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The Journal of Immunology, 2004, 173: 550-558.
Copyright © 2004 by The American Association of Immunologists

The Hybrid Cytomegalovirus Enhancer/Chicken {beta}-Actin Promoter along with Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element Enhances the Protective Efficacy of DNA Vaccines1

Sanjay Garg2,3, Alp E. Oran2, Huiming Hon and Joshy Jacob4

Department of Microbiology and Immunology, and Emory Vaccine Center, Yerkes National Primate Research Center, Emory University, Atlanta, GA 30329


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
DNA vaccines represent a novel and powerful alternative to conventional vaccine approaches. They are extremely stable and can be produced en masse at low cost; more importantly, DNA vaccines against emerging pathogens or bioterrorism threats can be quickly constructed based solely upon the pathogen’s genetic code. The main drawback of DNA vaccines is that they often induce lower immune responses than traditional vaccines, particularly in nonrodent species. Thus, improving the efficacy of DNA vaccines is a critical issue in vaccine development. In this study we have enhanced the efficacy of DNA vaccines by adopting strategies that increase gene expression. We generated influenza-hemagglutinin (HA)-encoding DNA vaccines that contain the hybrid CMV enhancer/chicken {beta}-actin (CAG) promoter and/or the mRNA-stabilizing post-transcriptional regulatory element from the woodchuck hepatitis virus (WPRE). Mice were immunized with these DNA vaccines, and the influenza-HA-specific cellular and humoral immune responses were compared with a conventional, HA-encoding DNA vaccine whose gene expression was driven by the CMV immediate-early promoter (pCMV-HA). CAG promoter-driven DNA vaccines elicited significantly higher humoral and cellular immune responses compared with the pCMV-HA vaccine. DNA vaccines consisting of both CAG and WPRE elements (pCAG-HA-WPRE) induced the highest level of protective immunity, such that immunization with 10-fold lower DNA doses prevented death in 100% of the mice upon lethal viral challenge, whereas all mice immunized with the conventional pCMV-HA vaccine succumbed to influenza infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
An ideal vaccine is one that provides life-long immunity with a single immunization. Live attenuated virus vaccines are one of the most effective means of inducing protective immunity in a host without the need for booster immunizations. However, the risk of reversion of the attenuated virus back to a virulent strain and the instability of these live virus stocks make these vaccines potentially dangerous and hazardous in public vaccination programs. In contrast, DNA vaccines, like viral vectors, are capable of raising both humoral and cellular responses. Unlike live virus vaccines, they are extremely stable and relatively inexpensive to manufacture and store (1). The main limitation of DNA-based vaccines is their relative impotency; DNA vaccines require multiple boosts with high doses to raise responses comparable to that achieved from a single attenuated virus vaccination (2).

Many strategies have been used to enhance the potency of DNA vaccines. These include, but are not limited to, 1) better promoters/enhancers for increasing gene expression, such as elongation factor I{alpha} promoter or class I MHC promoter (3, 4); 2) vectors encoding both the Ag and immunomodulatory molecules, such as cytokines IL-2, GM-CSF, IL-12, and Flt-3 ligand (5, 6, 7, 8); 3) direct targeting of DNA vaccines to APCs (9, 10); and 4) vectors encoding Ags fused to molecules that facilitate Ag spreading and cross-presentation (11, 12, 13, 14, 15).

The human CMV immediate-early enhancer/promoter is the most commonly used promoter in DNA vaccines, but gene expression driven by CMV promoter has been shown to be down-regulated in the presence of IFN-{gamma} (4, 16), a cytokine commonly induced after DNA vaccination. The activity of this promoter is variable among different tissues and cell lines. Sawicki et al. (17) have shown that CMV enhancer/promoter is not transcriptionally active in most cell types in the mouse skin. This is problematic, because biolistic delivery of DNA vaccines into skin is an accepted mode of DNA vaccination for delivering Ags direct to the dendritic cells (DC)5 (1). Consequently, because of the need for more consistent and stronger gene expression, vectors containing a hybrid CMV enhancer coupled to a modified chicken {beta}-actin promoter (CAG), have been used. These vectors have been shown to drive high expression of Ag in various cell types and tissues, including skin (17). Another element that has been shown to enhance gene expression is the woodchuck hepatitis virus post-transcriptional response element (WPRE). WPRE is a 600-bp, noncoding, cis-acting RNA sequence that acts to increase the stability and extranuclear transport of mRNA to the cytoplasm, resulting in enhanced levels of mRNA for translation and greater protein production (18).

In this report we studied the relative magnitudes of protective cellular and humoral immune responses generated by DNA vaccines using either the CMV or the hybrid CAG promoter, with and without WPRE. We demonstrate that vaccine constructs using either CAG promoter or WPRE induced greater immune responses than the vaccine driven by CMV alone. The most significant difference was observed when both CAG and WPRE were used in the same vector; this vaccine elicited higher cellular and humoral responses and conferred better protection to a lethal viral challenge at a 10-fold lower dosage compared with the construct driven only by the CMV promoter.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Six- to 8-wk-old female BALB/c and C57BL/6 mice (Charles River Laboratories, Wilmington, MA) were used in these experiments. They were housed under pathogen-free conditions at Emory Vaccine Center (Atlanta, GA) and cared for under U.S. Department of Agriculture guidelines for laboratory animals.

DNA vaccines

DNA vaccine plasmids were constructed using routine molecular biology techniques. The backbone of all DNA vaccine constructs used in this study was derived from the CMV promoter-driven, {beta}-galactosidase ({beta}-gal)-encoding plasmid (pCMV-lacZ; Clontech, Palo Alto, CA). The pCMV-lacZ plasmid was modified by replacing the CMV promoter with the CAG promoter (a gift from Dr. C. Lois, Caltech, Pasadena, CA; pCAG-lacZ). The lacZ cassette was excised by restriction digest with NotI and replaced with either transmembrane hemagglutinin from A/PR/8/34 (HA) or HA-WPRE. Four different DNA vaccines were constructed: pCMV-HA, pCMV-HA-WPRE, pCAG-HA, and pCAG-HA-WPRE. In addition, we generated a pCAG-lacZ WPRE plasmid. All plasmids were sequenced for cloning accuracy. The ability of different DNA vaccines to express the encoded protein was tested by in vitro transient transfection into the human embryonic kidney cell line 293T.

