Electroporation Enables Plasmid Vaccines to Elicit CD8+ T Cell Responses in the Absence of CD4+ T Cells

Kelley Dayball, James Millar, Mark Miller, Yong Hong Wan and Jonathan Bramson
J Immunol October 1, 2003, 171 (7) 3379-3384; DOI: https://doi.org/10.4049/jimmunol.171.7.3379

Abstract

In vivo electroporation dramatically enhances plasmid vaccine efficacy. This enhancement can be attributed to increased plasmid delivery and, possibly, to some undefined adjuvant properties. Previous reports have demonstrated CD8+ T cell priming by plasmid vaccines is strongly dependent upon CD4+ T cell help. Indeed, the efficacy of a plasmid vaccine expressing Escherichia coli β-galactosidase was severely attenuated in MHC class II-deficient (C2D) mice. To determine whether electroporation could compensate for the absence of CD4+ T cell help, C2D mice were immunized by a single administration of plasmid in combination with electroporation using two conditions which differed only by the duration of the pulse (20 or 50 msec). Both conditions elicited robust cellular and humoral responses in wild-type mice, as measured by IFN-γ ELISPOT, anti-β-galactosidase ELISA, and protection from virus challenge. In C2D mice, the cellular response produced by the vaccine combined with the 50-msec pulse, as measured by ELISPOT, was identical to the response in wild-type mice. The 20-msec pulse elicited a milder response that was approximately one-fifth that of the response elicited by the 50-msec pulse. By contrast, the 20-msec conditions provided comparable protection in both wild-type and C2D recipients whereas the protection elicited by the 50-msec conditions in C2D mice was weaker than in wild-type mice. Further investigation is required to understand the discordance between the ELISPOT results and outcome of virus challenge in the C2D mice. Nonetheless, using this technique to prime CD8+ T cells using plasmid vaccines may prove extremely useful when immunizing hosts with limiting CD4+ T cell function, such as AIDS patients.

Plasmid vaccination is emerging as a safe and cost-effective method for genetic immunization (1, 2). The basic form of the vaccine consists of two components: 1) a mammalian expression cassette and 2) the bacterial plasmid backbone. The importance of the plasmid backbone during vaccination has been attributed to the presence of specific immunostimulatory CG dinucleotide sequences within the bacterial DNA, known as CpG motifs (3). DNA sequences containing CpG motifs have been shown to activate T and B cells and promote maturation of dendritic cells, increasing their Ag presentation capacity. When the CpG sequences contained within an immunization plasmid were removed or methylated, the vaccine activity was dramatically reduced, supporting an important role for unmethylated CpG motifs in successful immunization by plasmid DNA vaccines (4, 5, 6).

Implementation of plasmid vaccines in humans has met with limited success, possibly due to inefficient gene transfer or weakened immunogenicity compared with rodents (7, 8, 9, 10). A number of methods have been developed to increase the efficiency of plasmid delivery and our particular interest is in vivo electroporation (11). Recent experiments have demonstrated that electroporation can greatly enhance plasmid vaccination and is associated with up to 1000-fold increased levels of gene expression (12, 13, 14, 15, 16). Additionally, electroporation displays an adjuvant quality, thus enhancement of plasmid vaccination using this technology may be due to a combination of increased gene expression and an undefined adjuvant effect (17).

CD4+ T cells provide critical support for CD8+ T cell priming, in part by conditioning dendritic cells for CD8+ T cell activation (18, 19, 20). The requirement for CD4+ T cells in this process can be overcome by ligation of CD40 on the dendritic cell or by virus infection. Plasmid vaccines produce their Ags within host cells leading to presentation on MHC class I and the development of robust CD8+ T cell responses similar to Ag production during viral infection. Unlike viral infection, activation of CD8+ T cells following plasmid vaccination appears to be critically dependent upon the presence of MHC class II and available CD4+ T cell help (21, 22, 23, 24). Thus, while viruses and plasmid DNA may share common features in terms of Ag production, they are clearly distinct with regard to their mechanisms of immune induction.

The requirement of CD4+ T cells for CD8+ T cell priming by plasmid vaccines could be partially compensated by coexpressing B7-1 in the vaccine inoculum or by treating mice with a CD40 agonist during immunization (21). Interestingly, gene gun-mediated delivery of a plasmid vaccine could elicit CD8+ T cell responses in the absence of CD4+ T cell help, suggesting that the adjuvant properties of plasmid delivery systems may provide unexpected benefits (25). In the present study, we sought to determine whether the putative adjuvant activity of electroporation could compensate for the absence of CD4+ T cell help in the activation of CD8+ T cells following plasmid immunization. Developing methods to overcome the CD4+ T cell dependence of plasmid vaccination is critical for application of these vaccines in patient populations where CD4+ T cell function is limiting, such as individuals with AIDS.

