Induction and characterization of humoral and cellular immune responses elicited via gene gun-mediated nucleic acid immunization

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21 (1996)

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Induction and characterization of humoral and cellular immune responses elicited via gene gun-mediated nucleic acid immunization Joel R. Haynes”, Deborah

H. Fuller, Dennis McCabe, William F. Swain, Georg Widera

Avragen, Inc., 8520 University Green, Middleton, WI 53562, USA

Contents

3 1. Nucleic acid vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..................................... 3 2. Gene gun-mediated DNA delivery in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 2.1. Elicitation of immune responses in rodents following gene gun-mediated delivery of antigen and cytokineencoding vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................................... 5 10 of responses elicited via gene gun-mediated DNA immunization .. ..... .... ...... ... ...... ..... .. ...... ...... . ........ 2.2. Characterization 13 3. Gene gun immunization of larger animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... .I ................................................. ......................... . . . . .......................................................................................16 4. Conclusions.. ................................................................................... References.. .......................................................................................16

1. Nucleic acid vaccines

Nucleic acid vaccines have the potential to mimic several characteristics of live attenuated viral or bacterial vaccines since they induce the de novo production of microbial antigens, leading to the presentation of correctly folded conformational determinants and the induction of MHC class I-restricted cytotoxic T lymphocyte (CTL) responses. Because plasmid DNA-based vaccines are noninfectious and incapable of replication, they may be regarded as an attractive alternative to the use of live attenuated or live recombinant viruses that generally carry a finite risk of pathogenicity. Recent activity in the development of candidate DNA vaccines has involved two parallel tracks based on the method of delivery. While the first reported DNA or genetic vaccine involved the intracellular delivery of an antigen-encoding plasmid vector to the skin of mice using a gene gun [l], subsequent DNA vaccine reports demonstrated *Corresponding

2. Gene gun-mediated

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The majority of reports describing the elicita-

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that intramuscular or intradermal inoculation of naked plasmid DNA was effective as well [2-141. Both methods elicit humoral, cellular and protective immune responses and represent an attractive strategy for developing a new generation of safe and effective vaccines for various infectious diseases. Gene gun vaccination technology, which relies on a specialized particle delivery device, offers the advantage of requiring minimal amounts of DNA and the capability of delivering this DNA intracellularly to the epidermis, a major immunological inductive site. In this article, we review the development of gene gun instrumentation and summarize progress in inducing and characterizing immune responses elicited via the intracellular delivery of DNA-coated gold particles to the epidermis.

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tion of humoral and cellular responses to specifc antigens following the parenteral administration of naked DNA expression vectors involved the direct intramuscular inoculation of DNA in saline. While this method results in the induction of immune responses to a wide variety of antigens. the mechanism of DNA uptake into skeletal muscle cells is relatively inefficient and poorly understood [lS]. In addition, recent data suggest that muscle transfection efficiencies in higher animals. such as ferrets and nonhuman primates. are considerably reduced relative to rodents [ 16,171. These observations, along with the fact that skeletal muscle is generally not considered a major immunological inductive site. suggest that alternative routes and methods of DNA vaccine delivery could result in considerably stronger responses using smaller quantities of DNA. While concern over the amount of DNA required to elicit specific responses may be largely one of economy. there is a limit to the amount of DNA that can be administered as a single DNA vaccine dose and still remain practical. The potential of gene gun-based gene transfer methods to effectively deliver DNA vaccines was recognized several years ago since this technology achieves the direct intracellular deposition of small quantities of DNA. While this advantage alone has the potential to dramatically reduce the amount of DNA required per immunization, the ability of gene guns to target the skin provides a simple means of delivering DNA to a major immunological inductive site [18,19]. The original gene gun concept is attributed to John Sanford who developed a method to accelerate microscopic tungsten particles coated with DNA directly into the interior of plant cells. This physical delivery method overcame many of the barriers typically associated with plant cell transformation [20,21]. While effective in plant cells and tissues, the original instrument was not practical for DNA delivery into the tissues of living animals. since gene transfer took place in an enclosed vacuum chamber and was mediated by a gun powder explosion. This device did. however. set the stage for improved instruments which were based on the original concept of accelerating a macroscopic projectile into a bar-

rier. resulting in the release of the DNA-coated microprojectiles and their subsequent penetration into the target tissue. A helium-powered version of the original gun powder device was later developed and enabled greater control over the delivery energy. facilitating particle delivery into various targets from cells in culture to intact plant or animal tissues [22]. In this version, rupture disks were employed to achieve the instantaneous release of pressurized helium, generating a pressure wave that accelerated a plastic disk, serving as a macrocarrier. into a steel mesh retaining screen. Upon impact with the screen, DNA-coated gold particles attached to the surface of the macrocarrier continued into the target cells and resulted in effective gene delivery in vitro [23]. A version of this instrument is commercially available from Bio-Rad (Hercules, CA) under the Biolistic trademark, but is limited to use in cultured cells or tissue specimens since DNA/particle delivery still occurs in a partial vacuum chamber. More recent modifications to the Biolistic device were implemented by Stephen Johnston, resulting in a hand-held wand version of the helium-powered instrument with the macrocarrier retaining screen positioned at the DNA/gold exit point in the end of the wand [24]. This design obviated the requirement for a vacuum chamber and enabled the wand to be placed in direct contact with various tissue target sites in vivo. Although this latter device is not commercially available, it has led to successful transgene expression in a number of tissues and cell types in vitro and in vivo [25] and to antigen-specific immune responses in vivo [1,26,27]. In parallel with the development of the Biolistic device, an alternative gene gun technology platform evolved under the Accell trademark ([28,29] Agracetus, Middleton, WI) and has been used to deliver numerous reporter, antigen and cytokine genes in vitro and in vivo [17,30-461. The original version of this technology employed a controlled electric discharge to accelerate a macrocarrier, consisting of an aluminized mylar membrane, into a retaining screen. This was the first demonstration of the use of a membranetype macrocarrier which resulted in a more effective release of DNA-coated gold particles