DNA immunizations

Mice were immunized with one of four HA-bearing DNA vaccines by i.m. injection. Briefly, mice were subdivided into groups (5–10 mice/group) based on the DNA vaccine and the dose of vaccination (10 and 100 µg DNA/mouse). HA-bearing DNA vaccines resuspended in 100 µl of sterile PBS were injected into the quadriceps femoris muscle of mice, 50 µl/hind leg.

Heterologous prime and boost

Respective groups of mice were immunized i.m. once with 10 or 100 µg of either pCMV-HA or pCAG-HA-WPRE DNA vaccine plasmids. Four weeks later, the DNA-primed mice were challenged with an i.v. injection of 1 x 106 PFU of recombinant modified vaccinia Ankara (MVA) expressing influenza HA (MVA-HA; a gift from Dr. B. Moss).

Influenza virus challenge and survival

After DNA vaccination, animals were challenged with the mouse-adapted influenza virus A/PR/8/34. Allontoic fluid containing influenza virus was diluted in sterile PBS with 0.2% BSA (fraction V; Sigma-Aldrich, St. Louis, MO), and a 50-µl volume containing 3x LD50 viral dose was applied into the nostrils of anesthetized mice. Intranasal inoculation of 3x LD50 influenza virus leads to 100% death in nonimmunized mice. After viral challenge, total body weight and survival were recorded on alternate days. Mice with weight loss exceeding 20% of their initial weight were euthanized.

ELISA for anti-HA Abs

On different intervals after immunization, we collected serum samples from each group of mice by retro-orbital bleeding. The influenza HA-specific, total IgG, IgG1, and IgG2a Abs were measured using a quantitative ELISA as previously described (19).

ELISPOT analyses

Splenocytes were harvested 2 wk after the final DNA booster immunization or 1 wk after viral challenge and processed as previously described (20). Cells were stimulated in the presence of H-2d-restricted class I HA peptide (IYSTVASSL) (21) or a pool of five class II peptides (SFERREIFPKE, HNTNGVTAACSH, CPKYVRSAKLRM, KLKNSYVNKKGK, and NAYVSVVTSNYNRRF) (22) prepared in complete RPMI 1640 (10% FCS, 2mM L-glutamine, 1000 U of penicillin G, and 1000 U of streptomycin) at a final concentration of 2 x 10–6 M total peptide. Complete RPMI 1640 containing 2 x 10–6 M Kd-binding irrelevant peptide derived from the HIV-1 envelope protein (IGPGRAFYR) (23) and PMA plus ionomycin were used as negative and positive controls, respectively. All peptide solutions were supplemented with 2 µg/ml anti-CD28 and anti-CD49d (37.51 and R1-2, respectively; BD Pharmingen, San Diego, CA). Spot-forming units were counted using a C.T.L. automated immunoscope (C.T.L., Cleveland, OH) and normalized for 106 splenocytes.

Luminescence assay for {beta}-gal in muscle tissue

Cohorts of C57BL/6 mice were immunized i.m. in the hind limbs with 5-µg (on the right hind limb) or 50-µg (on the left hind limb) doses of pCAG-lacZ-WPRE, pCMV-lacZ, or pCMV-HA. Four days later, muscle from the immunization site was removed and homogenized with a Tissue Tearor (Biospec Products, Bartlesville, OK) in 1 ml of lysis buffer containing 100 mM potassium phosphate, 0.2% Triton X-100, 1 mM DTT, 0.2 mM PMSF, and Complete Mini protease inhibitor mixture tablets (Roche, Indianapolis, IN). The homogenate was incubated at 48°C for 60 min to inactivate endogenous {beta}-gal activity and then centrifuged at 12,500 x g for 5 min. Twenty microliters of the muscle lysate was assayed for {beta}-gal activity using the Galacto-Light assay (Applied Biosystems, Bedford, MA); the reactions were conducted for 1 h, and luminescence was measured for 10 s in a TD20/20 luminometer (Turner Designs, Sunnyvale, CA). Activity was expressed as relative Turner light units per gram of muscle.

Flow cytometry assay for {beta}-gal+ DCs

Cohorts of C57BL/6 mice were immunized using a gene gun with gold bullets coated with either pCMV-lacZ or pCAG-lacZ-WPRE. Briefly, 0.5 µg of endotoxin-free plasmid DNA was coated per 0.5 µg of 1-µm gold beads (DeGussa-Huls, Ridgefield Park, NJ). The mice were lightly anesthetized and immunized on the shaved abdominal skin using a Helios gene gun (Bio-Rad, Hercules, CA) set at a discharge pressure of 400 {psi}. Each mouse was immunized with four nonoverlapping shots that corresponded to a total of 2 µg of plasmid DNA/mouse/immunization. At 2.5 days postimmunization, we harvested the draining superficial inguinal lymph nodes (iLNs), made single-cell suspensions by collagenase treatment, and then hypotonically loaded the bulk iLN cells with 1 mM fluorescein digalactopyranoside (FDG; Molecular Probes, Eugene, OR) to detect the DNA vaccine-encoded {beta}-gal (24, 25). After FDG loading, the cells were stained with anti-mouse CD11c-allophycocyanin (BD Biosciences, Mountain View, CA). Samples were collected on a FACSCalibur (BD Biosciences) and analyzed with FlowJo software.

Sample sizes and statistical analyses

For immunizations with different vaccine constructs and at different dosages we used 5–10 mice/group. Statistical differences between the groups were determined by Student’s t test, and a value of p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DNA vaccines

We generated a panel of DNA vaccines harboring full-length transmembrane HA-coding sequences derived from influenza virus, A/PR/8/34. We tested two promoters, the conventional CMV enhancer/immediate-early promoter (CMV) and the hybrid CMV enhancer/chicken {beta} actin promoter (CAG) in conjunction with the mRNA post-transcriptional regulatory element of the woodchuck hepatitis virus (WPRE) by generating four DNA vaccine constructs: pCMV-HA, pCMV-HA-WPRE, pCAG-HA, and pCAG-HA-WPRE (Fig. 1). The accuracy of cloning and protein expression for HA in each of these constructs was confirmed by in vitro transient transfection into the human embryonic kidney cell line 293T, followed by flow cytometry for surface HA expression (data not shown).