Materials and Methods

Plasmids

The plasmid pCMVβ, which expresses the Escherichia coli LacZ under the control of the CMV promoter, has been described previously (26). The control plasmid, pL018, that expresses firefly luciferase under the control of the CMV promoter, was constructed by replacing the LacZ gene in pCMVβ with firefly luciferase (provided by Inex Pharmaceuticals, Vancouver, British Columbia, Canada). Plasmids were prepared using Endo-Free Mega Prep kits from Qiagen (Valencia, CA) and resuspended in sterile, endotoxin-free water (Life Technologies, Grand Island, NY).

Cell culture

All plasticware used for cell culture was purchased from Falcon (BD Biosciences, Franklin Lakes, NJ). All cells were cultured in complete RPMI consisting of RPMI 1640 supplemented with 10% FBS, 2 mM l-glutamine, 50 μM 2-ME, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cell lines stably expressing β-galactosidase (β-gal)3 (E22) and OVA (E.G7-OVA) were maintained in complete RPMI containing 400 μg/ml G418.

Plasmid injections and histology

Female C57BL/6 mice were purchased from Charles River Breeding Laboratories (Wilmington, MA). MHC class II-deficient (C2D) mice (B6,129S-H2dlAb1-Ea), originally obtained from The Jackson Laboratory (Bar Harbor, ME), were bred in our animal facility under specific pathogen-free conditions. Plasmids were diluted to the appropriate concentration in endotoxin-free, sterile water. The inoculum was buffered by the addition of 10× PBS (Life Technologies). Groups of three to five mice were anesthetized and injected in both rear thighs with plasmid solution (1 μg/μl; 50 μl/thigh). When indicated, the injection site was spanned by two needles separated by 0.5 cm (two-needle array; BTX Division, Genetronix, San Diego, CA) and electroporated with six square wave pulses (field strength = 200 V/cm; pulse length = 20 or 50 msec) using the ECM 830 field generator (BTX Division, Genetronix). To examine histological changes within the injection site, muscle tissues were removed 5 days postimmunization, fixed in formalin, sectioned for histology, and stained with H&E.

ELISPOT assay

Spleens were harvested 14 days after immunization and tested for immune reactivity by ELISPOT assay following an 18 h stimulation with E22 cells (β-gal-positive) and E.G7-OVA (negative control) as described (27).

Anti-β-gal ELISA

Serum was obtained by cardiac puncture 14 days postimmunization. β-gal-specific IgG1 and IgG2a were measured as described previously (27).

Vaccinia challenge model

Mice were challenged with 5 × 106 PFU of a recombinant vaccinia virus expressing β-gal (Vacc-βgal). Five days later, the spleens and ovaries were harvested and homogenized in 2 ml of 1 mM Tris, pH 9.0. The homogenates were further disrupted by three consecutive “freeze/thaw” cycles. To determine virus titer in the homogenates, confluent CV-1 cells in 12-well plates were infected with serial dilutions of the homogenate and, 2 days later, plaques were visualized by staining with 0.1% crystal violet in 20% ethanol. An animal was considered “protected” when virus titers were more than or equal to one-tenth of the lowest titer measured in the control group.

In vivo luciferase and β-gal assays

Muscle tissues encompassing the injection site were harvested 48 and 120 h following plasmid injection. The tissues were snap-frozen and homogenized in Reporter Lysis buffer (Promega, Madison, WI) supplemented with protease inhibitor mixture (Roche, Basel, Switzerland). The homogenates were then cleared by centrifugation. Luciferase activity was measured using the Promega luciferase assay kit. β-gal activity in the homogenates was measured using the Galacto-Light assay kit (Applied Biosystems, Foster City, CA). Total protein was determined using the DC protein assay (Bio-Rad, Hercules, CA) and the data is presented as relative light units (RLUs)/mg total protein.

Statistical analysis

Statistical analysis of the results and area under the curve calculations were performed using MedCalc version 7.0.1.0 (MedCalc Software, Mariakerke, Belgium). Statistical significance was determined using the Wilcoxon Mann-Whitney test and the data was log transformed to stabilize variances. Probability values (p) > 0.05 were considered NS. Data is presented as mean ± SEM.

Results

Plasmid vaccination is severely attenuated in C2D mice

Previous experiments demonstrating the requirement of CD4+ T cell help for the generation of CD8+ T cell responses by plasmid vaccines relied upon chromium-release assays to demonstrate the defect. Although these assays are quite reliable, they are not very quantitative. We have used ELISPOT to directly quantify the number of Ag-specific IFN-γ-producing cells with minimal restimulation in vitro. Similar to our previous observations, only 40% of recipients responded to a single administration of pCMVβ (8 of 20; Fig. 1⇓) (28). A second administration of the vaccine 21 days later increased the efficacy to nearly 100% (Table I⇓; Fig. 1⇓). By contrast, two administrations of the plasmid vaccine into C2D mice only elicited a cellular response in 5 of 14 recipients (35%; Fig. 1⇓). This response rate was similar to that seen in wild-type animals following a single immunization, however the average number of spot-forming cells (SFCs) in wild-type mice that responded to a single immunization (119 ± 68) was higher than the number of SFCs in C2D mice responding to two administrations of pCMVβ (10 ± 1), although this difference was not significant.

