DNA vaccines in sheep: CTLA-4 mediated targeting and CpG motifs enhance immunogenicity in a DNA prime/protein boost strategy

DNA vaccines have proven to be an efficient means of inducing immune responses in small laboratory animals; however, their efficacy in large out-bred animal models has been much less promising. In addressing this issue, we have investigated the ability of ovine cytotoxic lymphocyte antigen 4 (CTLA-4) mediated targeting and ruminant specific CpG optimised plasmids, both alone and in combination, to enhance immune responses in sheep to the pro cathepsin B (FhCatB) antigen from Fasciola hepatica. In this study, CTLA-4 mediated targeting enhanced the speed and magnitude of the primary antibody response and effectively primed for a potent memory response compared to conventional DNA vaccination alone, which failed to induce a detectable immune response. While the CpG-augmentation of the CTLA-4 targeted construct did not further enhance the magnitude or isotype profile of the CTLA-4 induced antibody titres, it did result in the induction of significant antigen-specific, lymphocyte-proliferative responses that were not observed in any other treatment group, showing for the first time that significant cellular responses can be induced in sheep following DNA vaccination. In contrast, CpG-augmentation in the absence of CTLA-4 mediated targeting failed to induce a detectable immune response. This is the first study to explore the potential adjuvant effects of ruminant specific CpG motifs on DNA vaccine induced immune responses in sheep. The ability of CpG-augmented CTLA-4 mediated targeting to induce both humoral and cellular immune responses in this study suggests that this may be an effective approach for enhancing the efficacy of DNA vaccines in large out-bred animal models.     

Distinct immune responses of recombinant plasmid DNA replicon vaccines expressing two types of antigens with or without signal sequences

Here, DNA replicon vaccines encoding the Hc domain of botulinum neurotoxin serotype A (AHc) or the receptor binding domain of anthrax protective antigen (PA4) with or without signal sequences were evaluated in mice. Strong antibody and protective responses were elicited only from AHc DNA vaccines with an Ig κ signal sequence or tissue plasminogen activator signal sequence. Meanwhile, there were no differences in total antibody responses or isotypes, lymphocyte proliferative responses, cytokine profiles and protective immune responses with the PA4 DNA vaccines with or without a signal sequence. Therefore, use of targeting sequences in designing DNA replicon vaccines depends on the specific antigen.     

CTLA-4 blockade in combination with xenogeneic DNA vaccines enhances T-cell responses, tumor immunity and autoimmunity to self antigens in animal and cellular model systems

Xenogeneic DNA vaccination can elicit tumor immunity through T cell and antibody-dependent effector mechanisms. Blockade of CTLA-4 engagement with B7 expressed on APCs has been shown to enhance T cell-dependent immunity. We investigated whether CTLA-4 blockade could increase T-cell responses and tumor immunity elicited by DNA vaccines. CTLA-4 blockade enhanced B16 tumor rejection in mice immunized against the melanoma differentiation antigens tyrosinase-related protein 2 and gp100, and this effect was stronger when anti-CTLA-4 was administered with booster vaccinations. CTLA-4 blockade also increased the T-cell responses to prostate-specific membrane antigen (PSMA) when given with the second or third vaccination. Based on these pre-clinical studies, we suggest that anti-CTLA-4 should be tested with xenogeneic DNA vaccines against cancer and that special attention should be given to sequence and schedule of administration.     

Optimization of DNA vaccination immune responses in dairy cows: effect of injection site and the targeting efficacy of antigen-bCTLA-4 complex

The objectives of this study were to evaluate the effects of immunization site and antigen presenting cell targeting on cattle immune responses to DNA immunization. Cows were vaccinated with the plasmid expression vector pCI alone, pCI encoding the bacterial antigen beta-galactosidase (pCI-beta-gal) or pCI encoding bCTLA 4 fused to beta-gal (pCI-bCTLA-hIgG-beta-gal). The plasmids were delivered by intramuscular, intradermal, intramammary gland, or intra supramammary lymph node needle-injection. Both vaccines induced significant humoral and cellular immune responses. pCI-beta-gal elicited a higher IgG response than immunization with pCI-bCTLA-hIgG-beta-gal. Cows injected intramuscularly and intramammary had higher IgG and IgG-1 humoral responses than cows immunized intradermaly or in the lymph nodes. The injection site did not significantly affect the magnitude of the IgG2 and IgM antibody responses, although a trend similar to the IgG results was observed. The lymphocyte proliferation index was higher with pCI-beta-gal but was not affected by the injection site. These results suggest that in bovine, the injection site can affect immune responses but they do not provide evidence that bCTLA-4-hIgG-antigen targeting is effective in cattle.     

