transfer immunization by transplanting skin within 12 hours following intra-dermal administration of plasmid DNA. These findings suggested that keratinocytes and/or Langerhans cells might be the APC that are transfected.

Later reports indicated that the bone marrow derived dendritic cell is the principal APC involved in plasmid DNA immunization. The plasmid DNA could either transfect somatic cells (myocytes, keratinocytes) or bone marrow derived dendritic cells that infiltrate muscle or skin as a part of the inflammatory response to vaccination. In the former case, it is thought that somatic cells such as myocytes and keratinocytes are reservoirs for the antigen. They are transfected, the product of the reporter gene is expressed, released and taken up by dendritic cells (cross priming). In support of this mechanism of processing, Ulmer et al. [43, 47] were able to induce both antibody and CTL responses to influenza nucleoprotein in mice by transplanting transfected myoblasts. In support of bone marrow derived dendritic cells becoming directly transfected, it was reported that plasmid DNA could be isolated from lymph node derived and skin derived dendritic cells after intra-muscular or intra-dermal administration, respectively [48].

Adjuvanticity of Plasmid DNA

An immunologic adjuvant is an agent that can increase the rate, level and duration of an immune response to an antigen. An adjuvant that is added to some vaccines for human use is alum (aluminum hydroxide and/or aluminum phosphate), which creates a biased Th2 response. The immunostimulatory effect of bacterial DNA was reported by Tokunaga et. al.[49] and Yamamoto et al. [50]. They showed that a DNA-rich fraction extracted from Mycobacterium bovis exhibited strong anti-tumor activity, stimulated mouse NK activity and induced the production of interferon and macrophage activating factor. Bacterial DNA contains immunostimulatory sequences (ISS) or motifs that trigger innate immunity in the host. ISS consists of unmethylated CpG dinucleotides flanked by two 5’ purines and two 3’ pyrimidines. Sequence analysis of the ampicillin resistant gene of plasmid DNA vaccines indicated that it contained two repeats of a palindromic CpG hexamer 5’AACpGTT3’ [51]. This 6 base motif proved to be a potent adjuvant in mice. These ISS behave as an internal immunological adjuvant. They stimulate the production of cytokines, in particular gamma interferon and IL-12 that favor a Th1 response [50, 52-54]. CpG motifs are about 20 times more common in the bacterial than in the mammalian genome. Moreover, less than 5% of the cytosine residues in the CpG dinucleotides of bacterial DNA are methylated as compared to 70-90% of the cytosine residues in eukaryotic DNA. [28, 52, 53, 55]. Methylation of the cytosine residues in the plasmid DNA results in the loss of the immunostimulatory effect [52].

Th1 and Th2 Lymphocyte Responses

Plasmid DNA vaccines when injected intramuscularly or intradermally, would induce a Th1 response because the CpG motifs stimulate the production of IL-12 that favors the activation of Th1 lymphocytes. On the other hand, if the plasmid DNA is administered with a gene gun, a Th2 response is generated [28]. Reasons for a biased Th response based on the method of vaccine administration are yet unknown, but a number of factors that need further investigation exist. These include, the amount of DNA administered (less when using a gene gun), host MHC haplotype, nature of the processed antigen and route taken by administered DNA to arrive at its destiny (injection delivers DNA to extracellular space where it is taken up by cells; gene gun introduces DNA directly into cells) [56]

In a number of animal models of infection, dominant Th1 responses have been correlated with protection. Some of the reports will be mentioned. BALB/c mice are susceptible to Leishmania major because they are unable to generate a Th1 response unless they are given IL-12 [57]. Injection of BALB/c mice with a plasmid DNA vaccine containing gp63 reporter gene from L. major, induced a dominant Th1 response that was protective [58]. In the case of Influenza virus, intra-muscular injection of mice with a plasmid DNA vaccine encoding influenza NP induced a protective Th1 response [6, 59]. An enhanced pulmonary inflammatory response with a predominant Th2 pattern and an unfavorable outcome was observed in mice immunized with an inactivated vaccine and infected with Respiratory Syncytial Virus (RSV). However, a predominant Th1 protective response against RSV was induced in mice injected with a plasmid DNA vaccine [60, 61].

Moreover, a Th1 response rather than a Th2 response to a potential allergen appeared to abrogate an allergic reaction. Plasmid DNA encoding the house dust mite allergen (Der p 5) injected intra-dermally to rats inhibited specific IgE synthesis, histamine release and airway hyper-responsiveness upon aerosolized allergen challenge [62]. In another report, mice were first sensitized to the latex allergen (Hev b 5) with alum that is an adjuvant that favors a Th2 response. Injection of these mice with plasmid DNA encoding the Hev b 5 antigen resulted in a significant decrease in antigen-specific IgE levels [63]. Thus it appears that plasmid DNA vaccines can both prevent allergic responses and down regulate an ongoing Th2 response.