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  		FIGURE 1. HA-encoding DNA vaccines used in this study. a, The pCMV-HA vaccine contains the 0.8-kb CMV immediate-early enhancer/promoter and intron (SV40 splice donor/acceptor) for initiating transcription of the influenza transmembrane HA-coding sequence and the SV40 polyadenylation signal for termination of transcript, along with origin of replication and Ampr selection marker (not shown). pCMV-HA was generated by replacing the lacZ cassette in the pCMV-lacZ plasmid (Clontech) with the transmembrane HA-coding sequence from influenza virus, A/PR/8/34. b, The pCMV-HA-WPRE vaccine contains all elements of construct a plus 0.6 kbp of WPRE downstream of the HA gene cassette. c, The pCAG-HA vaccine contains the 1.6 kbp of hybrid chicken {beta}-actin promoter and CMV enhancer in place of CMV, with all other elements remaining same as in construct a. d, The pCAG-HA-WPRE vaccine contains all elements of construct c plus WPRE downstream of the HA gene.

 
Improved humoral responses with pCAG-HA-WPRE vaccinations

Lentiviruses using the CAG enhancer/promoter and WPRE elements have been shown to exhibit higher gene expression than CMV-driven vectors (26). We determined whether incorporation of these two elements into DNA vaccines would lead to enhanced immune responses. Conventional CMV-driven HA DNA vaccines in mice achieve optimal immune responses after i.m. immunization with 100 µg of DNA; doses delivered by i.m. at higher concentrations do not increase the immune response significantly beyond this level (27). Differences in the immune responses elicited by the DNA vaccines used in this study may be missed because of saturating immune responses induced by all four constructs at high dosage. Consequently, we vaccinated mice i.m. using a suboptimal dose of 10 µg of DNA and boosted animals at 4 and 8 wk with the same dose. During the course of the experiment we collected serum at various time points before and after each vaccination, as shown in Fig. 2a. Serum anti-HA IgG was measured by ELISA, and the levels of specific IgG were plotted over time (Fig. 2a). At the 10-µg dose, even after two booster immunizations, the conventional CMV promoter-driven vaccine (pCMV-HA) failed to generate any detectable anti-HA IgG (Fig. 2a), comparable to the control vector DNA-immunized group. In contrast, pCMV-HA-WPRE, pCAG-HA, and pCAG-HA-WPRE were able to raise a very low, but detectable, anti-HA response (Fig. 2a). For each group the serum anti-HA Ab concentration increased after each booster immunization. The magnitude of Ab responses was highest in mice immunized with pCAG-HA-WPRE. After the second boost at 8 wk postprime, the serum anti-HA IgG concentration was 14.33 ± 7.6 µg/ml (±SEM) for the pCAG-HA-WPRE group, whereas the anti-HA levels were 6.9 ± 2.6 and 2.25 ± 1.2 µg/ml for pCMV-HA-WPRE- and pCAG-HA-immunized mice, respectively. In the pCMV-HA-immunized group the mean serum anti-HA level was 0.04 ± 0.006 µg/ml. Statistical comparisons of each vaccine with pCMV-HA revealed that the addition of CAG (p < 0.0001), WPRE (p < 0.0001), or both CAG and WPRE (p < 0.0001) to the vaccine elicited significantly higher Ab responses.



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  		FIGURE 2. The pCAG-HA-WPRE-immunized mice exhibit enhanced Ab responses. a, Groups of mice were i.m. immunized with 10 µg of pCMV-HA, pCMV-HA-WPRE, pCAG-HA, pCAG-HA-WPRE, or control vector backbone DNA at wk 0, 4, and 8 as shown. After immunization, mice were bled three times at 4-wk intervals (primary, secondary, and tertiary bleeds) after each vaccination as depicted in the figure. The total influenza HA-specific IgG levels in the sera was measured by quantitative ELISA. The mean anti-HA Ab levels (±SEM) are shown. Each bar represents data from five mice. b, Mice were immunized with 100 µg of pCMV-HA or pCAG-HA-WPRE DNA and were boosted with the same dose at 4 wk as shown. Sera were collected from these mice on days 15 and 30 after primary immunization and 9 days after boost, and the anti-HA levels were determined.

 
Is the enhanced humoral immune response seen with the 10-µg immunization dose reproducible at higher DNA doses? To address this issue we immunized groups of mice with 100 µg of DNA. In these experiments we compared the conventional pCMV-HA with pCAG-HA-WPRE, the vaccine that gave the highest immune response at a 10-µg dose of DNA. Briefly, mice were immunized with a single vaccination of 100 µg of pCMV-HA or pCAG-HA-WPRE and boosted once with the same dose on day 30 (Fig. 2b). We then collected serum samples on days 15 and 30 after primary immunization or 9 days after boost (Fig. 2b) and measured the total anti-HA IgG levels in the serum samples by ELISA. Mice vaccinated with 100 µg (high dose) of pCAG-HA-WPRE showed significantly higher serum Ab responses than mice given an equivalent immunization with the same high dose of pCMV-HA at all time points analyzed (Fig. 2b). On day 30 after primary immunization, the Ab concentration in serum anti-HA IgG Ab for pCAG-HA-WPRE-immunized mice was 40.47 ± 9.66 µg/ml, whereas the serum of mice immunized with pCMV-HA contained 19.8 ± 4.39 µg/ml anti-HA Abs. These data show that pCAG-HA-WPRE induced almost 2-fold higher, statistically significant (p < 0.04) levels of anti-HA Abs than the conventional pCMV-HA vaccine even when comparing the responses at higher doses. Booster immunization increased the Ab levels even higher in both groups; 9 days after the secondary immunization, the Ab levels for pCAG-HA-WPRE and pCMV-HA were 62.5 ± 13.4 and 45.06 ± 12.7 µg/ml, respectively. Interestingly, the anti-HA IgG levels in the pCAG-HA-WPRE-immunized group 30 days after a single immunization were comparable to Ab levels achieved in the pCMV-HA group by two immunizations. The anti-HA levels were 40.47 ± 9.66 and 45.06 ± 12.8 µg/ml, respectively, and the difference between the two groups was not significant (p = 0.535). Thus, a single immunization with pCAG-HA-WPRE vaccine achieved Ab levels comparable to those achieved with two immunizations with pCMV-HA at the 100-µg dose.

Intramuscular immunization with DNA vaccines typically elicits a Th1-biased response to the Ag. One of the hallmarks of Th1 responses is the induction of IgG2a-biased humoral response. We examined whether the inclusion of CAG or WPRE elements in DNA vaccines altered the polarity of the immune responses induced. To this end, we immunized mice with 10 or 100 µg of pCMV-HA or pCAG-HA-WPRE and analyzed the isotype of their serum anti-HA Abs. All DNA vaccines elicited stronger HA-specific IgG2a levels relative to IgG1, a hallmark of a Th1 response, and the Th1 trend was maintained over the course of the study (data not shown). Thus, the Th1 bias was maintained in DNA vaccines driven by CAG promoter and WPRE elements.

