Reporter gene expression is greatly enhanced following EP-mediated delivery of pDNA.

Vectors encoding LacZ or GFP were injected into the left tibialis cranialis muscle of three woodchucks using the EP device with the electrode array, followed by electrical stimulation, and then into the right tibialis cranialis muscle of each woodchuck by HI. Four days later, all three woodchucks receiving the LacZ-expressing vector, pCMV-beta, by EP had distinctive blue coloration of the muscle tissue starting within 20 min of the X-gal detection reaction, which was indicative of marked LacZ expression, and it became maximal by 24 h in the reaction (Fig. 1A, bottom). In contrast, among woodchucks receiving pCMV-beta by non-EP injection, only one had a faint blue discoloration by 24 h (Fig. 1A, top), while muscles from the other two woodchucks had no evidence of LacZ expression at this time. Similarly, using the GFP expressing vector, the muscles of all three woodchucks receiving pEGFP by EP had the characteristic green fluorescent signal indicative of marked GFP expression by fluorescence microscopy (Fig. 1B, bottom). No significant fluorescent green signal was detected in muscles of the three woodchucks receiving pEGFP by non-EP injection (Fig. 1B, top). In addition, the delivery of a plasmid encoding the reporter gene, SEAP, with or without EP resulted in significantly higher serum levels with EP delivery (Fig. 1C). These results validate that the EP delivery of pDNA encoding both intracellular and secreted markers into the woodchuck tibialis cranialis muscle results in much more efficient gene expression than delivery by HI alone.

Fig. 1.

Fig. 1. Expression of reporter genes following non-EP or EP injection of pDNA into the tibialis cranialis muscle of woodchucks. (A) LacZ expression. Three woodchucks received a single dose of vector pCMV-beta (0.5 mg pDNA in 0.5 ml PBS) into the right tibialis cranialis muscle by HI alone (non-EP; top) and the same vector dose into the left tibialis cranialis muscle by EP injection (EP; bottom). LacZ expression in muscle tissue surrounding the injection sites was determined 4 days later by immunohistochemistry; results from one subject are shown. (B) GFP expression. Three woodchucks received a single dose of the vector pEGFP (0.5 mg pDNA in 0.5 ml PBS) into the right tibialis cranialis muscle by HI alone (non-EP; top) and the same vector dose into the left tibialis cranialis muscle by EP injection (EP; bottom). GFP expression in muscle tissue surrounding the injection sites was determined 4 days later by fluorescence microscopy; results from one representative subject are shown. Arrows in the bottom panel indicate the muscle tissue with strong fluorescent green signal. (C) SEAP expression. Woodchucks received a single dose of vector pgWIZ-SEAP (0.5 mg pDNA in 0.5 ml PBS) intramuscularly by HI or with EP (three animals per group). SEAP activity was determined as described in Materials and Methods. The arrow indicates the single dose of vector pgWIZ-SEAP administered at T0. The single asterisks indicate that the geometric mean serum SEAP activity levels in woodchucks that received vector pgWIZ-SEAP by EP were statistically significantly different at days 2 and 4 from those in woodchucks that received the same vector by non-EP injection (P < 0.05). Vertical lines (error bars) denote standard deviations.

WHsAg DNA vaccination by EP elicits anti-WHs antibody responses in woodchucks similar to those elicited by immunization with conventional WHsAg vaccine.

In the EP versus non-EP immunogenicity study in WHV-naïve woodchucks using pDNA expressing WHsAg, the initial immunization at T0 induced anti-WHs within 4 weeks in two of five (40%) and in four of five (80%) woodchucks in the EP low-dose and EP high-dose groups, respectively; each had transient low-level anti-WHs responses with titers ranging between 102 and 231 (Fig. 2). At the same time, anti-WHs was evident in one of five (20%) and in two of five (40%) woodchucks, respectively, following non-EP injection of WHsAg-pDNA (non-EP high-dose) and standard injection of conventional WHsAg vaccine (positive vaccine control). Woodchucks from the non-EP negative vaccine control group that received saline placebo had no detectable anti-WHs at any time throughout the study period. Thus, the higher dose of pIMS-310 given by EP rapidly induced detectable anti-WHs in more woodchucks than did the same construct dose by non-EP injection; however, at these early times, the difference in anti-WHs GMTs between these two groups was not statistically significant (P > 0.05).