On the other hand, in autoimmune diseases, a Th1 response appears to correlate with progression of the disease [64]. Waisman et al. [65] used a DNA vaccine coding for a T cell receptor to protect mice against experimental autoimmune encephalitis. Protection was associated with a decrease in the Th1, and an increase in the Th2 response.

CTL Response

The CTL response plays an important role in protection against intracellular parasites such as viruses, protozoa and bacteria, and tumor cells. Viruses are obligate intracellular parasites and depend on host cells to complete their life cycle. Because they depend on the host’s metabolic machinery, all viral proteins are accessible to the endogenous pathway of presenting/processing and subsequent activation of specific CTL. Virus activated CTL destroy virus infected cells. This has been reported for a number of viruses in animal models of infection including Lymphocytic Choriomeningitis Virus [66, 67], Influenza virus [68, 69] and orthopox virus [69]. CTL and/or protective responses were obtained when plasmid DNA vaccines were used to immunize animals to a number of viruses including Influenza virus [6, 25], Hepatitis B [70], Herpes Simplex virus [71, 72] Rabies [73] HIV [74], rota virus [75], Ebola virus [76] and Hepatitis C [77].

The immune response to intracellular protozoan parasites is complex and multi-factorial. Experiments using mouse models of infection revealed that CTL contributed to immunity against a number of protozoan parasites [78-85]. Malaria remains an overwhelming problem in tropical developing countries, with 200-300 million cases and 1-2 million deaths per year [86]. DNA vaccines against malaria tested in mice induced a specific CTL response and protection [87, 88].

Tuberculosis is another world health problem. WHO reports indicate that there are about 8 million new cases with 3 million deaths per year. Intracellular bacteria enter eukaryotic cells in a membrane–bound structure called a phagosome. Some, such as Mycobacteria remain and survive within the phagosome. Others such as Listeria escape from the phagosome into the cytosol of the infected cell. Mycobacterial antigens do not have access to the cytosol and can not be processed by the classical endogenous pathway. Yet, a number of studies reported that protective specific CTL to Mycobacterial antigens were obtained [89-93]. Different studies have shown BCG vaccination protection levels ranging from no protection at all to as high as 80% protection. Thus it appears that there is a need for an effective vaccine. Promising results were obtained by Tascon et al. [94] who reported a high degree of protection in mice immunized with a plasmid DNA vaccine containing the gene that codes for the 65Kda heat shock protein of M. tuberculosis.

Tumor cells may behave as professional APC that would specifically activate CTL, if they express a tumor peptide-MHC class I complex and co-stimulatory molecules. Weak or no tumor antigens have been detected on certain tumors. If such tumor cells are tranfected with plasmid DNA containing a tumor antigen gene then a specific CTL might be generated. Conry et al. [95] reported that a vaccine containing the gene that codes for the carcinoembryonic antigen was effective in generating a CTL response in mice and Graham et al. [96] reported that a plasmid vaccine containing the polymorphic epithelial mucin (PEM) gene protected mice against PEM-expressing tumor cells.

Antibody Production

There are 3 main ways by which antibodies contribute to immunity. Pathogen or toxin specific antibodies may neutralize a pathogen or toxin, respectively, some types of antibodies behave as opsonins enhancing phagocytosis and some activate the Complement System. All three mechanisms prevent, or minimize the chance of disease.

Conventional vaccines in use nowadays induce protective antibody responses. Plasmid DNA vaccines also induce the production of antibodies. Peak antibody levels are reached 4-12 weeks post-immunization [97, 98]. However, studies that have compared the humoral immune responses obtained in animals immunized with a DNA vaccine and a conventional vaccine or a sublethal dose of an infectious agent revealed that antibody levels achieved using the latter immunizing agents were higher. Boyle et al. [99] and Deck et al. [97] reported that Influenza virus antibody titers in mice immunized with a DNA vaccine were lower than that in mice immunized with live influenza virus. Similar results were obtained when mice immunized with DNA encoding a malaria surface protein and mice immunized with the malaria protein alone were compared [100]. These results might not be considered as a shortcoming of DNA vaccines since memory was established and elevated antibody levels would be expected on exposure to the infectious agent. Moreover, as mentioned earlier, the major aim in vaccinology was to prepare novel vaccines that would induce effective Th1 and CTL responses to intracellular parasites and tumors. DNA vaccines appear to do so, at least in animal models of infection.

In as much as antibody classes and sub-classes produced in an immune response to a DNA vaccine is concerned, it appears reasonable to expect (and this was observed in a number of studies mentioned in this review) that this would depend on the method of vaccine administration. Using a gene gun, a biased Th2 response would be obtained and the production of IgG1 and IgE antibodies would predominate. On the other hand, if the DNA vaccine were administered by injection, a biased Th1 response and the production of IgG2a would predominate.