Immunogenicity of CTLA4 fusion anti-caries DNA vaccine in rabbits and monkeys

Enhancement of mucosal and systemic immune responses is still a challenge for the application of DNA vaccine. Here, we show anti-caries DNA vaccines, pGJA-P and pGJA-P/VAX, encoding Streptococcus mutans antigens fused to cytotoxic T lymphocyte antigen-4 (CTLA4), which binds to B7 molecule expressed on the surfaces of antigen-presenting cells. Rabbits and monkeys were immunized via intranasal or intramuscular routes. The fusion vaccine induced accelerated and increased specific antibody responses in serum and saliva compared with non-fusion DNA vaccine in rabbits. Significant specific serum IgG and salivary IgA levels could be detected in fusion vaccine-immunized monkeys. Therefore, this study demonstrates that fusing antigens to CTLA4 results in enhancing immune efficacy and strongly suggests that it may represent a promising approach to prevent dental caries or other mucosal infectious diseases. These findings also suggest that CTLA4 fusion anti-caries DNA vaccine may be effective immunogen in primates.     

Site-directed immune responses in DNA vaccines encoding ligand-antigen fusions

One of the key limitations to DNA vaccines is lack of efficacy. We found that the spleen was a superior injection site to the dermis or muscle for inducing immune responses. To target sites of immune induction more practicably, antigen (human IgG1) was fused with two ligands, L-selectin (L-SEL-hIg) or CTLA4 (CTLA4-hIg) the receptors of which are found on high endothelial venule cells in lymph nodes and antigen presenting cells, respectively. Antibody and lymphocyte proliferative responses were increased. We now show that dimerization is critical for this enhancement, presumably because of avidity considerations. The hinge of hIgG3 can replace that of hIgG1 as a dimerization moiety. Fusion of other antigens e.g. ovalbumin and a malaria antigen AMA-1 have confirmed that CTLA4 induces an enhanced antibody response. Notably, in a challenge model, we have shown that CTLA4 also improves efficacy.     

Enhancing therapeutic HPV DNA vaccine potency through depletion of CD4CD25 T regulatory cells

Therapeutic human papillomavirus (HPV) vaccines targeting E6 and/or E7 antigens represent an opportunity to control HPV-associated lesions. We have previously generated several therapeutic DNA vaccines targeting HPV-16 E7 antigen and generated significant antitumor effects. Since regulatory T cells (Tregs) play an important role in suppressing immune responses against tumors by immunotherapy, such as DNA vaccines, we tested if the therapeutic effects of a DNA vaccine encoding E7 linked to heat shock protein 70 (Hsp70) can be improved by a strategy to deplete Tregs using a anti-CD25 monoclonal antibody (PC61) in vaccinated mice. We found that administration of PC61 prior to vaccination with E7/Hsp70 DNA was capable of generating higher levels of E7-specific CD8⁺ T cells compared to the control antibody, leading to significantly improved therapeutic and long-term protective antitumor effects against an E7-expressing tumor, TC-1. Thus, a strategy to deplete CD4⁺CD25⁺ Tregs in conjunction with therapeutic tumor antigen-specific DNA vaccine may represent a potentially promising approach to control tumor. The clinical implications of our study are discussed.     

A single DNA immunization in combination with electroporation prolongs the primary immune response and maintains immune memory for six months

Recombinant protein vaccines and vaccines using killed or inactivated pathogens frequently require multiple vaccinations to induce protective immune responses which may be of relatively short duration. Furthermore, increasing concern regarding adverse local and systemic reactions to injected vaccines is driving the quest for vaccine formulations, which induce protective immunity following a single administration. Vaccine studies frequently evaluate immune responses and disease protection within a relatively short interval following primary and secondary immunizations and, therefore, fail to address the duration of immunological memory or protection. DNA vaccines offer a unique opportunity to enhance the duration of immune responses through their capacity for prolonged antigen expression. The route of DNA vaccination and the method of plasmid delivery are critical factors, which can determine transfection efficiency and the duration of vaccine antigen production. Studies were completed which demonstrated that a single intramuscular DNA vaccination, when combined with electroporation, significantly enhanced the onset and duration but not the magnitude of the primary antibody response. A secondary protein vaccination was performed 6 months after the primary DNA immunization. A significant (p < or = 0.0001) correlation was observed between both the magnitude (r2 = 0.40) and duration (r2 = 0.74) of the primary antibody response and the occurrence of a secondary antibody response. Therefore, an effective primary DNA vaccination has the potential to significantly prolong the duration of an antibody response and possibly reduce the frequency of revaccination.     