The pCAG-HA-WPRE-vaccinated mice were better protected from lethal influenza challenge than the pCMV-HA-vaccinated mice

Protection against influenza infection in mice is primarily mediated by antiviral Abs, and upon lethal challenge, naive mice die within 5–7 days. We compared the extent of protection to viral challenge conferred by the different DNA vaccines at 10- and 100-µg doses. We immunized mice with 10 µg of pCMV-HA, pCMV-HA-WPRE, pCAG-HA, or pCAG-HA-WPRE and boosted them with the same dose at 4 and 8 wk, as shown in Fig. 2a. Seven days after the second booster vaccination, mice were challenged intranasally with a lethal dose (3x LD50) of influenza A/PR/8/34 virus. Survival and weight loss were monitored daily until day 12 postchallenge (Fig. 3a). Mice were euthanized when weight loss exceeded 20% of initial body weight. Upon live viral challenge, all control mice (immunized with vector backbone only) succumbed to the infection and died by day 5. The pCMV-HA-immunized group upon challenge exhibited drastic weight loss (averaging 20%), and by day 7, 100% of the mice died or had to be euthanized due to excessive weight loss (Fig. 3a). The group immunized with pCAG-HA and pCMV-HA-WPRE fared slightly better, with weight loss averaging 9.6 ± 3.5% in all pCAG-HA mice and 6.94 ± 3.9% in all pCMV-HA-WPRE mice (Fig. 3a). By day 12 postinfection, the survival was 50% in pCAG-HA and 40% in pCMV-HA-WPRE mice. The most dramatic effect was observed in mice immunized with pCAG-HA-WPRE; no significant weight loss was observed in this group, and 100% of the mice survived lethal viral challenge (Fig. 3a). The data clearly show that pCAG-HA-WPRE induces better protective immunity than all other DNA vaccines tested.



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  		FIGURE 3. The pCAG-HA-WPRE-immunized mice exhibited decreased weight loss and enhanced survival upon lethal viral challenge. a, Groups of mice were vaccinated with 10 µg of pCMV-HA, pCMV-HA-WPRE, pCAG-HA, pCAG-HA-WPRE, or control vector backbone DNA and boosted at 4 and 8 wk. Seven days after the secondary boost the animals were infected intranasally with a lethal dose (3x LD50) of influenza virus, A/PR/8/34 (H1N1). The mice were observed for survival and weight loss for the next 12 days; mice whose weight loss exceeded 20% of their initial body weight were euthanized. The survival plot from these mice and the peak percent change in body weight (day 5 postinfection) are shown in the left and right columns, respectively. b, Groups of mice were immunized with 100 µg of pCMV-HA, pCAG-HA-WPRE, or vector DNA and were boosted once at 4 wk. Seven days after the boost, the mice were infected with influenza virus, and survival and weight loss were monitored as described in a. Each experiment represents the average of five mice per group, and the error bars represent the SEM.

 
We also determined weight loss and survival in mice immunized with a single dose of 100 µg of DNA vaccine. Briefly, cohorts of mice were immunized i.m. with pCMV-HA or pCAG-HA-WPRE DNA vaccines, and 30 days later, the mice were challenged with 3x LD50 influenza virus. A single immunization with 100 µg of either pCMV-HA or pCAG-HA-WPRE was sufficient to protect 100% of the mice from influenza-induced death (Fig. 3b). The protection, as measured by weight loss, was better in mice immunized with pCAG-HA-WPRE, and it correlated with the higher Ab responses. At 5 days postinfection, the pCMV-HA-immunized mice lost, on the average, 4.5% of their body weight, whereas mice immunized with pCAG-HA-WPRE did not lose any weight, but instead gained, on the average, 2.6% of their body weight (Fig. 3b), generating a total effective difference of 7.1%. At both the low and high immunization doses, the differences in percent body weight change between the pCMV-HA- and pCAG-HA-WPRE-immunized mice were statistically significant (p < 0.001).

Improved CD4 and CD8 T cell responses to pCAG-HA-WPRE relative to pCMV-HA

From the data presented (Fig. 2) it is clear that pCAG-HA-WPRE DNA vaccine elicits significantly greater humoral responses than the conventional pCMV-HA vaccine. We compared the CD4 and CD8 T cell responses elicited by these two DNA vaccines in a DNA-prime-DNA-boost approach. Briefly, groups of mice were i.m. immunized with 100 µg of DNA and 4 wk later were revaccinated with 100 µg of DNA. Splenocytes were harvested 9 days later, and HA-specific T cell responses were measured by ELISPOT assays for IFN-{gamma} and IL-4 production (Fig. 4). HA-specific CD8 and CD4 T cell responses were higher in pCAG-HA-WPRE than pCMV-HA in the immunized mice. The pCAG-HA-WPRE DNA vaccine elicited significantly stronger IFN-{gamma} responses for both class I (p < 0.001) and class II (p = 0.002) HA peptides compared with the pCMV-HA vaccine. The data presented demonstrate that the pCAG-HA-WPRE vaccine elicited significantly higher CD4 and CD8 T cell responses than the conventional pCMV-HA vaccine.



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  		FIGURE 4. Homologous DNA immunization. HA-specific CD4 and CD8 T cell responses were greater in pCAG-HA-WPRE-immunized mice. Cohorts of BALB/c mice were immunized with 100 µg of pCMV-HA, pCAG-HA-WPRE, or vector DNA and were boosted once at 4 wk. Nine days later, the mice were sacrificed, and their splenocytes were analyzed for IFN-{gamma} (a and c) and IL-4 (b and d) production by ELISPOT assay after stimulation with immunodominant MHC class I-restricted (a and b) and class II-restricted (c and d) HA peptides. Data are represented as the mean ± SEM for five mice per group.

