Protective efficacy of a DNA influenza virus vaccine is markedly increased by the coadministration of a Schiff base-forming drug

Abstract

Effective vaccination against heterologous influenza virus infection remains elusive. Immunization with plasmid DNA (pDNA) expressing conserved genes from influenza virus is a promising approach to achieve cross-variant protection. However, despite having been described for more than a decade, pDNA vaccination still requires further optimization to be applied clinically as a standard vaccination approach. We have recently described a simple and efficient approach to enhance pDNA immunization, based on the use of tucaresol, a Schiff base-forming drug. In this report we have tested the ability of this drug to increase the protection conferred by pDNA vaccination against influenza virus infection. Our results demonstrate that a significant protection was achieved in two strains of mice by using the combination of pDNA and tucaresol. This protection was associated with an elevated humoral and cellular response and a switch in the type of the T helper cell (Th) immune response from type 2 to type 1. This vaccine combination represents a promising strategy for designing a clinical study for the protection from influenza and similar infections.

FIG. 1.

Tucaresol enhances the protective efficacy of HA- and P-NP-based pDNA vaccines against influenza virus infection. C57BL/6 mice were immunized twice with a gene gun and then challenged intranasally with a LD₅₀. Mice from groups treated with tucaresol received 1 mg of tucaresol subcutaneously 24 h after pDNA immunization. (A) Percent survival among mice after virus challenge. (B to F) Body weight (grams) after virus challenge. Dead mice are indicated by 0-g weight. Numbers above the abscissa represent the number of live mice out of the total number of mice per group. P equals 0.039 for P+Tuc compared to either P-NP+Tuc or P-HA+Tuc vaccination, and P is >0.05 for all other comparisons.

FIG. 2.

Tucaresol enhances the protective efficacy of P-NP-based pDNA vaccines against influenza virus infection in BALB/c mice. Mice were immunized twice with a gene gun and then challenged intranasally with a LD₅₀. Mice from groups treated with tucaresol received 1 mg of tucaresol subcutaneously 24 h after pDNA immunization. (A) Percent survival after virus challenge. (B to D) Body weight (grams) after virus challenge. Mouse death is indicated by 0-g weight. Numbers above the abscissa represent the number of live mice out of the total number of mice per group. P is <0.001 for control compared to P-NP+Tuc, P equals 0.017 for P-NP+Tuc compared to P-NP vaccination, and P equals 0.23 for control compared to P-NP vaccination.

FIG. 3.

Marked enhancement of influenza virus-NP-specific cytotoxicity is induced by administrating tucaresol and P-NP pDNA vaccine. Effector cells are derived from splenocytes of mice immunized with tucaresol only, P-NP, or P-NP+Tuc. Four weeks after the last immunization, pooled splenocytes from five mice of each immunization group were pulsed with the NP 8.147 peptide or the control peptide and cultured as described in Materials and Methods for 6 days. Thereafter, their cytotoxic activity was measured in a standard ⁵¹Cr release assay at the indicated effector-to-target ratios using P815 cells as targets.

FIG. 4.

Tucaresol enhances the production of Th1-associated antibody response and inverts the ratio of Th1 to Th2 induced by pDNA gene gun immunization. (A) The ratio of IgG2a to IgG1 was determined by specific ELISA and measured for each mouse. The concentration of anti-NP-specific antibodies was measured by ELISA at 405-nm optical density. Isotype-specific secondary antibodies were used to detect IgG2a (B) and IgG1 (C) concentrations in sera. Each symbol represents one mouse. Sera from 10 mice were tested per each group. P values are indicated in the upper right quadrant of each panel.