Fig. 2.

Fig. 2. Serum antibody response to WHsAg following non-EP or EP injection of pIMS-310 into the tibialis cranialis muscle of WHV-negative woodchucks. Groups of five animals received administrations at weeks 0, 4, and 8. (A) Non-EP negative vaccine control group. Woodchucks received saline (0.5 ml PBS) in one muscle site by HI injection alone. Anti-WHs titers were determined by ELISA (assay cutoff value, ≥101 U/ml). (B) Positive vaccine control group. Woodchucks received a conventional protein vaccine (10 μg of alum-adsorbed WHsAg in 0.5 ml PBS) in one muscle site by HI. (C) Non-EP high-dose group. Woodchucks received pIMS-310 (1.0 mg in 0.5 ml PBS) in two separate muscle sites (total of 2.0 mg pDNA per dose) by HI alone. (D) EP low-dose group. Woodchucks received pIMS-310 (0.5 mg in 0.5 ml PBS) in one muscle site (total of 0.5 mg pDNA per dose) by EP. (E) EP high-dose group. Woodchucks received pIMS-310 (1.0 mg in 0.5 ml PBS) in two separate muscle sites (total of 2.0 mg pDNA per dose) by EP. (F) Comparison of anti-WHs GMTs between experimental groups. Arrows indicate the three doses of saline, conventional protein vaccine, or pIMS-310 administered at weeks 0, 4, and 8. Anti-WHs GMTs in the non-EP negative vaccine control group were significantly lower than those in the non-EP high-dose group at week 9, in the EP high-dose group between weeks 6 and 14, and in the positive vaccine control group between weeks 5 and 14 (P < 0.05). Anti-WHs GMTs in the non-EP high-dose group were significantly lower than those in the EP high-dose group between weeks 11 and 14 and in the positive vaccine control group at week 6 and again between weeks 9 and 14 (P < 0.05). Anti-WHs GMTs in the EP low-dose group were significantly lower than those in the EP high-dose group at weeks 12 and 14, in the positive vaccine control group at week 9, and again between weeks 11 and 14 (P < 0.05). The anti-WHs GMT in the EP high-dose group was significantly lower than that in the positive vaccine control group at week 9 (P < 0.05).
Following the second immunization at week 4, all five of the woodchucks (100%) in the EP high-dose group remained or became positive for anti-WHs, and four of five (80%) woodchucks in the EP low-dose and non-EP high-dose groups remained or became anti-WHs positive (Fig. 2). Variation was noted in the magnitude of anti-WHs response among individual woodchucks in each group receiving pDNA as well as in the positive vaccine control group (with 100% now anti-WHs positive) (Fig. 2). Among woodchucks receiving WHsAg-pDNA, the peak GMTs for anti-WHs during the observation period were highest in the EP high-dose group at 7 weeks postimmunization (pi) (333 U/ml), followed by that in the EP low-dose (230 U/ml; week 6) and non-EP high-dose groups (154 U/ml, week 5). The anti-WHs GMT in the positive vaccine control group (463 U/ml; week 6) was higher than that in the EP high-dose group but waned more thereafter, and at 8 weeks pi the antibody response was similar to slightly lower than that in the EP high-dose group (235 versus 248 U/ml; P > 0.05).
Following the third immunization at week 8, anti-WHs titers increased again in all (100%) woodchucks from the EP high-dose group, with a maximum titer at 12 weeks pi (409 U/ml), which waned thereafter (Fig. 2). Anti-WHs titers increased and then waned in three of five (60%) woodchucks from the EP low-dose group (note that one of these woodchucks had no detectable anti-WHs throughout the study) and in four of five (80%) woodchucks from the non-EP high-dose group. Based on the maximum anti-WHs responses for non-EP high-dose and EP low-dose groups at 5 weeks (154 U/ml) and 6 weeks pi (230 U/ml), respectively, the anti-WHs GMTs for the EP high-dose group were significantly higher (P P
As indicated above, the kinetics of anti-WHs development differed among the immunized groups, with variable peak GMT anti-WHs responses occurring anywhere between 5 and 12 weeks pi and with these ranging between 154 and 841 U/ml (Fig. 2). The anti-WHs response waned thereafter in all woodchucks, and at the end of the study (week 14), in order of magnitude, these were highest in the positive vaccine control group (334 U/ml) and comparable to the EP high-dose group (288 U/ml), followed by the EP low-dose (126 U/ml) and non-EP high-dose groups (113 U/ml). None of the woodchucks from any of the groups had detectable WHsAg or anti-WHc in serum (data not shown).