Duration and Strength of the Immune Response

Results of studies have indicated that long lasting immunity is attained when a DNA vaccine is used. Akbari et al. [101] reported that antigen-specific CD4⁺ T-lymphocytes remained elevated for up to about 10 months following immunization with a DNA vaccine, and Gurunathan et al. [102] reported long term antigen- specific Th1 activity in mice immunized with a DNA vaccine containing a gene that coded for a Leishmania antigen. CTL responses and antibody levels were observed for up to about 17 months in mice immunized with a DNA vaccine containing a reporter gene coding for an influenza virus protein and a DNA vaccine containing a reporter gene coding for hepatitis B protein, respectively [103, 104].

The observed prolonged duration of the immune response in the above-mentioned studies was probably due to the persistence of the antigen produced in the host. Wolff et al. [105] detected antigen in muscle for up to more than 1 year, and influenza virus nucleoprotein [103] was detected in the dermis at one month, post plasmid DNA inoculation.

An immunization regimen that may result in an optimal immune response is to prime the host using the DNA vaccine and subsequently boosting with the antigen. Letvin et al. [106] primed rhesus monkeys with plasmid DNA containing HIV env gene. Subsequent boosters were done using a combination of the plasmid DNA and HIV env protein. Strong CTL and neutralizing antibody activity were obtained.

Selection of the Reporter Gene

Antigenic components of infectious agents may induce a protective, a partially protective or a non-protective immune response. It is therefore important to select the proper reporter gene or combination of reporter genes to be used in the preparation of a plasmid DNA vaccine. Sedegah et al. [107] reported that 56% of mice immunized intramuscularly with plasmid DNA containing the circumsporozoite protein (CSP) gene were protected against subsequent challenge with Plasmodium yoelii. Better survival rates (82-90%) were reported by Doolan et. al [88] who immunized mice with plasmid DNA containing genes that encode CSP and the 17Kda hepatocyte erythrocyte protein.

One of the virulence regulatory genes of Salmonella typhimurium is the mouse virulence gene A (MviA) [108]. Lena Kalfayan, a graduate student in the author’s (AMA) laboratory prepared plasmid DNA containing the gene that encodes MviA. BALBc mice were given primary and booster intramuscular injections of this vaccine and were subsequently challenged with100LD50 of S. typhimurium. Fifty percent of the mice survived at 10 day post-challenge and 34% were still alive 55 days later. None of the mice in the control groups survived by 8 days post-challenge. On the other hand, another graduate student in the author’s laboratory (Daad Farhat) prepared plasmid DNA containing the gene that encodes pilin, one of the virulence factors of Pseudomonas aeruginosa. BALBc mice were given primary and booster injections of this vaccine and were subsequently challenged with 2 LD50 of P. aeruginosa. All mice in the control groups died within 24 hours post-challenge. Fifty percent of the immunized mice survived at 2 days post-challenge, and none of the mice survived thereafter. Anti-pilin antibodies were detected in sera of immunized, non-challenged mice at 15, 30 and 45 days post-immunization. One of the reasons for obtaining only partial protection in both studies might be that the immune response generated against the product of the reporter gene in each case was not sufficient to neutralize the infection. Possibly the neutralization of more than one virulence factor in each case, is required to obtain full protection and work in progress deals with the use of more than one reporter gene for each of the pathogens, each encoding for a different virulence factor.

REPORTER GENE ENCODED PRODUCTS THAT MODULATE/ORIENT THE IMMUNE RESPONSE TO AN ENCODED ANTIGEN

Plasmid DNA constructs containing reporter genes that encode MHC molecules, co-stimulatory molecules, or cytokines have been prepared. Unlike prokayotes, eukaryote genes contain introns. To avoid the inclusion of introns in the plasmid, the DNA containing the reporter gene is prepared using functional m-RNA (introns had been removed) as a template and reverse transcriptase. The gene in question is then PCR-amplified and ligated to the plasmid [20].

A tumor cell may behave both as a professional APC and a target for CTL. To behave like a professional APC, tumor cells should express tumor antigen in association with MHC molecules, and co-stimulatory molecules on their surface. Some tumors do not express, or have lost the ability to express MHC molecules. Moreover, the MHC phenotype expressed by some tumors may not have the proper motifs for high-affinity binding of the tumor peptide [109-111]. Wahl et al. [113] using a murine system, transfected a tumor in vivo with an allogeneic MHC class I gene. They used the poorly immunogenic BL6 melanoma, which is a highly invasive tumor incapable of inducing immunity and lacking the expression of MHC class I and II molecules. Allogenization of the BL6 tumor resulted in enhanced generation of specifically active CTL.