 
Higher T cell responses in pCAG-HA-WPRE-immunized mice upon heterologous boost with a recombinant poxvirus-encoding HA

We also compared the T cell responses elicited by the two DNA vaccines in a heterologous prime boost (DNA prime, poxvirus boost) approach. Priming the immune system with DNA vaccinations, followed by boosting with recombinant poxvirus, have been shown to significantly amplify the level of cellular immune responses primed by DNA vaccines (28). Briefly, groups of mice were immunized once with either 10 µg (low dose) or 100 µg (high dose) of pCMV-HA or pCAG-HA-WPRE DNA vaccines, and 4 wk later animals were boosted with 1 x 106 PFU of recombinant MVA encoding influenza HA (MVA-HA). Ten days after the MVA-HA boost, splenocytes were harvested, and IFN-{gamma} and IL-4 ELISPOTs were performed (Fig. 5). When mice were primed with high dose (100 µg) DNA vaccines and boosted with MVA-HA, there was no significant difference in cytokine-secreting cells between the two groups of mice. However, when mice were primed with low dose (10 µg) DNA, pCAG-HA-WPRE mice had significantly higher frequencies of IFN-{gamma}- and IL-4-secreting T cells for both class I and class II HA peptides compared with pCMV-HA mice (p < 0.01 for class I; p < 0.001 for class II). Moreover, the responses were IFN-{gamma}-biased in both T cell compartments, suggesting a Th1 bias that was in full agreement with the IgG2a bias described above.



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  		FIGURE 5. Heterologous prime-boost. HA-specific IFN-{gamma} ELISPOTS in mice challenged with recombinant MVA bearing influenza-HA Ag. Cohorts of mice were immunized once with either 10 µg (a and b) or 100 µg (c and d) of pCMV-HA, pCAG-HA-WPRE. or control vector DNA and were challenged i.v. 4 wk later with 2 x 106 PFU of recombinant HA-encoding MVA (MVA-HA). Ten days after the MVA-HA challenge, HA-specific, class I-restricted CD8 (a and c) and class II-restricted CD4 (b and d) responses were quantitated by ELISPOT assay. Data presented are the mean ± SEM for five mice per group.

 
CAG/WPRE-driven expression vectors give greater expression than CMV-driven expression vectors in vivo

Based upon the data presented in Figs. 1–4, it is clear that the pCAG-HA-WPRE vaccine elicited significantly higher humoral and cellular responses than the pCMV-HA vaccine. We determined whether these increased immune responses elicited were due to enhanced gene expression in vivo. Previous reports show that the use of the CAG promoter and the WPRE element in lentiviral vectors leads to higher gene expression (26). To determine whether the incorporation of CAG and WPRE elements into DNA vaccines resulted in enhanced gene expression in vivo, we generated two {beta}-gal-encoding DNA vaccines, pCAG-lacZ-WPRE and pCMV-lacZ, and immunized two groups of C57BL/6 mice i.m. Each mouse received pCAG-lacZ-WPRE, pCMV-lacZ, or pCMV-HA at 5- and 50-µg doses in the right and left hind limbs, respectively. Four days later, we harvested muscle tissues and measured {beta}-gal activity using the Galacto-Light chemiluminescence assay (Fig. 6a). Significantly higher {beta}-gal expression was observed in the muscle tissue of mice immunized with pCAG-lacZ-WPRE (p < 0.05).



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  		FIGURE 6. In vivo Ag expression from pCAG-driven, WPRE-enhanced DNA vaccine is greater than that from the conventional pCMV promoter-driven vaccine. a, Immunization with pCAG-lacZ-WPRE results in significantly increased expression over a conventional CMV-driven vector in vivo. Cohorts of mice were i.m. immunized with pCAG-lacZ-WPRE (n = 5), pCMV-lacZ (n = 5), or a control DNA (pCMV-HA; n = 2). Each mouse received doses of 5 µg of DNA in the right hind limb and 50 µg of DNA in the left hind limb. Four days later, the muscle at the site of immunization was excised and homogenized. Lysates were assayed for {beta}-gal activity by the Galacto-Light chemiluminescence system. Data are graphed as relative light units per gram of excised muscle. *, p < 0.05, significant difference between the 50-µg dose groups. b, Cohorts of mice were immunized in the abdominal skin using a gene gun with pCMV-lacZ, pCAG-lacZ-WPRE, or control empty vector DNA; 2.5 days postimmunization, the draining inguinal LNs were isolated, and the frequency of CD11c+ {beta}-gal+ DCs was quantitated by flow cytometry. Representative flow plots are shown. b, The absolute numbers of CD11c+ {beta}-gal+ DC per draining inguinal lymph node for the two immunization groups are plotted. The total number of {beta}-gal+ DC in the pCAG-lacZ-WPRE-immunized group was significantly higher (p = 0.029). c, The levels of {beta}-gal expression in DC, as measured by the geometric mean fluorescence intensity (GMFI), are shown for mice immunized with pCMV-lacZ and pCAG-lacZ-WPRE. DCs from the pCAG-lacZ-WPRE DNA vaccine-immunized group expressed significantly higher {beta}-gal than those from the pCMV-lacZ-immunized group (p = 0.0068).

 
We also analyzed expression of the two {beta}-gal-encoding plasmid DNAs in DCs in vivo. To target DCs, we used gene gun immunization to deliver the DNA to skin-resident DCs. Gene gun delivery directly transfects skin DCs at the site of immunization, and these Ag-bearing DC migrate to the draining superficial iLNs (25, 29). Immunization with a {beta}-gal-encoding DNA plasmid will lead to {beta}-gal-marked DC that migrate to the LNs (29). The levels of in vivo-expressed {beta}-gal in the LNs can then be assayed by flow cytometry using the fluorescent substrate, FDG (25). Briefly, cohorts of mice were gene gun immunized with pCMV-lacZ, pCAG-lacZ-WPRE, or control pCMV empty vector DNA. We harvested the draining iLNs 2.5 days later and assayed for {beta}-gal expression by flow cytometry. CD11c+ DCs from the LNs of pCAG-lacZ-WPRE-immunized mice exhibited a significantly higher frequency (p = 0.0494; Fig. 6a) and absolute number (p = 0.029; Fig. 6b) of {beta}-gal+ DC compared with mice that received pCMV-lacZ. The level of {beta}-gal expression per cell, as measured by the geometric mean fluorescence intensity, was also significantly higher in the pCAG-lacZ-WPRE group (Fig. 6c; p = 0.006). These data clearly show that pCAG-lacZ-WPRE exhibited greater expression in vivo than pCMV-lacZ.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Conventional expression vectors used in vaccine strategies incorporate two fundamental features that drive the expression of encoded Ag in vivo. These include a strong promoter, usually CMV, and a polyadenylation sequence (30, 31, 32, 33). The use of these conventional DNA vaccines in vivo elicits both humoral and cellular immune responses, albeit at low levels. Clearly, there is a need to enhance the potency of these vaccines. To amplify the immune response, DNA vaccine-primed animals must be boosted, whether it be by further DNA vaccinations (27) or by a live recombinant virus boost (28, 34, 35). Booster immunizations are required to raise immune responses to levels sufficient to protect against live pathogen challenge (as reviewed in Ref.36).