The administration of pIMS-310 to woodchucks by non-EP or EP injection was safe based on comparable patterns of body weight changes among these groups and compared to those in the non-EP negative vaccine control and positive vaccine control groups (data not shown). No hepatic flare reactions were observed in immunized groups based on the assay of liver enzyme activities in blood and complete blood counts (data not shown), and based on these measurements it was concluded that there was no evident toxicity related to DNA vaccine administration by non-EP or EP injection.

The results described above demonstrate that the EP delivery of a high dose of pDNA expressing WHsAg clearly was superior to the same vector dose administered by non-EP injection in regard to the magnitude and sustainability of the induced antibody response. Furthermore, responses to EP administration were dose dependent in the range tested, and at the higher dose the anti-WHs response pattern was comparable to that observed following immunization with a subunit WHsAg vaccine. Both types of vaccine (high dose of EP-administered pDNA-WHsAg and conventional WHsAg protein) elicited anti-WHs antibodies in all woodchucks with similar titers and duration throughout most of the study.

WHsAg DNA vaccination by EP induces significant T-cell proliferative responses in woodchucks similar to immunization with conventional WHsAg vaccine.

Based on the unique potential for the use of pDNA vaccines as an additional therapeutic modality for chronic HBV infection, vaccine potency and immunogenicity was judged here not simply by anti-WHs titer and duration (which is indeed important) but also on the balance of these anti-WHs responses relative to other immune response components, such as the magnitude and type of T-cell responses elicited (Th1 versus Th2), as typified previously in murine species. Regarding overall T-cell activation following immunizations, it was found that in vitro T-cell responses to purified WHsAg protein particles were correlated generally with the anti-WHs responses (Fig. 3). Prior to immunization at T0, T-cell responses were undetectable with SIs below the assay cutoff (≥3.1). Although serum anti-WHs antibodies were detected in several woodchucks early on, corresponding T-cell responses were not evident in the first week following the initial immunization with pIMS-310 or conventional WHsAg vaccine (since WHsAg is considered a T-cell-dependent antigen, like HBsAg, the apparent dissociation described above likely results because the anti-WHs ELISA is more sensitive than the T-cell proliferation assay being used).

Fig. 3.