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upon impact with the retaining screen. The electric discharge version of the Accell gene delivery system resulted in effective gene delivery to cultured cells in vitro, rodent skin and other cells in vivo [30,32,34-37,40-421, and skin in larger animals such as ferrets and rhesus monkeys [17,45]. However, despite the successful induction of both transgene activity and immune responses in vivo using this instrument (see below), further development of the electric discharge technology for clinical use was discontinued since it was deemed impractical. As an alternative to the use of an explosive electric discharge, the Accell device was redesigned, resulting in a simplified, hand-held instrument utilizing compressed helium to accelerate DNA-coated gold particles in the absence of a macrocarrier [47]. In this design, DNA-laden gold particles are coated on the inner wall of short cartridges of Tefzel tubing and are stable in this form for at least one year at ambient temperatures. When loaded into the device, a bolus of pressurized helium is directed through the cartridge, entraining the gold particles in the gas flow and directing them through the barrel of the instrument, into the target tissue. The inner curvature of the barrel results in a significant broadening of the beam of gold particles to achieve a penetration pattern approximately 1 cm in diameter. In addition, the design of the barrel and the use of fixed spacers between the barrel and the target tissue causes the majority of the helium shock wave to be exhausted to the sides, minimizing tissue impact. As a result of this design and the ability to adjust the pressure and amount of gold delivered per shot, this device can be used for gene delivery in targets ranging from monolayer and suspension tissue culture cells, to skin and organ tissues of large animals. In as much as a variety of means have been devised to accelerate DNA-coated gold particles into target cells, the end result is the direct deposition of DNA within the cytoplasm or nucleus of individual target cells. Once in the cell, the DNA behaves in a manner similar to that seen following the delivery of DNA by other nonviral transfection methods such as electroporation, calcium phosphate precipitation or the

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use of cationic lipids or DEAE-dextran. Transient gene expression has been observed following gene gun-mediated delivery into cultured cells and organ explants in vitro [23,30,48,49] and in various tissues in vivo, such as the liver and skin [25,30,32,50]. The skin expression studies were notable from the point of view that essentially all of the expression was in the epidermis. lo-20% of the epidermal keratinocytes stained positive for gene expression following DNA/ gold delivery in mouse skin in vivo [25,30], even when particle penetration extended into the dermal layers. This suggests that low delivery energies that achieve particle penetration into the epidermis only can still achieve near maximal levels of gene expression, while minimizing the possibility for long-term maintenance of DNA in more permanent dermal tissues. This characteristic may be of importance when considering safety issues associated with the administration of DNA vaccines in healthy individuals. While gene gun delivery results in transient expression in most target tissues, longer term expression has been observed following gene gun delivery into muscle cells [32], consistent with many of the observations made following intramuscular DNA inoculation in saline. Longterm expression in muscle cells is likely due to the lack of cell division and dilution of DNA rather than chromosomal integration. This is not to say that chromosomal integration cannot occur following gene gun-mediated DNA delivery, since in vitro gene gun experiments in cultured cells resulted in integration frequencies of 1 X lo-” to 1 X 10-j [23,30,48] when selectable marker genes were delivered. Whether or not integration occurs in vivo remains to be determined but may not be an important issue if exfoliating cells, such as those found in the epidermis are the primary target for vaccine administration. 2.1. Elicitation of immune responses in rodents following gene gun-mediated delivery qf antigen and cytokine-encoding vectors The first reported demonstration of the induction of immune responses in vivo following naked DNA administration was by Tang et al. [l]

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and involved the use of the helium wand Biolistic device to deliver plasmid DNA to mouse ear epidermis. Humoral immune responses specific for human growth hormone (hGH) and human al-antitrypsin (hAAT) were elicited in several strains of mice using either the human p-actin promoter or the immediate early promoter from human cytomegalovirus (hCMV) to drive antigen expression. While the actual amount of DNA delivered in each immunization was not reported, typical DNA coating procedures generally result in the delivery of only 1 or 2 ,ug of DNA per shot. Thus, the hGH- and hAATspecific responses seen following one immunization, and boosted following receipt of a second immunization, developed following delivery of much less DNA than generally required in many of the subsequent intramuscular DNA vaccination reports. The ease with which immune responses were elicited in the above gene gun study was likely due to the use of efficient expression vectors, as well as to the direct intracellular delivery of these vectors. Promoter comparison studies following gene gun delivery in vivo demonstrated that the hCMV promoter was superior to all other viral and cellular promoters tested in a number of tissues, including the epidermis [31,32]. Indeed, the hCMV promoter, with and without its associated intron A sequence [51], has been the promoter of choice for most DNA vaccine studies, regardless of the DNA delivery method. Eisenbraun et al. [34] also demonstrated the induction of immune responses to hGH following use of the electric discharge Accell instrument. After delivery of as little as 80 ng of a hCMVhGH expression vector to the abdominal skin of mice, hGH-specific IgG responses were observed in 100% of animals that received at least 0.16 mg of gold per shot. While lesser amounts of gold resulted in poor responses, varying the DNA from 16 ng to 4000 ng per shot had little effect, showing that the delivery of as few as 300 copies of plasmid per gold particle was sufficient to elicit vigorous humoral responses. Despite the apparent lack of a DNA dose-response relationship, more recent experiments showed a direct correlation between the level of input DNA and resultant hGH expression. Reducing the amount