Targeting of nucleoprotein to chemokine receptors by DNA vaccination results in increased CD8+-mediated cross protection against influenza

Vaccination is at present the most efficient way of preventing influenza infections. Currently used inactivated influenza vaccines can induce virus-neutralizing antibodies that are protective against a particular influenza strain, but hamper the induction of cross-protective T-cell responses to later infections. Thus, influenza vaccines need to be updated annually in order to confer protection against circulating influenza strains. This study aims at developing an efficient vaccine that can induce broader protection against influenza. For this purpose, we have used the highly conserved nucleoprotein (NP) from an influenza A virus subtype H7N7 strain, and inserted it into a vaccine format that targets an antigen directly to relevant antigen presenting cells (APCs). The vaccine format consists of bivalent antigenic and targeting units, linked via an Ig-based dimerization unit. In this study, NP was linked to MIP-1α, a chemokine that targets the linked antigen to chemokine receptors 1, 3 and 5 expressed on various APCs. The vaccine protein was indirectly delivered by DNA. Mice were vaccinated intradermally with plasmids, in combination with electroporation to enhance cellular uptake of DNA. We found that a single DNA vaccination was sufficient for induction of both antibody and T cell responses in BALB/c mice. Targeting of nucleoprotein to chemokine receptors enhanced T cell responses but not antibody responses. Moreover, a single dose of MIP1α-NP conferred protection in BALB/c mice against a lethal challenge with an H1N1 influenza virus. The observed cross-protection was mediated by CD8⁺ T cells.     

A simple approach for enhanced immune response using engineered dendritic cell targeted nanoparticles

In this study, we demonstrate a simple strategy for enhanced immune response using a two-component dendritic cell (DC) targeted antigen delivery system. One component consists of a recombinant bifunctional fusion protein (bfFp) used for DC targeting, whereas, the other component is made of biotinylated PLGA nanoparticles that encapsulate the antigen. The fusion protein (bfFp) made of a truncated core-streptavidin fused to anti-DEC-205 single chain antibody (scFv) was mixed with ovalbumin-loaded biotinylated NPs that were formulated using biotin–PEG (2000)–PLGA, and the combination, bfFp functionalized NPs was used for DC targeted antigen delivery. In vitro DC uptake studies revealed a 2-fold higher receptor-mediated uptake of bfFp functionalized NPs when compared to non-targeted NPs. Immunization of the mice with the bfFp functionalized NPs in conjunction with DC maturation stimulus (anti-CD40 mAb) enhanced OVA-specific IgG and IgG subclass responses. Splenocytes of these mice secreted significantly higher levels of Th1 (IFN-γ and IL-2) cytokines upon ex vivo restimulation with OVA. The promising outcomes of the bfFp functionalized DC targeted system support its use as a versatile vaccine delivery system for the design of monovalent or polyvalent vaccines.     

Tailoring subunit vaccine immunogenicity: maximizing antibody and T cell responses by using combinations of adenovirus, poxvirus and protein-adjuvant vaccines against Plasmodium falciparum MSP1

Subunit vaccination modalities tend to induce particular immune effector responses. Viral vectors are well known for their ability to induce strong T cell responses, while protein-adjuvant vaccines have been used primarily for induction of antibody responses. Here, we demonstrate in mice using a Plasmodium falciparum merozoite surface protein 1 (PfMSP1) antigen that novel regimes combining adenovirus and poxvirus vectored vaccines with protein antigen in Montanide ISA720 adjuvant can achieve simultaneous antibody and T cell responses which equal, or in some cases surpass, the best immune responses achieved by either the viral vectors or the protein vaccine alone. Such broad responses can be achieved either using three-stage vaccination protocols, or with an equally effective two-stage protocol in which viral vectors are admixed with protein and adjuvant, and were apparent despite the use of a protein antigen that represented only a portion of the viral vector antigen. We describe further possible advantages of viral vectors in achieving consistent antibody priming, enhanced antibody avidity, and cytophilic isotype skew. These data strengthen the evidence that tailored combinations of vaccine platforms can achieve desired combinations of immune responses, and further encourage the co-administration of antibody-inducing recombinant protein vaccines with T cell- and antibody-inducing recombinant viral vectors as one strategy that may achieve protective blood-stage malaria immunity in humans.     