FIGURE 1.
FIGURE 1.

Cellular response following immunization without electroporation. Wild-type (WT) mice and MHC C2D mice were immunized once (1X; n = 20) or twice 21 days apart (2X; n = 17 for WT and n = 13 for C2D) with pCMVβ. Fourteen days after the final immunization, the cellular response was measured by ELISPOT. Each point represents the result from a single mouse. The p values were determined using a Wilcoxon test.

View this table:
Table I.

Summary of effector responses following immunization

Electroporation increases the efficacy of plasmid vaccines in wild-type mice

Previous reports have demonstrated that electroporation can improve the activity of plasmid vaccines in wild-type mice. Although most reports use similar field strengths for electroporation (200 V/cm), they differ in the number of pulses, pulse length, and orientation of the field. We tested a number of these conditions using a luciferase reporter plasmid (pL018). Luciferase was chosen as a reporter rather than β-gal because background levels of luciferase-like activity in muscle tissue are negligible (50–100 RLUs), whereas background levels of β-gal activity in the muscle are ∼100-fold higher (10,000–20,000 RLUs). Mice were injected with 5 μg of pL018 followed by a number of different electroporation conditions and gene expression in the muscle was measured 5 days later. Two pulse lengths were evaluated: 20 and 50 msec. When six pulses were applied to the muscle in a single direction, similar levels of gene expression were measured; 2.9 ± 1.0 × 106 RLU with the 20-msec pulse and 3.1 ± 0.9 × 106 RLU with the 50-msec pulse. This activity was 100-fold greater than the gene expression in the nonelectroporated muscle 2.5 ± 3.7 × 104 RLU. Increasing the number of pulses did not improve gene expression nor did reorientation of the field (data not shown). Thus, for all additional experiments, we used similar conditions (six pulses of 200 V/cm in a single direction), but we evaluated two different pulse lengths.

β-gal activity was readily measurable in the muscle at 48 and 120 h following administration of the vaccine plasmid, pCMVβ, using both conditions (Fig. 2⇓A). The activity in tissues treated with 20-msec pulses was slightly higher (40–60%) than the corresponding tissues in the 50-msec group, but this difference was not significant. Histological analysis of muscle tissues 5 days following plasmid injection revealed a strong inflammatory infiltrate in the tissues receiving the plasmid vaccine in combination with electroporation (Fig. 2⇓, B and C) whereas there was no evidence of infiltration at this time in tissues injected with plasmid without electroporation (Fig. 2⇓D). Under these conditions, cellular responses could be measured by ELISPOT in 100% of the electroporated mice (Table I⇑; Fig. 3⇓). Again, there was no significant difference between the two groups, however the average number of SFCs in the 50-msec group (296 ± 55) was greater than the 20-msec group (242 ± 44; NS). Similarly, all electroporated mice developed Abs against β-gal (Table I⇑) whereas only 1 of 18 mice in the nonelectroporated group exhibited Abs to β-gal. The humoral response in the electroporated group was composed primarily of IgG2a, consistent with a type 1 immune response. Again, the average Ab levels in the 50-msec group (5105 ± 994 U of IgG2a and 882 ± 365 U of IgG1) were higher than the 20-msec group (2973 ± 758 U of IgG2a and 197 ± 67 U of IgG1; NS) (Fig. 4⇓).

FIGURE 2.
FIGURE 2.

Gene expression and histology in muscle tissues injected with pCMVβ followed by electroporation. pCMVβ was injected i.m. followed by electroporation using either a pulse length of either 20 or 50 msec. A, Muscle tissues were harvested 48 and 120 h later (six muscles per group) and the level of β-gal production was assayed biochemically. B–D, Histological sections of muscle tissue 5 days following immunization with pCMVβ in combination with electroporation using the 20-msec pulse (B) or the 50-msec pulse (C) or no electroporation (D)

FIGURE 3.
FIGURE 3.

Cellular response following immunization with electroporation. Wild-type (WT) mice and MHC C2D mice were immunized by a single i.m. injection with pCMVβ followed electroporation using either a pulse length of either 20 msec (n = 14 for WT and n = 11 for C2D) or 50 msec (n = 17 for WT and n = 10 for C2D). A control group was included that received the vaccine without electroporation (no EP; n = 20). Fourteen days after the final immunization, the cellular response was measured by ELISPOT. Each point represents the results from a single mouse. The p values were determined using a Wilcoxon test.

FIGURE 4.
FIGURE 4.