of input DNA from 500 ng to 0.7 ng per shot resulted in a 500-fold decrease in protein expression. However, consistent with the earlier study. geometric mean titers between animals that received the 500 ng and 0.7 ng doses differed by only 2.5-fold, showing that maximal levels of antigen expression need not be achieved to elicit vigorous responses (T. Roberts, J. Haynes, unpublished). Further data in support of the concept that low levels of antigen expression in the epidermis are sufficient for the elicitation of significant immune responses came from the study by Barry et al. [27], in which the Biolistic helium wand device was used to immunize mice with libraries of expression constructs derived from Mycoplusma pulmonis. Expression libraries in this case were formed by fusing M. pulmonis DNA fragments onto the last exon of an hGH expression vector using all three reading frames. Immunization of mice with two different libraries consisting of approximately 3 X 10” members each (of which 500 should be in frame) resulted in complete protection following challenge, with no detectable lung lesions nor culturable mycoplasma. Since smaller libraries containing only 69 members each yielded no protection, it is likely that a small minority of gene fragments in each of the larger libraries were responsible for the observed protection. These findings are consistent with the above data showing that subnanogram quantities of antigen expression vectors can elicit significant responses following gene gun delivery to the epidermis. The hGH immunization study of Eisenbraun et al. [34] using the electric discharge gun also demonstrated the importance of the depth of gold particle penetration. Peak levels of both antigen expression and IgG responses were correlated with DNA/gold delivery specifically to the epidermis. The use of higher delivery energies to achieve penetration into dermal tissues not only resulted in disruption of the epidermal layer but significantly reduced both antigen expression and humoral responses. These findings were consistent with earlier reporter gene studies demonstrating that the vast majority of expression following skin delivery of DNA/gold was found in the epidermis [25,30]. While these data

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indicate that the epidermis is the ideal target site for gene gun-mediated DNA delivery to the skin, the capability of achieving DNA transfer exclusively within the epidermis by adjusting the delivery energy may be important from a safety standpoint, since epidermal targeting should minimize long-term DNA maintenance as mentioned above. Preliminary studies examining the presence of both DNA and expressed protein in the skin of mice following epidermal DNA/gold delivery, using either the electric discharge or helium pulse devices, are consistent with DNA delivery into an exfoliating tissue. hGH and hepatitis B surface antigen (HBsAg) expression in the skin of mice peaked at 24 h following delivery and gradually diminished with time. While hGH activity was barely detectable 10 days following delivery, HBsAg expression was seen for as much as three weeks, which may be due to the stability of HBsAg particles that form following HBsAg gene expression in mammalian cells (G. Widera, unpublished). PCR analysis of the kinetics of DNA disappearance showed a peak DNA signal immediately following delivery which disappeared within seven days (M. Ford, J. Haynes, unpublished). A cursory examination of other tissues of the treated animals did not yield a signal, consistent with little trafficking of DNA from the original delivery site. However, it remains to be determined whether DNA delivered to the epidermis can migrate to draining lymph nodes via trafficking Langerhans cells. While no direct evidence for this exists, the maintenance of a small amount of DNA in Langerhans or other dendritic cells within draining lymph nodes is a possible explanation for the longevity of responses elicited via gene gun delivery to the epidermis (see below). Humoral immune responses elicited via gene gun immunization against antigens such as hAAT, influenza nucleoprotein and HBsAg are. stable for extended periods of time following administration of the last immunization in the regimen. Fig. 1 shows an example of this phenomenon, where six mice received three DNA vaccinations containing 2 pug of an HBsAg expression vector using the electric discharge Accell device. Immune responses were observed in

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Weeks Fig. 1. HBsAg-specific antibody responses in mice following receipt of gene gun-mediated DNA immunizations at weeks 0.4 and 6. Immunizations contained 2 pg of a CMV-HBsAg expression vector and were delivered to the abdominal epidermis in the form of two tandem shots using the electric discharge Accell device. Each shot contained 0.5 mg of gold and 1.0 pg of DNA. Antibody responses were quantified using the Abbott AUSAB ELISA kit and quantification panel.

all animals following the primary immunization and 5 of 6 animals exhibited antibody titers that were higher than the 0.01 IU/ml level considered protective in humans [52]. HBsAg-specific antibody responses reached their peak following the second immunization at 4 weeks and were 2-4 orders of magnitude higher than the protective standard in all animals. These responses did not increase further following a second booster immunization but remained at elevated levels for more than one year following the final immunization. The absence of a booster response following the third immunization may be due to the already elevated responses following the second immunization and the short time frame between the second and third immunizations. Preliminary data from rhesus monkey trials indicate that significant enhancements in booster responses can be obtained by lengthening the resting period between immunizations (see below). In a direct comparison of the humoral responses elicited against hGH, hAAT and influenza NP using the electric discharge Accell gene gun and intramuscular inoculation tech-

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niques. high-titered responses were elicited against all three antigens in all animals tested, using as little as 16 ng of DNA per gene gun immunization [35]. Gene gun immunizations containing lO- and lOO-fold more DNA did not result in stronger responses, indicating that this level of DNA was sufficient to elicit the strongest responses obtainable using this approach. In contrast, immune responses elicited against these antigens following intramuscular inoculation of the same vectors were considerably weaker, even with the injection of as much as 5000-fold more DNA. Similar results were seen following examination of CTL responses in these same animals. This study also demonstrated the equivalence of the older electric discharge technology and the more recent helium pulse Accell gene gun, in that identical hGH-specific antibody titers were elicited, regardless of the device. Recent investigation of the number of DNA doses required to elicit peak responses for an HBsAg DNA vector showed that a single immunization in mice elicits responses equivalent to those seen following two immunizations spaced four weeks apart. Fig. 2 shows the HBsAg-specific antibody responses in two groups of animals that received one and two gene gun immunizations, respectively. Although the two groups of animals are from separate experiments using different guns, a single helium pulse gene gun immunization elicited antibody titers that were several log,,,-fold higher than the 01 IUiml protective standard and equal to the titers observed in a second group of animals that received two electric gun immunizations. While similar results have been observed for the influenza virus hemagglutinin (HA) expression vector (Harriet Robinson, personal communication), a single immunization will not suffice for all expression constructs since the influenza NP vector reproducibly requires two immunizations to elicit peak responses [35]. Differences in dosage requirements for various vectors may reflect differences in the length of transgene expression following delivery. Because NP expression is toxic for most cells, NP expression in the epidermis following DNA/gold delivery may be of a shorter duration, consistent with the requirement for two doses. In addition to humoral immune responses