Co-expression of granulocyte-macrophage colony-stimulating factor with antigen enhances humoral and tumor immunity after DNA vaccination

Granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to enhance humoral and tumor immunity resulting from DNA immunization. The genes encoding GM-CSF and antigen were cloned onto the same plasmid backbone, but separate promoters drove expression of each gene. beta-Galactosidase was used as the model antigen to generate antibody responses while the human tumor antigen, MAGE-1, was used to monitor tumor resistance. Immunization with a DNA vaccine co-expressing GM-CSF and beta-gal resulted in higher antigen-specific IgG responses than immunization with antigen encoding plasmid alone or co-inoculated with GM-CSF expressing plasmid. Similarly, DNA vaccines expressing both MAGE-1 antigen and GM-CSF were more effective in protecting against B16-MAGE-1 melanoma. However, both GM-CSF co-expressing DNA vaccines and co-inoculation with plasmids encoding the cytokine or antigen enhanced the generation antigen-specific IFN-gamma and IL-6 responses. These results demonstrate that co-expressing both GM-CSF and antigen on a DNA vaccine enhances humoral and tumor immune responses.     

Vaccination of mice with a cocktail DNA vaccine induces a Th1-type immune response and partial protection against Schistosoma japonicum infection.

Several defined vaccine candidate antigens of Schistosoma japonicum have shown promise in large animal vaccination experiments. However, vaccination of mice in the laboratory with either single recombinant antigens or DNA encoding forms of the individual antigens has so far failed to induce significant protection against S. japonicum cercarial challenge infection as judged by worm reduction, although specific antibodies were generated. This is in contrast to the results achieved using radiation-attenuated vaccines which are highly protective. Even in large animal vaccination experiments, the protection levels obtained with single defined antigens were far below those achieved using the attenuated vaccines. One possible interpretation is that the immune responses induced by single antigen vaccination may not be strong enough to combat the challenging infection. We, therefore, carried out mouse vaccination experiments using a cocktail DNA vaccine comprising four DNA plasmids encoding four different S. japonicum antigens, Sj62, Sj28, Sj23 and Sj14-3-3, respectively. We, also investigated whether co-injection of the mouse IL-12 encoding plasmid with the cocktail DNA vaccine was able to enhance the Th1 responses and hence the protective immunity. Three intramuscular injections of the cocktail DNA vaccine induced a significant Th1-type cellular response with high level of IFN-gamma production by splenocytes upon in vitro stimulation with recombinant antigens. Importantly, significant IgG antibody responses were also induced against crude worm antigens. In two out of three experiments, significant resistance (34-37 and 44-45%, respectively) was demonstrated while another experiment did not show any protection against S. japonicum cercarial challenge infection. Co-injection of the IL-12 encoding DNA did not further enhance these responses, nor the level of resistance, compared with the cocktail DNA alone.     

A DNA vaccine encoding VP22 of herpes simplex virus type I (HSV-1) and OprF confers enhanced protection from Pseudomonas aeruginosa in mice

Pseudomonas aeruginosa antimicrobial resistance is a major therapeutic challenge. DNA vaccination is an attractive approach for antigen-specific immunotherapy against P. aeruginosa. We explored the feasibility of employing Herpes simplex virus type 1 tegument protein, VP22, as a molecular tool to enhance the immunogenicity of an OprF DNA vaccine against P. aeruginosa. Recombinant DNA vaccines, pVAX1-OprF, pVAX1-OprF-VP22 (encoding a n-OprF-VP22-c fusion protein) and pVAX1-VP22-OprF (encoding a n-VP22-OprF-c fusion protein) were constructed. The humoral and cellular immune responses and immune protective effects of these DNA vaccines in mice were evaluated. In this report, we showed that vaccination with pVAX1-OprF-VP22 induced higher levels of IgG titer, T cell proliferation rate. It also provided better immune protection against the P. aeruginosa challenge when compared to that induced by pVAX1-OprF or pVAX1-VP22-OprF DNA vaccines. Molecular mechanistic analyses indicated vaccination with pVAX1-OprF-VP22 triggered immune responses characterized by a preferential increase in antigen specific IgG2a and IFN-γ in mice, indicating Th1 polarization. We concluded that VP22 is a potent stimulatory molecular tool for DNA vaccination when fused to the carboxyl end of OprF gene. Our study provides a novel strategy for prevention and treatment of P. aeruginosa infection.     