In this study we describe the use of a modified DNA vaccine to enhance gene expression levels in vivo. This DNA vaccine contains a hybrid CAG promoter and a cis-acting WPRE element. We show increased cellular and humoral immune responses against HA in mice immunized with DNA vaccine containing the CAG promoter and WPRE compared with a vaccine that contains the CMV enhancer/promoter. This enhancement was observed for both low (10 µg) and high (100 µg) immunization doses. Earlier work with CAG-driven expression vectors noted heightened expression in a wide variety of cell types compared with CMV-driven vectors (17, 37). Likewise, WPRE improved the level of expression of reporter genes in cells with DNA bearing this regulatory element (18, 37). Viral vectors encoding both elements show the greatest expression (38, 39), indicating that both elements can act synergistically to boost the level of Ag. Earlier vaccination studies using DNA vaccines that include either CAG or WPRE have been performed and compared with conventional CMV vectors (37, 40, 41), and each has ascribed increased immune responses to increased Ag expression. In this study we describe for the first time the synergistic effects of both these elements in a single DNA vaccine.

We demonstrate in this study that in our experimental design the use of CAG and WPRE in DNA vaccines successfully induced protective immune responses at doses that the conventional CMV promoter-driven DNA vaccines did not. We selected the doses of 10 and 100 µg of DNA for immunization to evaluate the comparative protective efficacy of two different constructs based on a very thorough and extensive review of all the reports describing different doses of DNA vaccine used for HA via the i.m. route of immunization (42, 43, 44). We also tested 20- and 40-µg doses, and the responses observed were intermediate between those of the 10- and 100-µg doses (data not shown). Increased protection, which is associated with greater humoral and cellular responses, was observed in mice immunized with DNA vaccines containing CAG or WPRE. The greatest synergy was seen when Ag was expressed under the control of both WPRE and the CAG promoter. This enhanced protection is a reflection of the increased expression of Ag in vivo by DNA vaccines using the CAG promoter and WPRE in lieu of CMV promoter alone.

Vaccines encoding influenza A/PR/8/34 HA under the control of the CAG promoter, WPRE, or both act to boost the level of cellular, humoral, and protective responses over that of CMV promoter-driven DNA vaccines. The greatest differences were seen at low doses, where DNA vaccines containing CAG and WPRE show the greatest protection against live virus challenge relative to CMV-based vaccines. High dose DNA vaccinations conferred 100% survival to a lethal dose of influenza, but, unlike the pCMV-HA vaccine group, mice that were vaccinated with pCAG-HA-WPRE showed no weight loss during the course of the infection. In contrast, mice immunized with pCMV-HA showed, on the average, 4.5% weight loss by day 5 and recovered soon thereafter. This suggests that mice immunized with a high dose (100 µg) of pCMV-HA were not completely immune to the effects of influenza challenge and experienced low grade infection after challenge. This is an important difference, because vaccines that protect against illness as well as mortality make better vaccines.

The most likely explanation for the differences in the immune responses elicited by pCMV- and pCAG-driven DNA vaccines is that the WPRE-enhanced, pCAG vectors result in greater Ag expression of Ag than pCMV in vivo. This hypothesis was supported by increased {beta}-gal expression in the DCs of mice gene gun vaccinated with pCAG-lacZ-WPRE than in those given the conventional pCMV-lacZ vaccines. In addition, the suppressive effects of IFN-{gamma} on the expression of reporter genes driven off the CMV promoter may explain why CAG promoters elicit larger immune responses in vivo. Another possibility for the observed increase in the immune response with the CAG promoter and WPRE could presumably be attributed to the presence of CpG and other immunostimulatory motifs that are missing in the CMV promoter, but there is no precedent in the literature to support this, and further studies to identify their presence in these elements are warranted.

In conclusion, by using a stronger promoter and a powerful noncoding post-transcriptional response element, we were able to amplify the immune response beyond what is seen with conventional CMV-based vaccines. The end result is a DNA vaccine that elicits protective immunity at a 10-fold lower dose. Our data also show that there is a threshold level of Ag expression that is necessary for eliciting protective immune responses. The CAG- and WPRE-driven vaccines can achieve this Ag threshold at lower doses of DNA, whereas the conventional CMV-driven vaccines fail to do so. By lowering the amount of DNA used for immunization, historic concerns with DNA vaccines, such as the risks posed by integration events into the host genome or the induction of autoimmune disease, are minimized. Whether the CAG- and WPRE-enhanced DNA vaccines can induce effective protective immunity in large animal models, such as nonhuman primates, remains to be investigated.


   Acknowledgments
 
We thank Dr. Bernard Moss for MVA-HA, Dr. Jacqueline Katz for influenza stocks, Dr. Harriet Robinson for advice, and Karin Smith for excellent mouse colony management.


   Footnotes
 
1 This work was supported by grants from the U.S. National Institutes of Health and the Emory Center for AIDS Research. Back

2 S.G. and A.E.O. contributed equally to this work. Back

3 Current address: Division of Viral and Rickettsial Diseases, Influenza Branch, National Centers for Infectious Diseases, Mail Stop G-16, Centers for Disease Control and Prevention, Atlanta, GA 30333. Back

4 Address correspondence and reprint requests to Dr. Joshy Jacob, Emory Vaccine Center, 954 Gatewood Road, Atlanta, GA 30329. E-mail address: jjacob3{at}emory.edu. Back

5 Abbreviations used in this paper: DC, dendritic cell; FDG, fluorescein digalactopyranoside; {beta}-gal, {beta}-galactosidase; HA, hemagglutinin; iLN, inguinal lymph node; MVA, modified vaccinia Ankara. Back