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Fig. 2. Comparison of HBsAg-specific immune responses in mice that received either one or two gene gun-mediated HBsAg DNA immunizations. One group of five mice received a single HBsAg DNA immunization at week 0 using the helium pulse Accell device. Immunizations were administcrcd as two tandem shots to the abdominal epidermis in which each shot contained 1.0 ,ug of DNA and 0.5 mg of gold. A second group of three mice received two immunizations using the electric discharge Accell device at weeks 0 and 4 in which each immunization consisted of two shots containing 1 pg of DNA and 0.5 mg of gold per shot. HBsAgspecific antibody levels were measured at weeks 0, 4 and 6.

described above, protective responses have also been observed in rodents following gene gun immunization. Fynan et al. [36] demonstrated that primary and booster immunizations containing 0.4 pug of a vector encoding influenza HA resulted in the induction of protective immunity in 95% of immunized mice, as determined by a lethal challenge using mouse-adapted influenza virus. While gene gun-immunized mice exhibited little in the way of symptoms following challenge, multiple groups of other animals that received parenteral DNA inoculations via a variety of routes (intramuscular, intradermal, intraperitoneal and subcutaneous) exhibited significantly more pronounced symptoms, including weight loss and fur ruffling, despite the administration of as much as 300 ,ug of DNA per immunization. It is interesting to note that titration of the DNA dose in the gene gun immunizations resulted in dramatically reduced protection with DNA doses lower than 0.4 hug, despite the above reported studies demonstrating high-titered antibody responses using as little as 16 ng of DNA per

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immunization. This may be due to the particular HA vector used which contained the hCMV immediate early promoter but did not include the intron A region. An additional HA vector containing the complete CMV intron A promoter resulted in high titered responses using only 4 ng of DNA in a single, primary immunization (Harriet Robinson, personal communication: ]441). Protective responses were also observed in mice following helium pulse gene gun immunizations with vectors encoding either the VP4, VP6 or VP7 proteins of murine rotavirus [37]. Each DNA separately elicited rotavirus-specific CTL and antibody responses. Virus neutralizing responses were also seen following immunization with either VP4- or VP7-encoding DNAs. In addition, protective immunity, as measured by challenge with 100 50% infectious doses of virus, was elicited following immunization with each DNA, even in the absence of neutralizing antibodies following VP6 DNA immunization. An additional immunization and protection study using the electric discharge Accell gun was reported by Zarozinski et al. [39] using a lymphocytic choriomeningitis virus (LCMV) challenge model in mice. Gene gun immunizations using a vector encoding only the LCMV nucleoprotein were employed to test for the induction and protective effect of CTL responses in the absence of neutralizing antibodies to the LCMV glycoprotein. A single gene gun immunization with the NP vector resulted in nearly a two log,,, reduction in LCMV titers following intraperitoneal virus challenge. A greater than three log,, reduction in virus titers was observed in animals that received two or three gene gun immunizations, with only 1 of 4 animals in each of two experiments exhibiting any detectable virus following challenge. Protection was attributed to the induction of memory CTLs which could be detected prior to challenge in a limiting dilution assay following in vitro stimulation. In contrast to the gene gun LCMV immunization above, Martins et al. [53] reported protection against LCMV infection in only 50% of animals receiving three 200 pg doses of a similar CMV-NP DNA vaccine administered by intramuscular injection. Unlike the gene gunimmunized animals above, neither NP-specific

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antibody nor CTL responses were detected in any animals vaccinated by intramuscular DNA inoculation. Interestingly, in the LCMV gene gun immunization of Zarozinski et al. [39], gene gun immunization prior to a lethal intracranial LCMV challenge which normally results in immunopathological disease and death in naive mice, resulted in either enhanced immunopathogenesis or protection, depending on the individual animal. Immunopathogenesis following intracranial LCMV infection is mediated by a CD8’ T cell response so it is not clear why the prior induction of LCMV NP-specific CTL responses would be protective in approximately 30% of the animals and accelerate symptoms by approximately 2 days in the others. In a similar intracranial LCMV challenge study following intramuscular NP DNA vaccination, Yokoyama et al. [54] reported the induction of CTL responses and protection in approximately 50% of vaccinated animals that received either one, two or three 100 pug doses of an LCMV NP DNA vaccine. In this latter study there was no report of enhanced immunopathogenesis upon challenge following intramuscular DNA vaccination. Additional data showing that gene gun-elicited CTL responses can confer protective immunological effects were reported by Irvine et al. [38]. Mice that were immunized with a p-galactosidase expression construct (pCMVI&gal), using the helium pulse Accell device, developed antigen-specific humoral and CTL responses that conferred protection upon challenge via injection with /3-galactosidase-expressing CT26 tumor cells. Protection in this case was mediated by cellular immunity, as shown by the adoptive transfer of splenocytes from immune animals following in vitro restimulation. In this case, adoptive transfer of immune cells resulted in a reduction of established pulmonary nodules in mice with already established metastases. Interestingly, direct gene gun vaccination using the pCMVI@gal vector in mice with already established lung metastases had no effect. This observation suggests that the P-galactosidase-specific cellular responses were not elicited rapidly enough or were below the minimum threshold required to cause regression. On the other hand, regression of established metastases was seen