Fig. 3. Group responder rates for T-cell responses to WHsAg and selected WHs peptides. GRRs for WHsAg and for WHs peptides S1, S7/8, S11, S12/13, S18, and S21 are presented in the top graph. The GRRs for the polyclonal activator ConA, used as a positive control for cell proliferation, are displayed for comparison. The number of woodchucks was five in each group. T-cell response was positive if the SI was ≥3.1. The single asterisks indicate that the GRR for the EP high-dose group was statistically different from those for the non-EP high-dose and non-EP negative vaccine control groups for WHsAg and WHs peptides S18 and S21 at 12 weeks pi (P < 0.05). The double asterisk indicates that the GRR for the positive vaccine control group was statistically different from those for the non-EP high-dose and non-EP negative vaccine control groups for WHs peptide S18 at 12 weeks pi (P < 0.05). The relative positions of the WHs peptides in the pre-S1, pre-S2, and S regions of the viral envelope protein are displayed in the bottom graph. The conventional WHsAg vaccine (protein vaccine) consists of 22-nm subviral particles that contain the L, M, and S proteins (pre-S1, pre-S2, and S regions of WHsAg). Vector pIMS-310 (DNA vaccine) contains a DNA sequence that encodes only the M and S proteins (pre-S2 and S regions of WHsAg). S1, N terminus of WHsAg; S21, C terminus of WHsAg.
Following the second immunization (week 4), one of five (20%) and two of five (40%) woodchucks from the EP low-dose and EP high-dose groups, respectively, had detectable T-cell responses to WHsAg (SIs ≥ 3.1) (Fig. 3). In contrast, none (0%) of the woodchucks from the non-EP high-dose group had detectable T-cell responses at this time. Three of five (60%) woodchucks from the positive vaccine control group had detectable T-cell responses to WHsAg at this time, whereas no responses were evident in the five (0%) woodchucks from the non-EP negative vaccine control group throughout the study. Thus, the T-cell responses detected were indeed specific to the WHsAg expressed from pIMS-310 or present within the protein vaccine. One week following the third immunization (week 8), T-cell responses to WHsAg were evident in one of five (20%) woodchucks each from the non-EP high-dose and EP low-dose groups, in two of five (40%) woodchucks from the EP-high-dose group, and in three of five (60%) woodchucks from the positive vaccine control group (Fig. 3). By week 12, four of five (80%) woodchucks from the EP high-dose group and two of five (20%) woodchucks each from the EP low-dose and positive vaccine control groups had positive T-cell responses to WHsAg. T-cell responses remained or became undetectable in all (0%) woodchucks from the non-EP high-dose group. Note that the observed animal- and group-associated variability, and the differences in WHsAg-specific T-cell responses, were not a result of individual variation in overall PBMC responsiveness, because overall proliferation to stimulation with polyclonal lymphocyte activators such as ConA (Fig. 3) (and recombinant human IL-2 for T cells and lipopolysaccharide for B cells; data not shown) were the same for all groups, with 100% of animals responding robustly and comparably at each time point.
T-cell responses of immunized woodchucks to WHsAg were analyzed further for fine specificity using selected WHs peptides that represent important protective epitopes (34) within the pre-S1, pre-S2, and S regions of the viral envelope protein (Fig. 3). None of the peptides recalled T-cell responses in any of the woodchucks prior to immunization or immediately following the first immunization, and woodchucks in the non-EP negative vaccine control group remained negative for responses throughout the study period. Woodchucks from the positive vaccine control group developed T-cell responses to WHs peptides S1 and S7/8 following the second immunization at GRRs (for week 5) between 20 and 60%. Both peptides correspond to sequences that are located within the pre-S1 region (L protein) of the WHV envelope (Fig. 3). The conventional WHsAg vaccine consists of 22-nm subviral particles that do contain small amounts of the L protein, whereas vector pIMS-310 contains a DNA sequence encoding only the M and S proteins (pre-S2 and S regions of WHsAg). Also following the second immunization, WHs peptide S11 detected T-cell responses in woodchucks from the EP high-dose and positive vaccine control groups at the same GRR (40%). T-cell responses to this peptide were absent from woodchucks from the non-EP high-dose and EP low-dose groups. WHs peptides S12/13, S18, and S21 induced T-cell responses in 40, 60, and 40%, respectively, of woodchucks each from the EP high-dose and positive vaccine control groups. Although T-cell responses to these peptides were observed occasionally in woodchucks from the EP low-dose group, the overall responder rates were lower; i.e., WHs peptides S12/13 and S21 recalled T-cell responses in 20% of woodchucks, and S18 in 40% of woodchucks, from this group. T-cell responses to these three peptides were absent from woodchucks from the non-EP high-dose group.

