In vivo electroporation restores the low effectiveness of DNA vaccination against HER-2/neu in aging

Abstract

Experimental evidence has been provided that cancer vaccines are less effective at older age than in young adults. In this study, we evaluated the possibility to recover the low effectiveness of DNA immunization against HER-2/neu increasing plasmid uptake by cells from old mice through electroporation with the aim to enhance the activation of specific immune responses. Young and old Balb/c mice received two immunizations with a pCMV-ECDTM DNA plasmid using plasmid intramuscular injection followed by electroporation (IM + E) or plasmid intramuscular injection alone (IM), and successively, they were challenged with syngeneic HER-2/neu overexpressing TUBO cells. Young mice were completely protected whereas less than 60% protection was observed in old mice after IM immunization. IM + E immunization completely protected old mice against a TUBO cell challenge. The protection was associated with increased transgene expression in the site of immunization and with the induction of both humoral and cell-mediated immunity in old mice. We conclude that the effectiveness of anticancer DNA vaccination in old ages may be improved increasing plasmid uptake and transgene expression through electroporation, suggesting the relevant role of the first steps of the immunization process in the success of cancer vaccines at older age.

Fig. 1

Kinetics of expression of HER-2/neu mRNA in plasmid DNA injection site. Total RNA was isolated at 3, 6, 9, and 24 h from DNA immunization from the tibial muscle and analyzed for the expression of HER-2/neu mRNA through real-time RT-PCR. Values were normalized with β-actin. Data shown are representative of one of the two independent experiments

Fig. 2

Effect of immunization with pCMV ECDTM plasmid on tumor incidence (left) or mean tumor diameter (right) in young and old Balb/c mice challenged with syngeneic TUBO cells. Young and old mice received two immunizations with pCMV ECDTM DNA plasmid or with pCMV and, 2 weeks ago, were challenged with TUBO cells. Eight mice for each group were used for each experiment. Data shown are representative of one of the two independent experiments

Fig. 3

Effect of IM immunization with increased pCMV ECDTM plasmid concentrations on tumor incidence in old Balb/c mice challenged with syngeneic TUBO cells. Old mice received two immunizations with 100, 200, or 300 μg of pCMV ECDTM DNA plasmid and, 2 weeks ago, were challenged with TUBO cells

Fig. 4

Presence of anti p185^neu Abs in the sera of young and old mice after vaccination with pCMV ECDTM plasmid. Sera from mice immunized with pCMV ECDTM plasmid by IM or IM + E or with pCMV were collected before TUBO cell challenge. Specific p185 Sbp was evaluated by flow cytometry after indirect immunofluorescence and calculated as reported in Mat and Methods. Data shown are representative of one of the three independent experiments. *P at least <0.05 versus HER-2/neu IM old value; **P at least <0.05 versus respective young value; ^§ P < 0.05 versus HER-2/neu IM young value

Fig. 5

Cytotoxicity of spleen lymphocytes from young and old mice after vaccination with pCMV ECDTM plasmid. The cytotoxicity of spleen lymphocytes from mice immunized with pCMV ECDTM plasmid by IM or IM + E or with pCMV was evaluated after in vitro incubation with mytomicin-treated TUBO cells as reported in “Materials and methods.” The cytotoxicity was evaluated through an in vitro cytotoxic assay against TUBO tumor cells (top) or through the evaluation of CD107 on CD8⁺ T cells (bottom). Data shown are representative of one of the three independent experiments. Top *P al least < 0.05 versus control or HER-2/neu IM young values; **P at least <0.05 versus control or HER-2/neu IM old values; ^§ P < 0.05 versus young control; bottom *P at least <0.05 versus control young value; **P at least <0.05 versus control old value

Fig. 6

In vivo cytotoxicity after vaccination with pCMV ECDTM plasmid in young and old mice. Plasmid-immunized mice were injected i.v with 5 × 10⁶ splenocytes (target cells) from naïve transgenic mice pulsed with either HER-2 immunodominant or irrelevant peptides and stained with CFSE as detailed in M&M. Sixteen hours later, the splenocytes of immunized mice were analyzed by flow cytometry. The value in each panel represents the percentage of CFSE high and CFSE low target cells remaining in the spleen in a representative sample. The histogram shows the mean ± SD of two independent experiments