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following pCMVI@gal gene gun immunization coupled with systemic cytokine delivery using either human rIL-2. mouse rIL-6, human rIL-7 or mouse rIL-12 at doses that had no effect in the absence gene gun treatment. These data demonstrate the potential of coupling gene gun immunizations with either cytokine administration or direct cytokine gene codelivery to modulate or augment antigen-specific immune responses. Additional evidence for the ability of cytokines to augment immune responses elicited following gene gun-mediated DNA immunization was reported by Conry et al. [55]. In this study, delivery of a granulocyte macrophage colony stimulating factor (GM-CSF) vector to the epidermis of mice using the helium pulse Accell gun three days prior to immunization with a vector encoding carcinoembryonic antigen (CEA), resulted in significantly enhanced CEAspecific antibody and proliferative T cell responses. These data are consistent with the known stimulatory effects of GM-CSF on epidermal Langerhans cells and provide evidence for the direct involvement for epidermal Langerhans cells in mediating responses elicited via vector delivery to the epidermis. Consistent with the transient expression of antigen in the epidermis and the time required for stimulation of Langerhans cells with GM-CSF. simultaneous delivery of the CEA and GM-CSF vectors resulted in no positive effect on CEA-specific immunity. In fact, simultaneous vector delivery largely abrogated the induction of CEA-specific humoral and cellular responses. Whether the inhibition of CEA immune responses via simultaneous CEA / GM-CSF vector delivery was due to vector competition or some other mechanism is not known at this time In as much as cytokine vector delivery to the epidermis may be a means of augmenting or modulating immune responses to antigens encoded by nucleic acid vaccines, the augmentation of tumor-specific immune responses can be achieved by gene gun delivery of cytokine expression vectors directly into tumor tissues in vivo or into the skin overlying subcutaneous tumors [40,41]. Using the electric discharge Accell device, Sun et al. [40] showed that in vivo delivery of a human IL-6 vector into an existing

tumor resulted in reduced methylcholanthreneinduced fibrosarcoma growth. In addition, a combination of murine tumor necrosis factor alpha (TNF-CX) and interferon-y vectors inhibited the growth of a renal carcinoma (Renca) tumor model, and treatment with murine IL-2 and interferon-y constructs prolonged the survival of Renca tumor-bearing mice. Thus, direct cytokine gene delivery in vivo may play an important role in either modulating or augmenting existing responses or new responses elicited as a result of antigen gene delivery, either by gene gun or intramuscular inoculation [56]. While direct cytokine gene delivery is one means of augmenting antigen-specific immune responses, the development of chimeric antigenencoding expression vectors provides an alternative strategy for enhancing responses to defined epitopes. Schodel et al. [57] demonstrated that chimeric hepatitis B core antigen (HBcAg) particles containing a malaria circumsporozoite (CS) epitope, between amino acid positions 75 and 81 of the HBcAg, stimulated vigorous CSspecific antibody responses and protection against Plasmodium berghei challenge in mice. While these chimeric particles were produced in an engineered Salmonella host strain and purified prior to vaccination, it follows that the direct in vivo delivery of a recombinant expression vector encoding chimeric HBcAg may be an effective means of augmenting immune responses to specific epitopes that may be poorly immunogenic in their native configuration. In one such experiment, a chimeric vector encoding HBcAg with a 15 amino acid HIV-l gp120 V3 loop insertion between amino acids positions 75 and 81 was delivered to the epidermis of mice using the helium pulse Accell instrument. HIV-l gp120 V3 loop-specific responses in these animals were enhanced 200- to 500-fold relative to animals that received a dedicated vector encoding the native HIV-l gp120 (D. Fuller and J. Haynes, unpublished). 2.2. Characterization of responses elicited via gene gun-mediated DNA immunization In addition to the measurement of humoral, cellular and protective immune responses in

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rodents and larger animals (see below), data are emerging regarding the types of responses elicited following gene gun-mediated DNA immunization. The first report of cytokine responses following gene gun vaccination involved the delivery of HIV-l gp120 expression vectors in mice [42] in which gp120 antigen expression was relatively low due to poor vector performance. The low levels of antigen production following DNA delivery to the epidermis were consistent with the delayed appearance and weak nature of gpl20-specific humoral responses. Interestingly, while gp120 antibody responses were not observed until after the second booster immunization, gpl20-specific CTL responses were seen following the primary immunization. CTL responses peaked following the first boost and declined with increasing antibody responses. These data are consistent with a qualitative progression in the nature of the gpl20-specific immunity due to the apparent reciprocity between the humoral and cytotoxic cellular responses and were confirmed by examining cytokine production patterns in antigen-stimulated splenocytes after increasing numbers of immunizations. These data showed that interferon-y production peaked following the first boost, similar to CTL responses and declined thereafter with the appearance of IL-4 production and humoral responses, consistent with a progression from a Thl to a Th2 response with successive immunizations. Further, examination of IgG subclass responses showed a low level of both IgGl and IgG2a early in the regimen, with enhancement of IgGl responses at the time of increasing antibody and IL-4 production (D. Fuller and J. Haynes, unpublished). Additional data showing that this qualitative progression was a function of the number of immunizations rather than time, came from experiments showing that memory CTL responses in animals immunized only once were constant for as long as a year but were significantly reduced in animals immunized multiple times. Additional cytokine and IgG subclass data involving gene gun immunizations using the influenza NP gene have also been completed [43]. In this study, both gene gun and intramuscular immunizations were employed, using

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1,ug and 100 pg of DNA, respectively, per immunization. Despite a lOO-fold difference in the amount of DNA inoculated, essentially identical endpoint IgG titers, CTL activity and interferon-y responses were observed following the primary and first booster immunizations between the gene gun and intramuscular groups. However, following the second boost, interferony production dramatically decreased in both groups of animals, being replaced by strong IL-4 production in the gene gun animals only. Little if any IL-4 activity was observed in animals immunized via intramuscular inoculation. While the endpoint IgG titers were similar between both groups, the antibody responses in the gene gun animals were mainly of the IgGl subclass, while the intramuscular immunizations resulted in a preponderance of IgG2a antibodies. The subclass ratios in individual animals were constant throughout the regimen and were independent of the fluctuations in the cytokine profiles, unlike that seen in the HIV-l gp120 immunization described above. These data demonstrate that intramuscular inoculation led to Thl-like responses due to elevated IgG2a levels, production of interferon-y, CTL activity and lack of IL-4. However, the gene gun responses were more difficult to categorize due to the presence of significant interferon-y and CTL activity, on the one hand and elevated IgGl antibodies and increasing IL-4 production with successive immunizations, on the other. This is reminiscent of the Thl to Th2 shift described above for gp120, but without the fluctuations in the IgG subclass profiles. The antibody subclass and cytokine production patterns in the gene gun animals immunized with NP DNA were essentially identical to those observed in control animals immunized with inactivated influenza virus in Freund’s adjuvant, showing that the interferon-y to IL-4 fluctuations and preponderance of IgGl responses may have been more a function of the antigen rather than the method of immunization. The only qualitative difference was that gene gun immunization resulted in significant NP-specific CTL activity while the Freund’s immunizations did not. Further data showing that the types of responses following gene gun immunization may be