Protective efficacy of a DNA vaccine construct encoding the VP2 gene of infectious bursal disease and a truncated HSP70 of Mycobacterium tuberculosis in chickens

Infectious bursal disease (IBD) is an acute, infectious, immunosuppressive disease affecting young chicken worldwide. The etiological agent IBD virus (IBDV) is a double stranded RNA virus with outer capsid protein VP2 of IBDV is the major antigenic determinant capable of inducing neutralizing antibody. DNA vaccines encoding VP2 has been extensively studied achieving only partial protection. However, the efficacy of DNA vaccines against IBDV can be augmented by choosing a potential molecular adjuvant. The goal of the present study is to evaluate the immune response and protective efficacy of a DNA vaccine encoding the C-terminal domain of the heat shock protein 70 (cHSP70) of Mycobacterium tuberculosis gene genetically fused with the full length VP2 gene of IBDV (pCIVP2-cHSP70) in comparison to a ‘DNA prime-protein boost’ approach and a DNA vaccine encoding the VP2 gene (pCIVP2) alone. The results indicate that both pCIVP2-cHSP70 and ‘DNA prime-protein boost’ elicited humoral as well as cellular immune responses. Chickens in the pCIVP2-cHSP70 and ‘DNA prime-protein boost’ groups developed significantly higher levels of ELISA titer to IBDV antigen compared to the group immunized with pCIVP2 alone (p < 0.01). However, significantly higher levels of lymphocyte proliferative response, IL-12 and IFN-γ production were found in the pCIVP2-cHSP70 group compared to ‘DNA prime-protein boost’ group. Additionally, chickens immunized with pCIVP2-cHSP70 and ‘DNA prime-protein boost’ vaccines were completely protected against the vvIBDV whereas pCIVP2 DNA vaccine alone was able to protect only 70%. These findings suggest that the truncated C-terminal HSP70 mediated DNA vaccine genetically fused with the VP2 gene construct stimulated both humoral and cell mediated immune responses and conferred complete protection against IBDV. This novel strategy is perhaps a seminal concept in utilizing HSP70 as an adjuvant molecule to elicit an immune response against IBD affecting chickens.     

Enhancing DNA immunization by targeting ASFV antigens to SLA-II bearing cells

One of the main criticisms to DNA vaccines is the poor immunogenicity that they confer on occasions, at least in large animals. Confirming this theory, immunization with plasmid DNA encoding two African swine fever virus genes in frame (pCMV-PQ), failed in inducing detectable immune responses in pigs, while it was successful in mice. Aiming to improve the immune responses induced in swine, a new plasmid was constructed, encoding the viral genes fused in frame with a single chain variable fragment of an antibody specific for a swine leukocyte antigen II (pCMV-APCH1PQ). Our results clearly demonstrate that targeting antigens to antigen professional cells exponentially enhanced the immune response induced in pigs, albeit that the DNA vaccine was not able to confer protection against lethal viral challenge. Indeed, a viremia exacerbation was observed in each of the pigs that received the pCMV-APCH1PQ plasmid, this correlating with the presence of non-neutralizing antibodies and antigen-specific SLA II-restricted T-cells. The implications of our discoveries for the development of future vaccines against African swine fever virus and other swine pathogens are discussed.     

Genetic adjuvants for DNA vaccines

The relatively low efficacy of DNA vaccines in inducing immune responses, especially in large animal species and humans, has impaired their practical use. Despite considerable effort expended on improving DNA vaccine delivery, only minute amounts of Ag are available for immune induction following DNA vaccination. Two complementary strategies have been used to improve and modulate the immune response induced by DNA vaccines: (i) supplementing DNA vaccines with plasmids encoding cytokines and (ii) targeting the Ag encoded by DNA vaccine through genetically fusing the Ag to molecules binding cell surface receptors. This paper reviews recent progress in these two areas and possible mechanisms responsible for the observed effects.     