Ab response following immunization with electroporation. Wild-type (WT) mice were immunized by a single i.m. injection with pCMVβ followed electroporation using a pulse length of either 20 or 50 msec. A control group was included that received the vaccine without electroporation (no EP). Fourteen days later, sera were harvested to measure anti-β-gal Abs. Each bar is representative of 16–18 mice (mean ± SEM). The p values were determined using a Wilcoxon test.

To measure protective immunity, mice were challenged with a recombinant vaccinia virus that expresses β-gal (Vacc-βgal). Immunization with a single injection of pCMVβ in the absence of electroporation afforded some degree of protection; 2 of 10 vaccinated mice had virus titers that were reduced by almost 2 logs compared with control (Fig. 5⇓). All of the mice in the 50-msec group exhibited protection from challenge and 4 of 10 were completely clear of virus. Similarly, 7 of 10 mice in the 20-msec group were protected from challenge and 2 of 10 were completely free of virus. The average titers (log10 PFU) in the 50-msec group (3.7 ± 0.5) were similar to the 20-msec group (4.6 ± 0.6).

FIGURE 5.
FIGURE 5.

Protection against virus challenge following vaccination of wild-type mice. Wild-type mice were immunized by a single i.m. injection with pCMVβ followed electroporation using either a pulse length of either 20 msec (n = 10) or 50 msec (n = 10). An additional group was included that received the vaccine without electroporation (no EP; n = 10). As a control, mice were immunized with a plasmid expressing luciferase (pLuc) followed by electroporation using either pulse length and the data for both groups of control mice are presented in the same column (20/50 msec; n = 10). Fourteen days after the final immunization, the mice were inoculated with Vacc-βgal. The results shown represent the titer of Vacc-βgal in the spleens and ovaries of individual mice 5 days after challenge. An animal was considered protected when virus titers were less than one-tenth of the lowest titer measured in the control group. The p values were determined using a Wilcoxon test.

Electroporation enables plasmid vaccines in MHC C2D mice

Immunization of C2D mice using the 50-msec pulse length elicited cellular responses in 100% of recipients and the average number of SFC (286 ± 58) was identical to the number in wild-type mice (296 ± 55) (Fig. 3⇑) demonstrating that electroporation can fully overcome the requirement of MHC class II presentation to CD4+ T cells for CD8+ T cell expansion by plasmid vaccines. By contrast, the CD8+ T cell response elicited by the vaccine in combination with the 20-msec pulse length was significantly weaker (64 ± 21; Fig. 3⇑) than both the 50-msec conditions in C2D (p < 0.01) and the 20-msec condition in wild-type mice (p < 0.01). Thus, although both conditions had relatively comparable impact on the CD8+ T cell response in wild-type hosts, only the 50-msec conditions could fully overcome the requirement for CD4+ T cell help in CD8+ T cell expansion.

Surprisingly, when vaccine efficacy was measured by protection from virus challenge, the 20-msec condition elicited a response in C2D mice comparable to wild-type mice despite lower frequencies of IFN-γ-secreting cells at the time of challenge; 70% of recipients were protected, 26% were fully protected from challenge and mean titers were quite similar (4.6 ± 0.6 log10 PFU in wild-type vs 4.9 ± 0.5 log10 PFU in C2D). (Table I⇑; Fig. 6⇓). In contrast, the protective response elicited by the 50-msec conditions in C2D was weaker than in wild-type mice. Less than 50% of C2D mice immunized using the 50-msec conditions were protected from challenge compared with 100% of wild-type mice. The average titers in the immunized C2D were also higher (5.8 ± 0.5 log10 PFU) than wild-type (3.7 ± 0.5 log10 PFU; p < 0.02). As expected, a single injection of plasmid vaccine in C2D mice without electroporation provided no protection against challenge with Vacc-βgal.

FIGURE 6.
FIGURE 6.

Protection against virus challenge following vaccination of MHC C2D mice. MHC C2D mice were immunized by a single i.m. injection with pCMVβ followed electroporation using either a pulse length of either 20 msec (n = 19) or 50 msec (n = 18). An additional group was included that received the vaccine without electroporation (No EP; n = 7). As a control, mice were immunized with a plasmid expressing luciferase (pLuc) followed by electroporation using either pulse length and the data for both groups of control mice are presented in the same column (20/50 msec; n = 9). Fourteen days after the final immunization, the mice were inoculated with Vacc-βgal. The results shown represent the titer of Vacc-βgal in the spleens and ovaries of individual mice 5 days after challenge. An animal was considered protected when virus titers were less than one-tenth of the lowest titer measured in the control group. The p values were determined using a Wilcoxon test.