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controlled more by the identity of the antigen rather than the method of immunization were obtained from an experiment examining the antibody subclass and cytokine responses following successive gene gun immunizations using a HBsAg expression vector (Fig. 3). These data demonstrate that successive HBsAg gene gun immunizations result in the elicitation of both interferon-y, as well as IL-4 production, with no evidence for qualitative shifts with successive immunizations. In addition, enhanced levels of IgG2a antibodies were observed relative to IgG 1.

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unlike the data from the prior NP and gp120 DNA. In a different series of analyses, Justewicz et al. 1441 demonstrated that antibody-forming cells (AFCs) specific for influenza virus could be found in the spleen and bone marrow 33 days following a single immunization with an influenza hemagglutinin (HA) DNA vaccine administered using the electric discharge device. Similar hemagglutination-inhibition antibody titers were observed in animals that received either one or two immunizations since antibody titers con0.7

‘!! [Antibody

0.6 zo.5 6 0.4 t 6 0.3 O 0.2 0.1 P

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Fig. 3. Measurement of HBsAg-specific antibody. GIL and cytokine responses in three groups of five mice each following receipt of one, two and three DNA immunizations respectively. using the helium pulse Accell device. Each immunization contained 2 pg of CMV-HBsAg vector DNA and was delivered in the form of two tandem shots to the abdominal epidermis in which each shot contained 1 pg of DNA and 0.5 mg of gold. Immune responses were analyzed following the final immunization in each of the three groups of animals. Immunizations were administered at weeks 0, 4 and 6 in the group of animals that received the full three dose regimen. HBsAg-specifc antibody responses wcrc measured using the Abbott AUSAB ELISA kit and quantification panel while IgGl and IgG2a subclasses were detected in an ELISA using immobilized HBsAg antigen and IgG subclass-specific antihody conjugates from Southern Biotech. Interferon-y and IL-4 responses were measured in supernatants of HBsAg-stimulated splenocytes using ELISA kits from Endogen and Genzyme. respectively.

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tinued to rise in the animals that received only a single immunization. In addition, similar low levels of AFCs were found in the spleen and bone marrow of both groups. However, in response to a lethal challenge, both IgG- and IgAproducing AFCs became localized in lymph nodes of the upper and lower respiratory tracts, at levels similar to those seen in animals previously immunized with live virus, consistent with the extent of protection observed. In contrast, in animals immunized with an HA-containing subunit vaccine, fewer AFCs were generated in response to challenge and reduced levels of protection relative to the gene gun-immunized animals were observed. Thus, gene gun-mediated DNA immunization may mimic the actions of live vaccines by eliciting the appropriate numbers and types of AFCs (IgG and IgA) in the appropriate locations for viral clearance.

3. Gene gun immunization of larger animals One of the advantages of gene gun-based DNA vaccines is the potential to achieve effective antigen expression in the epidermis of larger animals with only modest increases in the amount of DNA required to elicit significant immune responses in rodents. The first demonstration of this was reported by Webster et al. [17] using the electric discharge Accell device to immunize ferrets with an influenza virus HA expression construct. Earlier intramuscular inoculation studies in ferrets showed that two or three 500 pg inoculations of pCMV/Hl DNA resulted in accelerated clearance of influenza virus strain A/PR/8/34 in nasal washes five days post challenge. While virus titers in immunized and control animals were similar (approximately 5.5-6.5 log,,, 50% egg infectious doses (EIDS,,)) three days post challenge, the animal that received three intramuscular DNA doses exhibited only 1.5 log,, EID,, of virus on day 5 relative to 6.2 log,,, EIDso in the control animal. The animal that received 2 intramuscular pCMV/HI inoculations showed 4.7 log,, EID,, on day 5 post challenge. In contrast, immunization of three ferrets with two 2.0 pg doses of pCMV/HI DNA using the gene gun resulted in complete protec-

13

tion, in that no virus was detectable in nasal secretions on day three following challenge. 2.0 pg of DNA was likely the minimum dose required for the observed protection with this particular vector since only one of three ferrets that received two 0.4 pg doses of the same DNA exhibited a similar level of protection. Further evidence for the enhanced efficacy of the gene gun approach in these animals came from analysis of neutralizing antibody activity in the sera of animals prior to challenge. No neutralizing activity was observed in animals that received intramuscular inoculations containing 500 pg of pCMV/Hl DNA but two of three ferrets that received the 2.0 pg doses via gene gun delivery exhibited significant prechallenge neutralizing activity. In addition, analysis of neutralizing activity in all ferrets post challenge showed that only the gene gun-immunized animals exhibited significant levels of cross-neutralizing activity. In addition to ferrets, vigorous immune responses were observed in pigs following delivery of vectors encoding the hepatitis B surface and core antigens using the helium pulse Accell device. Fig. 4, Panel A shows antibody responses in pigs following the receipt of two 3.0 ,ug doses of an HBsAg expression vector delivered to the epidermis of the ear or the undersurface of the tongue. HBsAg-specific antibody responses exceeding the 10 mIU/ml protective level in humans by at least lo-fold were observed in 4 of 5 pigs three weeks following the booster immunization. While one of the animals immunized on the undersurface of the tongue did not respond well, immune responses in the remaining four animals increased markedly during the three week period following the booster immunization, suggesting that immune responses may continue to evolve for a significant period of time following immunization, as observed in rodent experiments. Pigs also responded well to HBcAg following DNA immunization as shown in Fig. 4, Panel B. Two 1.5 ,ug doses of a CMV-HBcAg vector resulted in HBcAg-specific titers of > 1:12 000 in 4 of 4 animals. While data from this figure indicate that decreasing the DNA dosage to 0.25 pug results in poorer responses, preliminary data