Immunogenic characterization and protection against Streptococcus mutans infection induced by intranasal DNA prime–protein boost immunization

Mucosal immune responses act as the first line of defense against dental caries. In this study, an optimal vaccination strategy was developed to enhance anti-caries mucosal immune responses. Mice and rats were vaccinated intranasally firstly with plasmid pCIA-P encoding PAc antigen of Streptococcus mutans and then with rPAc, or with pCIA-P for twice, or with rPAc protein for twice, respectively. The potential of inducing mucosal and systemic immune responses to special antigens was measured by ELISA. In addition, antibody type, cytokine production and protection effectiveness against dental caries were also evaluated. Although all immunized groups developed immune responses, the antibody responses in the DNA prime–protein boost group were stronger compared with those elicited by either the DNA vaccine or the protein vaccine. In particular, the Th1-biased response that was established by the DNA immunization was diverted to Th1/Th2-mixed response following the rPAc protein boost. Moreover, protection against S. mutans challenge was obtained in the rats treated with the DNA prime–protein boost regimen, as shown by a significant reduction in dental caries lesion, compared with the control groups immunized with the DNA or protein only. All these findings may provide useful information about effective mucosal vaccines against dental caries.     

A DNA vaccine encoding a single-chain trimer of HLA-A2 linked to human mesothelin peptide generates anti-tumor effects against human mesothelin-expressing tumors

Mesothelin is highly expressed in a majority of ovarian cancer cells and is expressed at low levels in normal cells. Therefore, mesothelin represents a potential target antigen for ovarian cancer vaccine development. DNA vaccines employing single-chain trimers (SCT) have been shown to bypass antigen processing and presentation and result in significant enhancement of DNA vaccine potency. In the current study, we created a DNA vaccine employing an SCT targeting human mesothelin and characterized the ensuing antigen-specific CD8+ T cell-mediated immune responses and anti-tumor effects against human mesothelin-expressing tumors in HLA-A2 transgenic mice. Our results showed that vaccination with DNA employing an SCT of HLA-A2 linked to human mesothelin epitope aa540-549 (pcDNA3-Hmeso540-beta2m-A2) generated strong human mesothelin peptide (aa540-549)-specific CD8+ T cell immune responses in HLA-A2 transgenic mice. Vaccination with pcDNA3-Hmeso540-beta2m-A2 prevented the growth of HLA-A2 positive human mesothelin-expressing tumor cell lines in HLA-A2 transgenic mice in contrast to vaccination with DNA encoding SCT linked to OVA CTL epitope. Thus, the employment of SCT of HLA-A2 linked to the human mesothelin epitope aa540-549 represents a potential opportunity for the clinical translation of DNA vaccines against human mesothelin-expressing tumors, particularly ovarian cancer cells.     

A novel prototype device for electroporation-enhanced DNA vaccine delivery simultaneously to both skin and muscle

Electroporation (EP) of either muscle or skin has proven to be an efficient method for increasing DNA-based vaccine delivery and immunogenicity in small and large animals. Previous comparative studies in large animals suggest that intramuscular (i.m.) DNA EP delivery appears to favor cellular immunity, while intradermal (i.d.) EP delivery may favor humoral immunity. While current EP devices are primarily designed either for i.m. or i.d. delivery, we developed a novel prototype Dual-Depth Device (DDD) for EP-mediated simultaneous i.d. and i.m. delivery of DNA-based vaccines with an attempt to elicit superior antibody and cellular immune responses. We performed comparisons of DDD EP delivery with standard i.d. EP, standard i.m. EP, and combined delivery of i.d. and i.m. EP at separate sites, for the ability to induce antigen-specific immune responses. In a guinea pig model using a SynCon™ DNA vaccine encoding the influenza virus H5 hemaglutinin (H5HA), vaccination via DDD or combined delivery induced higher antibody titers than via either i.d. or i.m. delivery alone. In a mouse model using a DNA vaccine encoding the nucleoprotein (NP) of influenza H1N1, the resulting trend of antibody responses was similar to that detected in guinea pig study. Importantly, cellular immune responses in the DDD or combined delivery groups were significantly stronger than that in either i.d. or i.m. delivery groups. We conclude that EP-mediated DNA-based vaccine delivery to both skin and muscle is superior to delivery to either tissue alone for induction of antigen-specific antibody and cellular immunity.