Discussion

Plasmid DNA is currently being evaluated in AIDS patients as a potential genetic vaccine platform. The results from trials in HIV-infected individuals have been positive but not very robust (8, 9, 29). By contrast, a recent study investigating plasmid vaccines for HIV in normal volunteers demonstrated immune responses in >50% of the recipients (30). Although there are many possible explanations for the increased cellular responses in the latter study, one possibility is the reduced activity of plasmid vaccines due to the absence of adequate CD4+ T cell help in HIV-infected recipients. It is interesting to note that the responses in the normal volunteers were all CD4+ T cell-mediated.

The requirement for CD4+ T cells during CD8+ T cell priming by plasmid vaccines remains unclear. It has been proposed that CD8+ T cells are elicited by plasmid vaccines through cross-presentation of Ag which has been shown to be dependent upon CD4+ T cell help (18, 19, 20, 21). By contrast, virus vectors elicit CD8+ T cell responses independent of CD4+ T cell help, possibly through direct infection of APCs (19, 21, 31, 32, 33). Although this is a legitimate explanation, cross-presentation does not appear to be a major route for CD8+ T cell priming following i.m. injection of plasmid, which is the route used in the current study (34, 35). Indeed, CD8+ T cell responses elicited by electroporation of a plasmid using a muscle-specific promoter were reduced by >90% compared with a similar immunization using a constitutive promoter despite equivalent levels of Ag expression in the muscle (J. Bramson, unpublished data), so CD8+ T cells priming by plasmid vaccines appears to be largely dependent upon direct transfection of APCs. Even so, dendritic cells which are presenting Ag through the endogenous pathway are still dependent upon CD4+ T cell help (36, 37, 38). Thus, the ability of viruses to elicit a CD8+ T cell response in the absence of CD4+ T cell help is not likely to be a reflection of the pathway of MHC class I peptide processing (endogenous vs exogenous) but rather a reflection of the changes in the dendritic cells induced by the process of virus infection which are not conveyed by plasmid transfection.

In consideration of the idea that CpG motifs within the plasmid vector are responsible for the activity of plasmid vaccines, it is possible that the immunostimulatory pathways elicited by CpG DNA are different from those induced by virus infection. However, CpG oligos have been demonstrated to be highly potent adjuvants which can elicit CD8+ T cell responses when combined with protein Ag, even in the absence of CD4+ T cells (22, 24). The key difference between the adjuvant CpG oligonucleotide adjuvants and the plasmid DNA backbone is the nature of the nucleic acid itself. The oligonucleotides are generally constructed with a nuclease-resistant backbone that allows the oligonucleotide to persist for a considerable time in the host. By contrast, the plasmid DNA is degraded rapidly following i.m. injection (39). So the key difference in outcome following immunization with Ag mixed with CpG oligonucleotides or plasmid DNA may be the degree of dendritic cell stimulation provided by the nucleic acid backbone. Therefore, although the plasmid may have transfected local dendritic cells resulting in direct presentation of MHC class I peptides, the immunostimulatory signal may not be strong enough to overcome the requirement for additional CD4+ T cell stimulation.

With the idea in mind that immune stimulation, not Ag delivery, is the key factor limiting the ability of plasmid vaccines to elicit CD8+ T cells, the true benefit of electroporation under conditions of limiting T cell help may be the inflammation produced during the process. We have observed substantial cellular infiltrate within the muscle tissue for a period of at least 5 days following electroporation, independent of Ag (J. Bramson, unpublished data). Thus, the increased efficacy of electroporated plasmid vaccines in C2D animals may be a reflection of the heightened inflammatory state within the muscle tissue leading to increased recruitment and activation of APCs. Further evidence of adjuvant-like properties of electroporation come from studies demonstrating that electroporation can increase the immunogenicity of CTL epitope peptides (17). Because class I binding peptides do not require uptake for presentation, the increased activity is likely reflective of an adjuvant quality. Immunization with gene gun can also elicit CD8+ T cell responses in the absence of CD4+ T cells (25). Taken together, these data suggest that, in addition to enhancing gene delivery, the electroporation and gene gun technologies also provide adjuvant-like effects.

One issue that remains to be addressed is the apparent discrepancy between the ELISPOT results and protective immune function in the experiment using the C2D animals. Based on the robust β-gal-specific cellular response measured following electroporation of plasmid using the 50-msec pulse length in C2D mice, we expected to observe very strong protective immunity. However, the protective immune function was actually weaker than in wild-type mice where a similar number of β-gal-reactive T cells were measured. Furthermore, the protective activity of the plasmid vaccine delivered to C2D mice using the 20-msec conditions was comparable to the activity in wild-type mice despite a significant reduction in the cellular response. These results suggest that the T cells elicited by the two conditions may have distinct phenotypes. A number of publications have demonstrated that CD8+ T cells elicited in the absence of CD4+ T cells exhibit various dysfunctions (40, 41, 42, 43). Most recently, a publication demonstrated that CD8+ T cells primed in the absence of CD4+ T cells expand normally but fail to expand further upon secondary stimulation (41). Thus, it is possible that in our studies the expansion of the CD8+ T cell population following vaccinia challenge is required to control the infection. In the case of the 50-msec pulse in C2D mice, the CD8+ T cells that are present may be overstimulated and fail to expand upon virus challenge whereas the CD8+ T cells in the host immunized using the 20-msec pulse, which are fewer in number, have not lost this capability.