Following the third immunization (week 8), additional woodchucks in the positive vaccine control group developed T-cell responses to WHs peptides S11, S12/13, S18, and S21, with GRRs (at week 9) of 60, 60, 100, and 60%, respectively. T-cell responses in woodchucks from the EP high-dose group indicated GRRs of 40, 40, and 60% for WHs peptides S11, S12/13, and S21, respectively. Woodchucks from the EP low-dose group also had T-cell responses to these peptides but with lower GRRs than those observed for the EP high-dose group, ranging between 20 and 40%. WHs peptides S11, S18, and S21 recalled T-cell responses in one of five (20%) woodchucks from the non-EP high-dose group but no T-cell responses to S12/13. By the end of the study (week 12), T-cell responses to WHs peptides based on GRRs increased slightly in the EP low-dose and EP high-dose groups. GRRs for S18 and S21 finished at 80% in the EP high-dose group and was significantly higher than that in the non-EP high-dose group (0%) (P < 0.05). GRRs for T-cell responses to WHs peptides S11 and S12/13 achieved 60% in the EP high-dose group. The GRRs for the four peptides S11, S12/13, S18, and S21 generally were higher in the EP high-dose group than in the positive vaccine control group (range, 40 to 60%). Although woodchucks from the EP low-dose group had lower GRRs than the EP high-dose group, they were in fact comparable overall to those in the positive vaccine control group.

WHsAg DNA vaccination by EP results in significant expansion of WHsAg-specific CD4+ and CD8+ leukocytes with increased Th1 and decreased Th2 cytokine expression compared to that of conventional WHsAg vaccine.

WHsAg-specific T-cell proliferation was significant following both EP pDNA- and protein-based immunizations. These responses were further dissected in terms of T-cell function (Th and CTL) and Th cell skew (Th1 versus Th2). Accordingly, the expression of mRNAs for leukocyte surface markers (CD4 and CD8), Th1 cytokines (IFN-γ and TNF-α), and Th2 cytokines (IL-4 and IL-10) was measured to study the relative expansion of CD4 and CD8 leukocytes and the balance of Th1/Th2 immune responses. In the woodchuck model, this is accomplished by measuring increases in the expression of these mRNAs in PBMCs stimulated in vitro with WHsAg, since reagents are not available to evaluate all of these markers at the protein level. Increases in mRNA expression in immunized woodchucks were discerned first relative to mRNA expression in unstimulated PBMCs from the immunized woodchucks and then controlled further relative to the values for both unstimulated and WHsAg-stimulated PBMCs from the non-EP negative vaccine control group; this group provided relevant baseline measurements at each time point of the study, and overall they showed no evidence of increased mRNA markers at any time during the study period. WHsAg-specific increases were indicated by a 3.1-fold increase in mRNA expression from unstimulated PBMCs (an FI of ≥3.1 was considered an increase, i.e., a positive response).

Prior to immunizations, WHsAg-induced increases in the expression of leukocyte surface marker and cytokine mRNAs were absent in all woodchucks (FIs ≤ 3.1) (Fig. 4). Following the second immunization (week 4), the WHsAg-stimulated samples at week 5 for one of five (20%) woodchucks each from the EP low-dose, EP high-dose, and positive vaccine control groups had increased (positive) IFN-γ mRNA expression (FIs ≥ 3.1) (Fig. 4). CD4 mRNA was increased in one of five (20%) and three of five (60%) woodchucks, respectively, from the EP high-dose and the positive vaccine control groups. Increases in CD8 mRNA and mRNA for other cytokines were not evident at this time point. Following the third immunization (week 8), the WHsAg-stimulated samples at week 9 for two of five (40%) woodchucks from the EP low-dose group and for three of five (60%) woodchucks each from the EP high-dose and positive vaccine control groups had increased IFN-γ mRNA. Interestingly, the same three of five (60%) woodchucks from the EP high-dose group with increases in IFN-γ mRNA also had increased TNF-α mRNA, and two of them (40%) had increased CD8 mRNA. In the positive vaccine control group, one of five (20%) had increased CD8 mRNA and three of five (60%) had increased TNF-α mRNA. Woodchucks in the other groups had no WHsAg-specific increases in CD8 or Th1 mRNAs. Three of five (60%) woodchucks from the EP high-dose and positive vaccine control groups had WHsAg-specific increases in CD4 mRNA, whereas one of five (20%) woodchucks from the non-EP high-dose and EP low-dose groups had WHsAg-specific increases in CD4 mRNA. Regarding Th2 cytokines in WHsAg-stimulated cultures, increased IL-4 mRNA was observed in one of five (20%) woodchucks each from the EP high-dose and positive vaccine control groups. Increased IL-10 mRNA was observed in four of five (80%) woodchucks from the positive vaccine control group but only one of five (20%) woodchucks from both the EP and non-EP high-dose groups.