The best-characterized co-stimulatory molecules on APC are the structurally related glycoproteins B7.1 and B7.2 and their receptor on T-lymphocytes CD28. Immunization of mice with tumor cells transfected with the co-stimulatory B7.1 and tumor-associated antigen genes resulted in increased protection against tumor challenge [95, 114]. Wang et al. [115] established and transfected a human renal carcinoma cell line with the human B7.1 gene and studied the antitumor immune response in vitro. Transfected, but non-transfected tumor cells induced a significant T-lymphocyte proliferation in the Mixed Lymphocyte and Tumor Reaction assay. Another stimulatory interaction that regulates the immune response and has received recent attention is CD40 on APC, in particular expressed on B-lymphocytes, and its ligand CD154 expressed on activated T-lymphocytes [116, 117]. Plasmid DNA encoding CD154 and a plasmid encoding an antigen coadministered to mice resulted in increased antibody levels and CTL activity [118].

Cytokines are small soluble proteins produced by one cell that alter the properties of another cell. Many cell types including those of the immune system release them. Most cytokines have a multitude of different biological effects. It is beyond the scope of this review to cover the biological effects of all the cytokines and only some of them will be briefly mentioned. IL-2 is produced by T- lymphocytes (Th1) and it causes the proliferation of a number of cell types. T-lymphocytes (Th2) and mast cells produce IL-4, which induce CD4 lymphocyte differentiation to the Th2 sub-population, activates B-lymphocytes and causes a switch to IgE synthesis. GM-CSF is produced by macrophages and T-lymphocytes and stimulates the growth and differentiation of the myelomonocytic lineage. IL-12 produced by macrophages and B-Lymphocytes induces CD4 T lymphocyte differentiation to the Th1 sub-population.

Chow et al. [119] reported that there were marked increases of antibody responses and T lymphocyte proliferation in mice injected with plasmid DNA containing both the genes encoding for hepatitis B surface antigen (HbsAg) and IL-2. Augmented CTL responses in mice that were coinjected with plasmid DNA containing the IL-2 gene and hepatitis C virus (HCV) protein gene was reported by Geissler et al. [77]. Increased antibody and T-lymphocyte responses were observed in mice injected with Plasmid DNA containing the IL-4 and HCV core protein genes. However, the specific CTL response was decreased. Co-inoculation of mice with plasmid DNA GM-CSF and antigen genes resulted in the boosting of both cellular and humoral responses [120]. Co-inoculation of mice with plasmid DNA IL-12 and antigen genes resulted in enhanced Th1 and CTL responses, and a decrease in antibody production [121].

Thus it appears that orientation of the immune response according to needs can be accomplished by including MHC, costimulatory molecule, and/or cytokine genes in the plasmid DNA.

ADVANTAGES, CLINICAL TRIALS AND POTENTIAL LIMITATIONS OF PLASMID DNA VACCINES

Advantages

Plasmid DNA is non-infectious, does not replicate and encodes only the antigen of interest, as opposed to live attenuated vaccines or viral carrier systems. It does not contain heterologous protein components to which the host may respond. It induces both cell mediated (Th1 and CTL) and humoral immunity, which are long lasting.[41, 46, 122-124]. DNA vaccines induce in vivo expression of immunogens thus conserving the native conformation of epitopes. Conserving an appropriate tertiary structure of proteins is important for the induction of conformationally specific antibodies and cellular responses. They may be constructed to include more than one immunogen gene, thus potentially decreasing the number of vaccinations required in children [123-125]. They offer the possibility of generating effective immune responses against diseases such as malaria and HIV where other types of vaccines have failed [22, 107, 126]. Moreover, they may be safer to use than live attenuated vaccines especially in immunocompromised hosts [41,123, 124]. They are stable, easy to freeze dry and reconstitute, and can be manufactured inexpensively in large quantities at high levels of purity [22, 41, 122].

Clinical Trials

That plasmid DNA vaccines induce immune responses that can be modulated or oriented by using different means of administration and/or coadministering an immunomodulator gene has been established in animal studies. Clinical trials for Malaria (Naval Medical Institute), Influenza (Johns Hopkins University), HIV (University of Pennsylvania), colon cancer (University of Alabama), Hepatitis B (University of Wisconsin) and Herpes (University of Washington) plasmid DNA vaccines are underway [127]. Additional references for these trials can be found on the website [http//www.dnavaccine.com]. It is yet to be established if DNA plasmid vaccines operate in the same manner in humans. Some differences between mice and humans have been observed. Higher doses of plasmid DNA are needed to induce an immune response in humans. Immune responses are usually elicited in mice using 0.1-1ug of plasmid DNA administered by a gene gun and 10-100ug administered by injection [25]. In a clinical trial of a malaria plasmid DNA vaccine, 500-2500ug of plasmid DNA was needed to elicit a CTL response [128]. The 6 pair motif (AACGTT) in plasmid DNA that induces optimal stimulation in mice may not work equally well in humans. Different CpG motifs might operate in humans [129].