Further refinement of the electroporation conditions will be required to ensure maximal expansion of CD8+ T cell effectors and overcome the observed defects in CD8+ T cell function following priming in the absence of CD4+ T cells. Nonetheless, these results certainly support further investigation of electroporation as a method for delivering plasmid vaccines in CD4+ T cell-deficient hosts with the aim of developing an effective method for immunizing patients with immunodeficiency.

Acknowledgments

We thank Carole Evelegh and Robin Parsons for preparing the plasmids used in these experiments. We are also grateful to Greg Rekas and Robin Persaud for assistance in the viral titrations.

Footnotes

  • ↵1 This work was supported by grants from the Canadian Institutes of Health Research (MOP-42433, to J.L.B.) and, in part, by grants from the Hamilton Health Sciences Corporation and St. Joseph’s Hospital. J.B. was supported by an Rx&D Health Research Foundation/Canadian Institutes of Health Research Career Award in Health Research. Y.H.W. is a Canadian Institutes of Health Research New Investigator.

  • ↵2 Address correspondence to Dr. Jonathan Bramson, Department of Pathology and Molecular Medicine, McMaster University, Room HSC-4H21B, 1200 Main Street West, Hamilton, Ontario, L8N 3Z5, Canada. E-mail address: bramsonj{at}mcmaster.ca

  • ↵3 Abbreviations used in this paper: β-gal, β-galactosidase; C2D, class II-deficient; RLU, relative light unit; SFC, spot-forming cell.

  • Received April 14, 2003.
  • Accepted July 29, 2003.