Fig. 4.

Fig. 4. Group responder rates for leukocyte surface marker and Th1/Th2 cytokine mRNA expression. GRRs for CD4, CD8, IFN-γ, TNF-α, IL-4, and IL-10 mRNA expression are presented. The number of woodchucks was five in each group. Expression was considered positive if the FI was ≥3.1. The single asterisks indicate that the GRRs for the non-EP negative vaccine control group were statistically different from those for the EP high-dose group for CD8, IFN-γ, TNF-α, and CD4 mRNA expression at 12 weeks pi and from those for the positive vaccine control group for IL-4 mRNA expression at 12 weeks pi and for IL-10 mRNA expression at 9 and 12 weeks pi (P < 0.05). The double asterisks indicate that the GRRs for the non-EP high-dose group were statistically different from those for the EP high-dose group for IFN-γ, TNF-α, and CD4 mRNA expression at 12 weeks pi and from those for the positive vaccine control group for IL-4 and IL-10 mRNA expression at 12 weeks pi (P < 0.05). The triple asterisks indicate that the GRRs for the EP low-dose group were statistically different from those for the positive vaccine control group for IL-10 mRNA expression at 9 and 12 weeks pi (P < 0.05). The quadruple asterisks indicate that the GRR for the EP high-dose group was statistically different from that for the positive vaccine control group for IL-10 mRNA expression at 12 weeks pi (P < 0.05).

By the end of the study, WHsAg-stimulated samples at week 12 for two of five (40%) woodchucks from the positive vaccine control group had increased CD8, IFN-γ, and TNF-α mRNAs; one of four (25%), two of five (40%), and four of five (80%) woodchucks, respectively, from the non-EP high-dose, EP low-dose, and EP high-dose groups had increased CD8 mRNA. IFN-γ mRNA GRRs in the non-EP high-dose, EP low-dose, and EP high-dose groups were 25, 40, and 100%, respectively, and those for TNF-α mRNA were 0, 40, and 100%, respectively. The difference in GRRs for Th1 cytokine expression was statistically significant between the EP high-dose and non-EP high-dose groups (P < 0.05). The GRRs for the positive vaccine control and non-EP high-dose groups were not significantly different for CD8, IFN-γ, and TNF-α mRNAs. None of four (0%), two of five (40%), and five of five (100%) woodchucks from the non-EP high-dose, EP low-dose, and EP high-dose groups, respectively, had WHsAg-specific increases in CD4 mRNA at this time point, as did three of five (60%) woodchucks from the positive vaccine control group (P < 0.05; EP high-dose group versus non-EP high-dose group). Increased IL-4 mRNA was not evident in woodchucks from the non-EP high-dose group (0%) but was evident in one of five (20%) woodchucks from the EP low-dose and EP high-dose groups. In contrast, WHsAg-stimulated PBMCs from four of five (80%) woodchucks from the positive vaccine control group had increased IL-4 mRNA (P < 0.05; positive vaccine control group versus non-EP high-dose group). In addition, stimulated PBMCs from all five (100%) woodchucks from the positive vaccine control group had increased IL-10 mRNA, while only one of five (20%) woodchucks in the EP low-dose and EP high-dose groups had increased IL-10 mRNA, and there was no increased IL-10 mRNA in woodchucks (0%) from the non-EP high-dose group (P < 0.05; positive vaccine control versus all pDNA groups). These results indicate a greater Th2 skew in the WHsAg-specific T-cell responses in woodchucks receiving the subunit WHsAg vaccine. Overall, by the end of the study, woodchucks from the EP high-dose group had improved T-cell expression of CD8, IFN-γ, and TNF-α mRNAs in response to stimulation with WHsAg, thus indicating a greater Th1 skew in responses of woodchucks receiving EP immunizations with WHsAg-pDNA.



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