Potential Limitations

Despite their multiple advantages, plasmid DNA vaccines might not be devoid of some limitations and potential dangers. If plasmid DNA integrates into the host genome it may either activate oncogenes or suppress tumor suppressor genes, which may lead to a malignant transformation. However, it is believed that there is no reason to be concerned about this possible drawback. If the vaccine is administered intradermally, the transfected epidermal cells are lost within 10-14 days because of the normal sloughing of keratinized skin tissue. If administered intramuscularly, the transfected muscle cells are non-dividing and random insertion is more likely to occur in replicating cells in which DNA is actively being synthesized [21, 130]. Moreover, Nicholas et al. [42] were unable to detect integration of plasmid DNA into the host cell genome at 3,6,12 and 18 weeks after intramuscular administration.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by the presence of anti-double stranded DNA antibodies in patient’s serum. The injection of plasmid DNA may induce the production of anti-DNA antibodies and SLE [130]. On the other hand, one way by which immunological tolerance can be induced in adults is by administration of minute amounts of antigen. The minute amount produced by transfected cells may induce a state of tolerance rather than immunity [21, 123, 131].

The potential risks of using plasmid DNA encoding cytokines or co-stimulatory molecules should also be considered. The long-term effects of a continuous supply of a cytokine or a co-stimulatory molecule in the host are not yet known.

Possible harmful effects of bacterial ISS (CpG motifs) must also be considered. Schwartz et al. reported that CpG motifs in bacterial DNA caused inflammation in the lower respiratory tract [132, 133].

In conclusion, DNA vaccines are now being tested in Phase I and Phase II clinical trials, yet further safety studies should be undertaken and potential dangers should not be neglected. The manner by which they are to be administered, the amount of plasmid DNA to be administered, the number of boosters to be given and time interval between boosters need to be optimized.

REFERENCES

[1] Youmans, G. P., Youmans, A. S. J. Bacteriol., 1965, 89, 1291.

[2] Berry, L. J., Vennman, M. R. In Cellular Antigens, Nowotny, A., Ed., Springer-Verla, New York Inc., 1972, pp. 3-13.

[3] Eisenstein, T. K. Infect. Immun., 1975,12, 364.

[4] Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Ascadi, G., Jani, A., Felgner, P. L. Science, 1990, 247, 1465.

[5] Tang, D. C., De Vit, M., Johnston, S. A. Nature, 1992, 356,152.

[6] Ulmer, J. B., Donnelly, J. J., Parker, S. E., Rhodes, G. H., Felgner, P. L., Dwarki, V. J., Gromkowaski, S. H., Deck, R. R., De Witt, D. M., Friedman, A., Hawe, L. A., Leander, K. R., Martinez, D., Perry, H. C., Shiver, J. W., Montgomery, D. C., Liu, M. A. Science, 1993, 259, 1745.

[7] York, I. A., Rock, K. L. Annu. Rev. Immunol., 1996, 14, 369.

[8] Abdelnoor, A. M. Critical Reviews in Oncogenesis,, 1997, 381.

[9] Mach, B., Steimle, V., Martinez-Soria, E., Reith, W. Annu. Rev. Immunol., 1996, 14, 301.

[10] Watts, C. Annu. Rev. Immunol.,1997, 15, 821.

[11] Castellino, F. G., Zhong, G., Germain, N. Human Immunol., 1997, 54, 159.

[12] Romagnani, S., Immunol. Today, 1997, 18, 263.

[13] Bevan, M. J. Nature, 1987, 325, 192.

[14] Carbone, F. R., Bevan, M. J. J. Exp. Med., 1990, 171, 377.

[15] Kovacsovics-Bankowski, M., Clark, K., Benacerraf, B., Rock, K. L. Proc. Natl. Acad. Sci. USA, 1993, 90, 4942.

[16] Shen, Z.:Reznikoff, G.,; Dranoff, G., Rock, K. L. J. Immunol. 1997, 158, 2723.

[17] Kovacsovics-Bankowski, M., Rock, K. L. Science, 1995, 267,243.

[18] Reis, C., Sousa, E., Germain, R. N. J. Exp. Med., 1995, 182, 841.

[19] Norbury, C. C., Hewlett, L. J., Prescott, A. R., Shastri, N., Watts, C. Immunity, 1995, 3, 783.

[20] Glick, B. R., Pasternak, J. L. Molecular Biotechnology – Principles & Applications of Recombinant DNA, ASM Press: Washington, D. C., 1994.

[21] Simmonds, R. S., Shearer, M. H., Kennedy, R. C. Parasitol. Today, 1997, 13, 328,

[22] Spier, R. E. Vaccine, 1996, 14, 1285.

[23] Emery, A. E. H., Macolm, S. An Introduction to Recombinant DNA in Medicine, John Wiley and Sons, Ltd: West Sussex, 1995.