Received for publication November 4, 2003. Accepted for publication April 23, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Robinson, H. L., T. M. Pertmer. 2000. DNA vaccines for viral infections: basic studies and applications. Adv. Virus Res. 55:1.[Medline]
  2. Gurunathan, S., D. M. Klinman, R. A. Seder. 2000. DNA vaccines: immunology, application, and optimization. Annu. Rev. Immunol. 18:927.[Medline]
  3. Nishimura, Y., A. Kamei, S. Uno-Furuta, S. Tamaki, G. Kim, Y. Adachi, K. Kuribayashi, Y. Matsuura, T. Miyamura, Y. Yasutomi. 1999. A single immunization with a plasmid encoding hepatitis C virus (HCV) structural proteins under the elongation factor 1-{alpha} promoter elicits HCV-specific cytotoxic T-lymphocytes (CTL). Vaccine 18:675.[Medline]
  4. Harms, J. S., G. A. Splitter. 1995. Interferon-{gamma} inhibits transgene expression driven by SV40 or CMV promoters but augments expression driven by the mammalian MHC I promoter. Hum. Gene Ther. 6:1291.[Medline]
  5. Sedegah, M., W. Weiss, J. B. Sacci, Jr, Y. Charoenvit, R. Hedstrom, K. Gowda, V. F. Majam, J. Tine, S. Kumar, P. Hobart, et al 2000. Improving protective immunity induced by DNA-based immunization: priming with antigen and GM-CSF-encoding plasmid DNA and boosting with antigen-expressing recombinant poxvirus. J. Immunol. 164:5905.[Abstract/Free Full Text]
  6. Chow, Y. H., W. L. Huang, W. K. Chi, Y. D. Chu, M. H. Tao. 1997. Improvement of hepatitis B virus DNA vaccines by plasmids coexpressing hepatitis B surface antigen and interleukin-2. J. Virol. 71:169.[Abstract]
  7. Sailaja, G., S. Husain, B. P. Nayak, A. M. Jabbar. 2003. Long-term maintenance of gp120-specific immune responses by genetic vaccination with the HIV-1 envelope genes linked to the gene encoding Flt-3 ligand. J. Immunol. 170:2496.[Abstract/Free Full Text]
  8. Lew, A. M., B. J. Brady, B. J. Boyle. 2000. Site-directed immune responses in DNA vaccines encoding ligand-antigen fusions. Vaccine 18:1681.[Medline]
  9. Tachedjian, M., J. S. Boyle, A. M. Lew, B. Horvatic, J. P. Scheerlinck, J. M. Tennent, M. E. Andrew. 2003. Gene gun immunization in a preclinical model is enhanced by B7 targeting. Vaccine 21:2900.[Medline]
  10. Deliyannis, G., J. S. Boyle, J. L. Brady, L. E. Brown, A. M. Lew. 2000. A fusion DNA vaccine that targets antigen-presenting cells increases protection from viral challenge. Proc. Natl. Acad. Sci. USA 97:6676.[Abstract/Free Full Text]
  11. Hung, C. F., W. F. Cheng, C. Y. Chai, K. F. Hsu, L. He, M. Ling, T. C. Wu. 2001. Improving vaccine potency through intercellular spreading and enhanced MHC class I presentation of antigen. J. Immunol. 166:5733.[Abstract/Free Full Text]
  12. Hung, C. F., L. He, J. Juang, T. J. Lin, M. Ling, T. C. Wu. 2002. Improving DNA vaccine potency by linking Marek’s disease virus type 1 VP22 to an antigen. J. Virol. 76:2676.[Abstract/Free Full Text]
  13. Ross, T. M., Y. Xu, R. A. Bright, H. L. Robinson. 2000. C3d enhancement of antibodies to hemagglutinin accelerates protection against influenza virus challenge. Nat. Immunol. 1:127.[Medline]
  14. Wills, K. N., I. A. Atencio, J. B. Avanzini, S. Neuteboom, A. Phelan, J. Philopena, S. Sutjipto, M. T. Vaillancourt, S. F. Wen, R. O. Ralston, et al 2001. Intratumoral spread and increased efficacy of a p53-VP22 fusion protein expressed by a recombinant adenovirus. J. Virol. 75:8733.[Abstract/Free Full Text]
  15. Sasaki, S., R. R. Amara, A. E. Oran, J. M. Smith, H. L. Robinson. 2001. Apoptosis-mediated enhancement of DNA-raised immune responses by mutant caspases. Nat. Biotechnol. 19:543.[Medline]
  16. Gribaudo, G., S. Ravaglia, A. Caliendo, R. Cavallo, M. Gariglio, M. G. Martinotti, S. Landolfo. 1993. Interferons inhibit onset of murine cytomegalovirus immediate-early gene transcription. Virology 197:303.[Medline]
  17. Sawicki, J. A., R. J. Morris, B. Monks, K. Sakai, J. Miyazaki. 1998. A composite CMV-IE enhancer/{beta}-actin promoter is ubiquitously expressed in mouse cutaneous epithelium. Exp. Cell Res. 244:367.[Medline]
  18. Zufferey, R., J. E. Donello, D. Trono, T. J. Hope. 1999. Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J. Virol. 73:2886.[Abstract/Free Full Text]
  19. Feltquate, D. M., S. Heaney, R. G. Webster, H. L. Robinson. 1997. Different T helper cell types and antibody isotypes generated by saline and gene gun DNA immunization. J. Immunol. 158:2278.[Abstract]
  20. Sarawar, S. R., P. C. Doherty. 1994. Concurrent production of interleukin-2, interleukin-10, and {gamma} interferon in the regional lymph nodes of mice with influenza pneumonia. J. Virol. 68:3112.[Abstract/Free Full Text]
  21. Deng, Y., J. W. Yewdell, L. C. Eisenlohr, J. R. Bennink. 1997. MHC affinity, peptide liberation, T cell repertoire, and immunodominance all contribute to the paucity of MHC class I-restricted peptides recognized by antiviral CTL. J. Immunol. 158:1507.[Abstract]
  22. Gerhard, W., A. M. Haberman, P. A. Scherle, A. H. Taylor, G. Palladino, A. J. Caton. 1991. Identification of eight determinants in the hemagglutinin molecule of influenza virus A/PR/8/34 (H1N1) which are recognized by class II-restricted T cells from BALB/c mice. J. Virol. 65:364.[Abstract/Free Full Text]