J.R. Haynes et al. I Advanced

A

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Drug Delivery Reviews 21 (19Y6) 3-18

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Fig. 4. Elicitation of HBsAg- and HBcAg-specific antibody responses in pigs via gene gun-mediated DNA immunization. Panel A. HBsAg-specific immune responses in pigs following primary and booster immunizations spaced 4 weeks apart. Each immunization consisted of six tandem gene gun shots to the epidermis of the ear or the undersurface of the tongue. Each shot contained 0.25 mg of 1-3 micron gold and 0.5 pg of HBsAg vector DNA. Antibody responses were measured 4 weeks following the primary immunization and 1 and 3 weeks following the booster immunization. Each cluster of bars represents an individual animal. Panel B, HBcAg-specific immune responses in pigs following primary and booster immunization spaced 4 weeks apart. The 0.25 ,LL~ DNA immunizations consisted of one gene gun shot per immunization while the 1.5 pg DNA immunizations consisted of 6 gene gun shots per immunization. Each shot contained 0.25 mg of l-3 micron gold and 1 /*g of DNA per mg gold. Antibody responses were measured 4 weeks following the primary immunization and 2 weeks following the booster immunization. Each pair of bars represents an individual animal.

from more recent dose titrations indicate that immune responses to smaller doses of DNA can be dramatically improved by increasing the resting period between primary and booster immunizations (data not shown). In addition to pigs, gene gun-based DNA immunizations have yielded significant responses in rhesus monkeys as well. Fig. 5 shows the results of a two dose CMV-HBcAg DNA immunization experiment in mice and rhesus monkeys using the helium pulse Accell device to administer 2.0 pg and 3.0 pg does of DNA per immunization, respectively. Following a primary immunization, both species developed modest HBcAg-specific titers that increased to 1:24 OOO1:lOO 000 following a booster immunization. The extent to which the final titers were actually dependent on receipt of the booster immuniza-

tion is not known at this time. If preliminary results seen in rodents using certain antigen expression vectors are predictive of results in larger animals, it is conceivable that responses in larger animals will continue to develop for an extended period of time in the absence of boosting. CTL responses have also been reported in rhesus monkeys following the delivery of simian immunodeficiency virus expression vectors using the electric discharge device [45]. In this study, monkeys received clusters of immunizations at weeks 1 and 3, 11 and 13 and 21 and 23. While one group of four animals was immunized via gene gun as well as intramuscular and intraderma1 inoculation, a second group of three animals received gene gun immunizations only. Intramuscular and intravenous inoculations con-

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Drug Delivery Reviews 21 (1996) 3-18

Fig. 5. HBcAg-specific antibody responses in mice and rhesus monkeys following gene gun-mediated DNA immunization. Six mice and two rhesus monkeys each received primary and booster immunizations at weeks 0 and 4, respectively using an HBcAg expression vector driven by the hCMV intron A promoter. Each mouse immunization consisted of 2 tandem gene gun shots in which each shot contained 1.0 pg of vector DNA and 0.5 mg of 0.95 micron gold. Each monkey immunization consisted of three tandem gene gun shots in which each shot contained 1.0 pg of vector DNA and 0.25 mg of 1-3 micron gold. Antibody responses were measured by ELISA at weeks 4 and 6. Closed circles, mice; open circles. monkeys.

tained 500 pug of DNA per vector per immunization while gene gun inoculations contained 29 pg of DNA per vector per immunization. A total of five SIV expression vectors were employed, encoding both truncated and full length versions of the SIV envelope glycoprotein, as well as noninfectious SIV virus-like particles. 14 weeks following the first immunizations, all seven vaccinated animals exhibited env-specific CTL activity,while only two animals from the group that received all three routes exhibited gag-specific CTL activity. The lower level of gag CTL activity is likely related to the fact that only 1 of the 5 vectors delivered encoded the gag antigen. However, CTL activity specific for both env and gag persisted in all monkeys between the second and third clusters of vaccinations and was shown to be both CDS’ and MHC class I-restricted. In as much as the preliminary gene gun im-

munizations in larger animals described above yielded results indicating that this technology may provide a safe, effective and economical means of administering nucleic acid vaccines in the clinic, it should be noted that immunization regimens have yet to be optimized. Important variables to investigate include the site of epiderma1 delivery, the number and timing of doses, the number of gene gun shots per immunization and the total amount of DNA required. Although significant responses have been observed following delivery of vectors encoding a number of antigens, simple changes in the immunization regimen can result in dramatic improvements in immunization effectiveness. An example of this is shown in Fig. 6, in which data from two monkey immunizations using the separate helium pulse Accell device to deliver pCMVSIVenv vectors to the abdominal epidermis of rhesus macaques are presented. SIV gpl20-spe-

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16

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cific immune responses were monitored over a 16 week period but one group of animals received three immunizations at weeks 0, 6 and 13, while the second received only two immunizations at weeks 0 and 13. Surprisingly, geometric mean titers in the latter group were 11-fold higher than those observed in the animals that received an additional immunization over the same time frame. These data show that longer resting periods and fewer immunizations may be a simple means of markedly improving responses to epidermally administered DNA vaccines. Additional systematic studies to optimize the variables mentioned above will provide important data, setting the stage for future human clinical investigation of this emerging vaccine technology.

PI

Ulmer,

J.B., Donnelly,

J.J., Parker,

S.E., Rhodes,

G.H..