[24] Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N. Y., 1989.

[25] Fynan, E. F., Webster, R. G., Fuller, D. H., Haynes, J. R., Santoro, J. C., Robinson, J. L. Proc. Natl. Acad. Sci. U. S. A., 1993, 90, 11478.

[26] Vahlsing, H. L., Yankauckus, M. A., Sawday, M. J. Immunol. Methods, 1994, 175, 11.

[27] Davis, H. L., Michel, M. L., Whalen, R. G. Ann. N Y Acad. Sci. 1995, 772, 21.

[28] Tigh, H., Corr, M., Roman, M., Raz, E. Immunol. Today, 1998, 19, 89.

[29] Fuller, D. H., Haynes, J. R. AIDS Res. Hum. Retrovir., 1994, 10, 1433.

[30] Fuller, D. H., Murphey-Corb, M., Clements, J., Barnet, S., Haynes, J. R. J. Med. Primatol., 1996, 25, 236.

[31] Mustafa, F., Richmond, J. F., Fernandez-Larsson, R., Lu, S., Fredriksson, R., Fenyo, E. M., O’Connell, M., Johnson, E., Weng, J., Santoro, J. C., Robinson, H. L. Virology, 1997, 229, 269.

[32] Richmond, J. F., Mustafa, F., Lu, S., Santoro, J. C., Weng, J. C., O’Connell, M., Fenyo, E. M., Hurwitz, J. L., Montfiori, D. C., Robinson, H. L. Virology, 1997, 230, 265.

[33] Lu, S., Manson, K., Wyand, M., Robinson, H. L. Vaccine, 1997, 15, 920.

[34] Lu, S., Wyatt, R., Richmond, J. F., Mustafa, F., Wang, S,; Weng, J., Montefieri, D. C. Sodroski, J., Robinson, H. L. AIDS Res. Hum. Retrovir., 1998, 14, 151.

[35] Richmond, J. F., Lu, S., Santoro, J. C., Weng, J., Hu, S. L., Montifiori, D. C., Robinson, H. L. J. Virol. 1998, 72, 9092.

[36] Leitner, W. W., Seguin, M. C., Ballou, W. R., Seitz, J. P., Shultz, A. M., Sheehy, M. J., Lyon, J. J. Immunol,, 1997, 159, 6112.

[37] Fuller,D. H., Corb, M. M., Barnett, S., Steimer, K., Haynes, J. R. Vaccine, 1997, 15, 924.

[38] Mor, G. Biochem. Pharmacol., 1998, 55, 1151.

[39] Danko, I., Wolff, J. A. Vaccine, 1994, 12, 1499.

[40] Hilleman, M. R., Ann. N Y Acad. Sci., 1995, 772,1.

[41] Lodmell, D. L., Ray, N. B., Ewalt, L. C., Vaccine, 1998, 16, 115.

[42] Nichols, W. W., Ledwith, B. J., Manam, S. V. Ann. N Y Acad, Sci. 1995, 772, 30.

[43] Ulmer, J. B., Deck, R. R., Dewitt, C. M., Donnelly, J. I., Liu, M. A. Immunology, 1996, 89, 59.

[44] Agadjanyan, M. G., Kim, J. J., Trivedi, N., Wilson, D. M., Monzavi-Karbassi, B., Morrison, L. D., Nottingham, L. K., Dentchev, T., Tsai, A., Dang, K., Chalian, A. A., Maldonado, M. A., Williams, W. V., Weiner, D. B. J. Immunol., 1999, 162, 3417.

[45] Torres, C. A., Iwasaki, A., Barber, B. H., Robinson, H. L. J. Immunol., 1997, 158, 4529.

[46] Klinman, D. M., Sechler, J. M., Conover, J., Gu, M., Rosenberg, A. S. J. Immunol., 1998, 160, 2388.

[47] Ulmer, J. B., Deck, R. R., Dewitt, C. M., Fu, T. M., Donnelly, J. J., Caulfield, M. J., Liu, M. A. Vaccine, 1997, 15, 839.

[48] Casares, S., Inaba, K., Brumeanu, T. D., Steinman, R. M., Bona, C. A. J. Exp. Med., 1997, 186, 1481.

[49] Tokunaga, T., Yamamoto, H., Shimada, S., J. Natl. Cancer Inst., 1984, 72, 955.

[50] Yamamoto, S., Yamamoto, T., Kataoka, T. J. Immunol., 1992, 148, 4072.

[51] Sato, Y., Roman, M., Tighe, H. Science, 1996, 273, 352.

[52] Kreig, A. M., Yi, A. K., Matson, S. Nature, 1995, 374, 546.

[53] Klinman, D. M., Yamshchikov, G., Ishigatsubo, Y. J. Immunol., 1997, 158, 3635.