  23. Belyakov, I. M., L. S. Wyatt, J. D. Ahlers, P. Earl, C. D. Pendleton, B. L. Kelsall, W. Strober, B. Moss, J. A. Berzofsky. 1998. Induction of a mucosal cytotoxic T-lymphocyte response by intrarectal immunization with a replication-deficient recombinant vaccinia virus expressing human immunodeficiency virus 89.6 envelope protein. J. Virol. 72:8264.[Abstract/Free Full Text]
  24. Maris, C. H., J. D. Miller, J. D. Altman, J. Jacob. 2003. A transgenic mouse model genetically tags all activated CD8 T cells. J. Immunol. 171:2393.[Abstract/Free Full Text]
  25. Garg, S., A. Oran, J. Wajchman, S. Sasaki, C. H. Maris, J. A. Kapp, J. Jacob. 2003. Genetic tagging shows increased frequency and longevity of antigen-presenting, skin-derived dendritic cells in vivo. Nat. Immunol. 4:907.[Medline]
  26. Ramezani, A., T. S. Hawley, R. G. Hawley. 2000. Lentiviral vectors for enhanced gene expression in human hematopoietic cells. Mol. Ther. 2:458.[Medline]
  27. Robinson, H. L., C. A. Boyle, D. M. Feltquate, M. J. Morin, J. C. Santoro, R. G. Webster. 1997. DNA immunization for influenza virus: studies using hemagglutinin- and nucleoprotein-expressing DNAs. J. Infect. Dis. 176:(Suppl. 1):S50.
  28. Schneider, J., S. C. Gilbert, T. J. Blanchard, T. Hanke, K. J. Robson, C. M. Hannan, M. Becker, R. Sinden, G. L. Smith, A. V. Hill. 1998. Enhanced immunogenicity for CD8+ T cell induction and complete protective efficacy of malaria DNA vaccination by boosting with modified vaccinia virus Ankara. Nat. Med. 4:397.[Medline]
  29. Porgador, A., K. R. Irvine, A. Iwasaki, B. H. Barber, N. P. Restifo, R. N. Germain. 1998. Predominant role for directly transfected dendritic cells in antigen presentation to CD8+ T cells after gene gun immunization. J. Exp. Med. 188:1075.[Abstract/Free Full Text]
  30. Bohm, W., A. Kuhrober, T. Paier, T. Mertens, J. Reimann, R. Schirmbeck. 1996. DNA vector constructs that prime hepatitis B surface antigen-specific cytotoxic T lymphocyte and antibody responses in mice after intramuscular injection. J. Immunol. Methods 193:29.[Medline]
  31. Hartikka, J., M. Sawdey, F. Cornefert-Jensen, M. Margalith, K. Barnhart, M. Nolasco, H. L. Vahlsing, J. Meek, M. Marquet, P. Hobart, et al 1996. An improved plasmid DNA expression vector for direct injection into skeletal muscle. Hum. Gene Ther. 7:1205.[Medline]
  32. Manthorpe, M., F. Cornefert-Jensen, J. Hartikka, J. Felgner, A. Rundell, M. Margalith, V. Dwarki. 1993. Gene therapy by intramuscular injection of plasmid DNA: studies on firefly luciferase gene expression in mice. Hum. Gene Ther. 4:419.[Medline]
  33. Montgomery, D. L., J. W. Shiver, K. R. Leander, H. C. Perry, A. Friedman, D. Martinez, J. B. Ulmer, J. J. Donnelly, M. A. Liu. 1993. Heterologous and homologous protection against influenza A by DNA vaccination: optimization of DNA vectors. DNA Cell Biol. 12:777.[Medline]
  34. Gilbert, S. C., J. Schneider, C. M. Hannan, J. T. Hu, M. Plebanski, R. Sinden, A. V. Hill. 2002. Enhanced CD8 T cell immunogenicity and protective efficacy in a mouse malaria model using a recombinant adenoviral vaccine in heterologous prime-boost immunisation regimes. Vaccine 20:1039.[Medline]
  35. Gilbert, S. C., J. Schneider, M. Plebanski, C. M. Hannan, T. J. Blanchard, G. L. Smith, A. V. Hill. 1999. Ty virus-like particles, DNA vaccines and modified vaccinia virus Ankara; comparisons and combinations. Biol. Chem. 380:299.[Medline]
  36. Robinson, H. L.. 2003. Prime boost vaccines power up in people. Nat. Med. 9:642.[Medline]
  37. Xu, Z. L., H. Mizuguchi, T. Mayumi, T. Hayakawa. 2003. Woodchuck hepatitis virus post-transcriptional regulation element enhances transgene expression from adenovirus vectors. Biochim. Biophys. Acta 1621:266.[Medline]
  38. Klein, R. L., M. E. Hamby, C. F. Sonntag, W. J. Millard, M. A. King, E. M. Meyer. 2002. Measurements of vector-derived neurotrophic factor and green fluorescent protein levels in the brain. Methods 28:286.[Medline]
  39. Klein, R. L., M. E. Hamby, Y. Gong, A. C. Hirko, S. Wang, J. A. Hughes, M. A. King, E. M. Meyer. 2002. Dose and promoter effects of adeno-associated viral vector for green fluorescent protein expression in the rat brain. Exp. Neurol. 176:66.[Medline]
  40. Pertl, U., H. Wodrich, J. M. Ruehlmann, S. D. Gillies, H. N. Lode, R. A. Reisfeld. 2003. Immunotherapy with a posttranscriptionally modified DNA vaccine induces complete protection against metastatic neuroblastoma. Blood 101:649.[Abstract/Free Full Text]
  41. Tsukamoto, K., S. Saito, S. Saeki, T. Sato, N. Tanimura, T. Isobe, M. Mase, T. Imada, N. Yuasa, S. Yamaguchi. 2002. Complete, long-lasting protection against lethal infectious bursal disease virus challenge by a single vaccination with an avian herpesvirus vector expressing VP2 antigens. J. Virol. 76:5637.[Abstract/Free Full Text]
  42. Torres, C. A. T., K. Yang, F. Mustafa, H. L. Robinson. 2000. DNA immunization: effect of secretion of DNA-expressed hemagglutinins on antibody responses. Vaccine 18:805.
  43. Fynan, E. F., R. G. Webster, D. H. Fuller, J. R. Haynes, J. C. Santoro, H. L. Robinson. 1993. DNA vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations. Proc. Natl. Acad. Sci. USA 15:11478.
  44. Ulmer, J. B., R. R Deck, C. M. DeWitt, A. Friedman, J. J. Donnelly, M. A. Liu. 1994. Protective immunity by intramuscular injection of low doses of influenza virus DNA vaccines. Vaccine 12:1541.[Medline]



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