Felgner. P.L., Dwarki, V.J., Gromkowski, S.H.. Deck, R.R., Dewitt. CM., Friedman, A., Hawe, L.A.. Leander, K.R.. Martinez, D., Perry. H.C., Shiver, J.W. MontD.L. and Lui, M.A. (1993). Heterologous gomery. protection against influenza ing a viral protein. Science

PI

Wang.

B., Ugen,

by injection 259. 1745.

K.E.. Srikantan,V..

of DNA encod-

Agadjanyan.

M.G.,

Dang, K., Refaeli, Y., Sato. A.I.. Boyer, J., Williams, WV. and Weiner. D.B. (1993). Gene inoculation generates immune responses against human immunodeficiency virus type 1. Proc. Natl. Acad. Sci. USA 90, 4156.

I41Davis.

H.L.. Michel, M.-L. and Whalen, R.G. (1993). DNA-based immunization induces continuous secretion

of hepatitis B surface antigen and high levels of circulating antibody. Hum. Mol. Genet. 2. 1847-1851.

151Watanabe,

A.. Raz. E., Kohsaka, H., Tighe. H., Baird, S.M., Kipps, T.J. and Carson, D.A. (1993). Induction of antibodies to a kappa V region by gene immunization. J. lmmunol. 151. 2871.

IhI Robinson, 4. Conclusions Gene gun-mediated, intracellular delivery of small quantities of antigen expression vectors within the epidermis results in transient antigen expression and the induction of humoral, cellular and protective immune responses in a variety of animal models. The effectiveness of this vaccine technology in both rodents and larger animals is consistent with its reliance upon a physical, intracellular delivery process with minimal interspecies variation. Data from several studies have shown that not only the strength of resulting immune responses but also the quality of these responses may be modulated by variation of the number and timing of immunizations. Demonstration of the induction of both humoral and cellular immunity, as well as true vaccine protection, provides compelling evidence that nucleic acid vaccines delivered via particle delivery can mimic important characteristics of live vaccines, such as MHC class I antigen presentation and the presentation of correctly folded, three dimensional B cell epitopes.

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H.L.. Hunt, L.A. and Webster, R.G. (lY93). Protection against a lethal influenza virus challenge by immunization with a haemagglutinin-expressing plasmid DNA. Vaccine 11, 957. I71 Cox. G.J.. Zamh, T.J. and Bahiuk, L.A. (1993). Bovine herpesvirus 1: Immune responses in mice and cattle injected with plasmid DNA. J. Virol. 67, 5664.

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M., Schleef, M. and Whalen, R.G. (1994). Direct gene transfer in skeletal muscle: plasmid DNA-based immunization against the hepatitis B virus surface antigen. Vaccine 12, 1503. R.. Hobart. P. and Hoffman. PI Sedegah, M., Hedstrom, S.L. (1994). Protection against malaria by immunization with plasmid DNA encoding circumsporozoite protein. Proc. Natl. Acad. Sci. USA 91. 9866. Il()l Raz, E., Carson, D.A., Parker, S.E., Parr, T.B., Ahai. A.M.. Aichinger, G., Gromkowski, S.H., Singh, M., Lew. D., Yankauckas, M.A., Baird, SM. and Rhodes, G.H. (1994). Intradermal gene immunization: The possible role of DNA uptake in the induction of cellular immunity to viruses. Proc. Natl. Acad. Sci. USA 91, 9519. [Ill Donnelly, J.J.. Friedman, A., Martinez, D., Montgomery, D.L.. Shiver, J.W.. Motzel, S.L., Ulmer, J.B. and Liu. M.A. (1995). Preclinical efficacy of a prototype DNA vaccine: Enhanced protection against antigenic drift in influenza virus. Nat. Med. 1, 583. 1121Michel. M.-L.. Davis, H.L., Schleef, M., Mancini, M., Tiollais, P. and Whalen, R.G. (1995). DNA-mediated immunization to the hepatitis B surface antigen in mice: Aspects of the humoral response mimic hepatitis B viral infection in humans. Proc. Natl. Acad. Sci. USA 92, 5307. against leish[I31 Xu, D. and Liew, F.Y. (1995). Protection tnaniasis by injection of DNA encoding a major surface glycoprotein, gp63, of L. major. Immunol. 84, 173. [ 141 Conry, R.M., LoBuglio, A.F., Wright, M., Sumerel, L.. Pike, M.J., Johanning, F.. Benjamin. R., Lu. D. and Curiel. D.T. (1995). Characterization of a messenger

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cytomegalovirus (Towne) immediate-early gene on heterologous expression in mammalian cells. Nucleic Acids Res. 19. 3979. 1521 Courouce-Pauty. A.M.. Naret. C.. Ciancionni, C.. Adhemar, J.P. and Soulier, J.P.( 1978). In: Transplantation and Clinical Immunology. Proceedings of the Tenth International Course, Lyon. May 22-24, Excerpta Medica, Amsterdam, pp 77. (531 Martins, L.P., Lau. L.L., Asano. M.S. and Ahmed. R.( 1995). DNA vaccination against persistent viral infection. J. Virol. 69, 2574. [S4] Yokoyama, M., Zhang, J. and Whitton, J.L. (1995). DNA immunization confers protection against lethal lymphocytic choriomeningitis virus infection. J. Virol. 69. 2684. IS51 Conry. R.M.. Widera. G., LoBuglio. A.F., Fuller, J.T.. Moore. SE., Barlow, D.L., Turner, J.. Yang, N.-S. and Curie]. D.T. (1996). Selected strategies to augment polynucleotide immunization. Gene Ther., (in press). [Sh] Xiang. Z. and Ertl, H.C. (1995). Manipulation of the immune response to a plasmid-encoded viral antigen by coinoculation with plasmids expressing cytokines. Immunity 2. 129. 1571 Schodel. F., Wirtz, R., Peterson, D.. Hughes, J., Warren, R.. Sadoff, J. and Milich, D.(1994). Immunity to malaria clicitcd by hybrid hepatitis B virus core particles carrying circumsporozoite protein epitopes. J. Exp. Med. 180, 1037.