[54] Klinman, D. M., Barnhart, K. M., Conover, J. Vaccine, 1999, 17, 19.

[55] Lee, S. W., Sung, Y. C. Immunology, 1998, 94. 285.

[56] Barry, M. A., Johnston, S. A. Vaccine, 1997, 15, 788.

[57] Trinchieri, G. Annu. Rev. Immunol.1995, 13, 251.

[58] Xu, D., Liew, F. Y. Immunology, 1995, 84, 173.

[59] Ulmer, J. B., Deck, R. R., Dewitt, C. M., Freidman, A., Donnelly, J. J., Lui, M. A., Vaccine, 1994, 12, 1541.

[60] Waris, M. E., Tsou, C., Erdman, D. D., Zaki, S. R., Anderson, L. J. J. Virol. 1996, 70, 2852.

[61] Li, X., Sambhara, S., Li, C. X., Ewasyshyn, M., Parrington, M., Caterini, J., James, O., Cates, G., Du, R. P., Klein, M. J. Exp. Med.,1998, 188, 681.

[62] Hsu, C. H., Chua, K. Y., Tao, M. H., Lai, Y. L., Wu, H. D., Huang, S. K., Hseih, K. H. Nat. Med., 1996, 2, 540.

[63] Slater, J. E., Zhang, Y. J., Arthur-Smith, A., Colberg-Poley, A. J. Allergy Clin. Immunol., 1997, 99, S504.

[64] Segal, B. M., Klinman, D. M., Shevach, E. M. J. Immunol., 1997, 158, 5087.

[65] Waisman, A., Ruiz, P. J., Hirschberg, D. L., Gelman, A., Oksenberg, J. R., Brocke, S., Mor, F., Cohen, I.R., Steinman, L. Nat. Med., 1996, 2, 899.

[66] Kagi, D., Ledermann, B., Burki, K., Seiler, P., Odermatt, B., Olsen, K. J., Podack, E. R., Zinkernagel, R. M., Hangartner, H. Nature, 1994, 369, 31.

[67] Walsh, C. M., Matloubian, M., Liu, C. C., Ueda, R., Kurahara, C. G., Christensen, J. L., Huang, M. T., Young, J. D., Ahmed, R., Clark, W. R. Proc. Natl. Acad. Sci. USA, 1994, 91, 10854.

[68] Doherty, P. C. Curr. Top. Microbiol. Immunol., 1996, 206, 1.

[69] Mullbacher, A., Hla, R. t., Museteanu, C., Simon, M. M. J. Virol., 1999, 73, 1665.

[70] Davis, H. L., Michel, M. L., Mancini, M., Schleef, M., Whalen, R. G. Vaccine, 1994, 12, 1503.

[71] Manickan, E., Rouse, R. J. D., Yu, Z., Wire, W. S., Rouse, B. T. J. Immunol. 1995, 155, 259.

[72] Bourne, N., Stanberry, L. R., Bernstein, D. I., Lew, D. J. Infect. Dis., 1996, 173, 800.

[73] Xiang, Z. Q., Spitalnik, S., Tran, M., Wunner, W, H., Cheng, J., Ertl, H. C. J. Virology, 1994, 199, 132.

[74] Wang, B., Ugen, K. E., Srikantan,, V., Agadjanyan, M. G., Dang, K., Refaeili, Y., Sato, A. I., Boyer, J., Williams, W. V., Weiner, D. B. Proc. Natl. Acad. Sci. USA, 1993, 90, 4156.

[75] Herrmann, J. E., Chen, S. C., Fynan, E. F., Santoro, J. C., Greenberg, H. B., Wang, S., Robinson, H. L. J. Infect. Dis. 1996 , 174, S93.

[76] Xu, L., Sanchez, A., Yang, Z. Y., Zaki, S. R., Nabel, E. G., Nichol, S. T., Nabel, G. J., Nat. Med. 1998, 4, 37.

[77] Geissler, M., Gesien, A., Tokushige, K., Wands, J. R. J. Immunol. 1997, 158, 1231.

[78] Suzuki, Y., Remington,J. S. J. Immunol., 1988, 140, 3943.

[79] Weiss, W. R., Berzofsky, J. A., Houghten, R. A., Sedegah, M., Hollindale, M., Hoffman, S. L. J. Immunol., 1992, 149, 2103.

[80] Weiss, W. R., Sedegah, M., Beaudoin, R. L., Miller, L. H., Good, M. F. Proc. Natl. Acad. Sci. USA, 1988, 85, 573.

[81] Romero, P., Maryanski, J. L., Corrandin, G., Nussenzweig, R. S., Nussenzweig, V., Zavala, F. Nature, 1989, 341, 323.

[82] Tarleton, R. L., J. Immunol., 1990, 144, 717.