References

  1. ↵
    Srivastava, I. K., M. A. Liu. 2003. Gene vaccines. Ann. Intern. Med. 138:550.
    OpenUrlCrossRefPubMed
  2. ↵
    Leitner, W. W., H. Ying, N. P. Restifo. 1999. DNA and RNA-based vaccines: principles, progress and prospects. Vaccine 18:765.
    OpenUrlCrossRefPubMed
  3. ↵
    Krieg, A. M.. 2000. The role of CpG motifs in innate immunity. Curr. Opin. Immunol. 12:35.
    OpenUrlCrossRefPubMed
  4. ↵
    Klinman, D. M., G. Yamshchikov, Y. Ishigatsubo. 1997. Contribution of CpG motifs to the immunogenicity of DNA vaccines. J. Immunol. 158:3635.
    OpenUrlAbstract
  5. ↵
    Sato, Y., M. Roman, H. Tighe, D. Lee, M. Corr, M. D. Nguyen, G. J. Silverman, M. Lotz, D. A. Carson, E. Raz. 1996. Immunostimulatory DNA sequences necessary for effective intradermal gene immunization. Science 273:352.
    OpenUrlAbstract
  6. ↵
    Klinman, D. M., K. J. Ishii, D. Verthelyi. 2000. CpG DNA augments the immunogenicity of plasmid DNA vaccines. Curr. Top. Microbiol. Immunol. 247:131.
    OpenUrlPubMed
  7. ↵
    Le, T. P., K. M. Coonan, R. C. Hedstrom, Y. Charoenvit, M. Sedegah, J. E. Epstein, S. Kumar, R. Wang, D. L. Doolan, J. D. Maguire, et al 2000. Safety, tolerability and humoral immune responses after intramuscular administration of a malaria DNA vaccine to healthy adult volunteers. Vaccine 18:1893.
    OpenUrlCrossRefPubMed
  8. ↵
    Calarota, S., G. Bratt, S. Nordlund, J. Hinkula, A. C. Leandersson, E. Sandstrom, B. Wahren. 1998. Cellular cytotoxic response induced by DNA vaccination in HIV-1-infected patients. Lancet 351:1320.
    OpenUrlCrossRefPubMed
  9. ↵
    MacGregor, R. R., J. D. Boyer, K. E. Ugen, K. E. Lacy, S. J. Gluckman, M. L. Bagarazzi, M. A. Chattergoon, Y. Baine, T. J. Higgins, R. B. Ciccarelli, et al 1998. First human trial of a DNA-based vaccine for treatment of human immunodeficiency virus type 1 infection: safety and host response. J. Infect. Dis. 178:92.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Wang, R., D. L. Doolan, T. P. Le, R. C. Hedstrom, K. M. Coonan, Y. Charoenvit, T. R. Jones, P. Hobart, M. Margalith, J. Ng, et al 1998. Induction of antigen-specific cytotoxic T lymphocytes in humans by a malaria DNA vaccine. Science 282:476.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    Somiari, S., J. Glasspool-Malone, J. J. Drabick, R. A. Gilbert, R. Heller, M. J. Jaroszeski, R. W. Malone. 2000. Theory and in vivo application of electroporative gene delivery. Mol. Ther. 2:178.
    OpenUrlCrossRefPubMed
  12. ↵
    Mir, L. M., M. F. Bureau, J. Gehl, R. Rangara, D. Rouy, J. M. Caillaud, P. Delaere, D. Branellec, B. Schwartz, D. Scherman. 1999. High-efficiency gene transfer into skeletal muscle mediated by electric pulses. Proc. Natl. Acad. Sci. USA 96:4262.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    Aihara, H., J. Miyazaki. 1998. Gene transfer into muscle by electroporation in vivo. Nat. Biotechnol. 16:867.
    OpenUrlCrossRefPubMed
  14. ↵
    Babiuk, S., M. E. Baca-Estrada, M. Foldvari, M. Storms, D. Rabussay, G. Widera, L. A. Babiuk. 2002. Electroporation improves the efficacy of DNA vaccines in large animals. Vaccine 20:3399.
    OpenUrlCrossRefPubMed
  15. ↵
    Kalat, M., Z. Kupcu, S. Schuller, D. Zalusky, M. Zehetner, W. Paster, T. Schweighoffer. 2002. In vivo plasmid electroporation induces tumor antigen-specific CD8+ T-cell responses and delays tumor growth in a syngeneic mouse melanoma model. Cancer Res. 62:5489.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    Zucchelli, S., S. Capone, E. Fattori, A. Folgori, A. Di Marco, D. Casimiro, A. J. Simon, R. Laufer, N. La Monica, R. Cortese, A. Nicosia. 2000. Enhancing B- and T-cell immune response to a hepatitis C virus E2 DNA vaccine by intramuscular electrical gene transfer. J. Virol. 74:11598.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    Uno-Furuta, S., S. Tamaki, Y. Takebe, S. Takamura, A. Kamei, G. Kim, I. Kuromatsu, M. Kaito, Y. Adachi, Y. Yasutomi. 2001. Induction of virus-specific cytotoxic T lymphocytes by in vivo electric administration of peptides. Vaccine 19:2190.
    OpenUrlCrossRefPubMed
  18. ↵
    Schoenberger, S. P., R. E. Toes, E. I. van der Voort, R. Offringa, C. J. Melief. 1998. T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature 393:480.
    OpenUrlCrossRefPubMed
  19. ↵
    Ridge, J. P., F. Di Rosa, P. Matzinger. 1998. A conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell. Nature 393:474.
    OpenUrlCrossRefPubMed
  20. ↵
    Bennett, S. R., F. R. Carbone, F. Karamalis, R. A. Flavell, J. F. Miller, W. R. Heath. 1998. Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature 393:478.
    OpenUrlCrossRefPubMed
  21. ↵
    Chan, K., D. J. Lee, A. Schubert, C. M. Tang, B. Crain, S. P. Schoenberger, M. Corr. 2001. The roles of MHC class II, CD40, and B7 costimulation in CTL induction by plasmid DNA. J. Immunol. 166:3061.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    Cho, H. J., K. Takabayashi, P. M. Cheng, M. D. Nguyen, M. Corr, S. Tuck, E. Raz. 2000. Immunostimulatory DNA-based vaccines induce cytotoxic lymphocyte activity by a T-helper cell-independent mechanism. Nat. Biotechnol. 18:509.
    OpenUrlCrossRefPubMed
  23. ↵