[83] Tarleton, R. L., Koller, B. H., Latour, A., Postan, M. Nature, 1992, 356, 338.

[84] Kumar, S., Tarleton, R. L. Parasite Immunol., 1998, 20, 207.

[85] Denkers, E. Y., Yap, G., Scharton-Kersten, T., Charest, H., Butcher, B. A., Caspar, P., Heiny, S., Sher, A. J. Immunol., 1997, 159, 1903.

[86] Breman, J. G., Campbell, C. C., Bull. WHO, 1988, 66, 611.

[87] Sedegah, M., Jones, T. R., Kaur, M., Hedstrom, R., Hobart, P., Tine, J. A., Hoffman, S. L. Proc. Natl. Acad. Sci. USA, 1998, 95, 7648.

[88] Doolan, D. L., Hoffman, S. L. Parasit. Today, 1997, 13, 171.

[89] De Libero, G., Flesch, I., Kaufmann, S. H. Eur. J. Immunol., 1988,18, 59.

[90] Aldovini, A., Young, R. A. Nature, 1991, 351, 479.

[91] Stover, C. K., de la Cruz, V. F., Fuerst, T. R., Burlein, J. E., Benson, L. A., Bennet, L. T., Bansal, G. P., Young, J. F., Lee, M. H., Hatfull, G. F., Snapper, S. B., Barletta, R. G., Jacobs, W. R., Bloom, B. R. Nature, 1991, 351, 456.

[92] Winter, N., Lagranderie, M., Gangloff, S., Leclerc, C., Gheorghiu, M., Gicquel, B. Vaccine, 1995, 13, 471.

[93] Lalvani, A., Brookes, R., Wildinson, R. J., Malin, A. S., Pathan, A. A., Anderson, P., Dockrell, H., Pasvol, G., Hill, A. V. Natl. Acad. Sci. USA, 1998, 95, 70.

[94] Tascon, R. E., Colston, M. J., Ragno, S. Nat. Med., 1996, 2, 888.

[95] Conry, R. M., Widera, G., LoBuglio, A. F., Fuller, J. T., Moore, S. E., Barlow, D. L., Turner, J., Yang, N. S., Cureil, D. T. Gen. Ther., 1996, 3, 67.

[96] Graham, R. A., Burchel, J. M., Beverely, P., Taylor-Papadimitrio, J. Int. J. Cancer, 1996, 65, 664.

[97] Deck, R. R., DeWitt, C. M., Donnelly, J. J., Liu, M. A., Ulmer, J. B. Vaccine, 1997, 15, 71.

[98] Robinson, H. L., Boyle, C. A., Feltquate, D. M., Morin, M. J., Santoro, J. C., Webster, R. G. J. Infect. Dis., 1997, 176, S50.

[99] Boyle, C. M., Morin, M., Webster, R. G., Robinson, H. L. J. Virol.,1996, 70, 9074.

[100] Kang, Y., Calvo, P. A., Daly, T. M., Long, C. A. J. Immunol., 1998, 161, 4211.

[101] Akbari, O., Panjwani, N., Garcia, S., Tascon, R., Lowrie, D., Stockinger, B. J. Exp. Med., 1999, 189, 169.

[102] Gurunathan, S., Prussin, C., Sacks, D. L., Seder, R. A. Nat. Med., 1998, 4, 1409.

[103] Raz, E., Carson, D. A., Parker, S. E., Parr, T. B., Abai, A. M., Aichinger, G., Gromkowski, S. H., Singh, M., Lew, D., Yankauckus, M. A., Baird, S. M., Rhodes, G. H. Proc. Natl. Acad. Sci. USA, 1994, 91, 9519.

[104] Davis, H. L., Schirmbeck, R., Reimann, J.,Whalen, R. G. Hum. Gene Ther., 1995, 6, 1447.

[105] Wolff, J. A., Ludtke, J. J., Acsadi, G., Williams, P., Jani, A. Hum. Mol. Genet., 1992, 1, 363.

[106] Letvin, N. L., Montefiori, D. C., Yasutomi, Y., Perry, H. C., Davies, M. E., Lekutis, C., Alroy, M., Freed, D. C., Lord, C. I., Handt, L. K., Liu, M. A., Shiver, J. W. Proc. Natl. Acad. Sci. USA, 1997, 94, 9378.

[107] Sedegah, M., Hadstrom, R., Hobart, P. Proc. Natl. Acad. Sci. USA, 1994, 91, 9866.

[108] Benjamin, W. H., Wu, X.: Swords, W. E. Infect. Immun., 1996, 64, 2365.

[109] Garrido, F., Cabrera, T., Coucha, A., Glew, S., Ruiz-Cabello, F., Stern, P. L. Immunol. Today, 1993, 14, 491.

[110] Cabrera, T., Duggan-Keen, M., Cabello, F. R., Stern, P. L., Garrido, F.