    Maecker, H. T., D. T. Umetsu, R. H. DeKruyff, S. Levy. 1998. Cytotoxic T cell responses to DNA vaccination: dependence on antigen presentation via class II MHC. J. Immunol. 161:6532.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Wild, J., M. J. Grusby, R. Schirmbeck, J. Reimann. 1999. Priming MHC-I-restricted cytotoxic T lymphocyte responses to exogenous hepatitis B surface antigen is CD4+ T cell dependent. J. Immunol. 163:1880.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Yoshida, A., T. Nagata, M. Uchijima, Y. Koide. 2001. Protective CTL response is induced in the absence of CD4+ T cells and IFN-γ by gene gun DNA vaccination with a minigene encoding a CTL epitope of Listeria monocytogenes. Vaccine. 19:4297.
    OpenUrlCrossRefPubMed
  26. ↵
    MacGregor, G. R., C. T. Caskey. 1989. Construction of plasmids that express E. coli β-galactosidase in mammalian cells. Nucleic Acids Res. 17:2365.
    OpenUrlFREE Full Text
  27. ↵
    Bramson, J., K. Dayball, C. Evelegh, Y. H. Wan, D. Page, A. Smith. 2003. Enabling topical immunization via microporation: a novel method for pain-free and needle-free delivery of adenovirus-based vaccines. Gene Ther. 10:251.
    OpenUrlCrossRefPubMed
  28. ↵
    Bramson, J. L., K. Dayball, J. R. Hall, J. B. Millar, M. Miller, Y. H. Wan, R. Lin, J. Hiscott. 2003. Super-activated interferon-regulatory factors can enhance plasmid immunization. Vaccine 21:1363.
    OpenUrlCrossRefPubMed
  29. ↵
    Ugen, K. E., S. B. Nyland, J. D. Boyer, C. Vidal, L. Lera, S. Rasheid, M. Chattergoon, M. L. Bagarazzi, R. Ciccarelli, T. Higgins, et al 1998. DNA vaccination with HIV-1 expressing constructs elicits immune responses in humans. Vaccine 16:1818.
    OpenUrlCrossRefPubMed
  30. ↵
    MacGregor, R. R., R. Ginsberg, K. E. Ugen, Y. Baine, C. U. Kang, X. M. Tu, T. Higgins, D. B. Weiner, J. D. Boyer. 2002. T-cell responses induced in normal volunteers immunized with a DNA-based vaccine containing HIV-1 env and rev. AIDS 16:2137.
    OpenUrlCrossRefPubMed
  31. ↵
    Jooss, K., Y. Yang, K. J. Fisher, J. M. Wilson. 1998. Transduction of dendritic cells by DNA viral vectors directs the immune response to transgene products in muscle fibers. J. Virol. 72:4212.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    Ahmed, R., L. D. Butler, L. Bhatti. 1988. T4+ T helper cell function in vivo: differential requirement for induction of antiviral cytotoxic T-cell and antibody responses. J. Virol. 62:2102.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Rahemtulla, A., W. P. Fung-Leung, M. W. Schilham, T. M. Kundig, S. R. Sambhara, A. Narendran, A. Arabian, A. Wakeham, C. J. Paige, R. M. Zinkernagel, et al 1991. Normal development and function of CD8+ cells but markedly decreased helper cell activity in mice lacking CD4. Nature 353:180.
    OpenUrlCrossRefPubMed
  34. ↵
    Weeratna, R. D., T. Wu, S. M. Efler, L. Zhang, H. L. Davis. 2001. Designing gene therapy vectors: avoiding immune responses by using tissue-specific promoters. Gene Ther. 8:1872.
    OpenUrlCrossRefPubMed
  35. ↵
    Wang, R. F.. 2001. The role of MHC class II-restricted tumor antigens and CD4+ T cells in antitumor immunity. Trends Immunol. 22:269.
    OpenUrlCrossRefPubMed
  36. ↵
    Schnell, S., J. W. Young, A. N. Houghton, M. Sadelain. 2000. Retrovirally transduced mouse dendritic cells require CD4+ T cell help to elicit antitumor immunity: implications for the clinical use of dendritic cells. J. Immunol. 164:1243.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    Porgador, A., E. Gilboa. 1995. Bone marrow-generated dendritic cells pulsed with a class I-restricted peptide are potent inducers of cytotoxic T lymphocytes. J. Exp. Med. 182:255.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    Wan, Y., L. Lu, J. L. Bramson, S. Baral, Q. Zhu, A. Pilon, K. Dayball. 2001. Dendritic cell-derived IL-12 is not required for the generation of cytotoxic, IFN-γ-secreting, CD8+ CTL in vivo. J. Immunol. 167:5027.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    Barry, M. E., D. Pinto-Gonzalez, F. M. Orson, G. J. McKenzie, G. R. Petry, M. A. Barry. 1999. Role of endogenous endonucleases and tissue site in transfection and CpG-mediated immune activation after naked DNA injection. Hum. Gene Ther. 10:2461.
    OpenUrlCrossRefPubMed
  40. ↵
    Zajac, A. J., J. N. Blattman, K. Murali-Krishna, D. J. Sourdive, M. Suresh, J. D. Altman, R. Ahmed. 1998. Viral immune evasion due to persistence of activated T cells without effector function. J. Exp. Med. 188:2205.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    Janssen, E. M., E. E. Lemmens, T. Wolfe, U. Christen, M. G. von Herrath, S. P. Schoenberger. 2003. CD4+ T cells are required for secondary expansion and memory in CD8+ T lymphocytes. Nature 421:852.
    OpenUrlCrossRefPubMed
  42. ↵
    Bourgeois, C., H. Veiga-Fernandes, A. M. Joret, B. Rocha, C. Tanchot. 2002. CD8 lethargy in the absence of CD4 help. Eur. J. Immunol. 32:2199.
    OpenUrlCrossRefPubMed
  43. ↵
    Liu, H., S. Andreansky, G. Diaz, T. Hogg, P. C. Doherty. 2002. Reduced functional capacity of CD8+ T cells expanded by post-exposure vaccination of γ-herpesvirus-infected CD4-deficient mice. J. Immunol. 168:3477.
    OpenUrlAbstract/FREE Full Text
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools

Jump to section