Improvement of cytomegalovirus pp65 DNA vaccine efficacy by co-administration of siRNAs targeting BAK and BAX

Abstract

The efficacy of DNA vaccines may be improved by small interfering (si)RNA adjuvants targeting pro-apoptotic genes. The aim of the present study was to investigate the capacity of siRNAs targeting B-cell lymphoma 2 homologous antagonist killer (BAK) and B-cell lymphoma 2-associated X protein (BAX) to improve the efficacy of a cytomegalovirus (CMV) vaccine. BALB/c mice were divided into four groups (n=18 in each): unimmunized and immunized with pcDNA 3.1-pp65 expressing CMV 65 kDa matrix phosphoprotein and BAK + BAX siRNAs, pcDNA 3.1-pp65 and control siRNA, or control pcDNA 3.1 and BAK + BAX siRNAs. Immunizations were performed twice with an interval of 3 weeks. CMV-specific mouse splenocyte interferon (IFN)-γ secretion was assessed by ELISPOT; furthermore, an in vivo cytotoxic T lymphocyte assay was performed 2 weeks after the last immunization. After lethal CMV challenge of the mice, body weight, virus titers in the spleens and salivary glands as well as survival were recorded. The amount of splenocytes secreting IFN-γ in response to CMV pp65 peptides and specific lysis of peptide-pulsed target cells were significantly higher in mice administered pcDNA3.1-pp65 and BAK + BAX siRNAs than those in mice administered pcDNA3.1-pp65 and control siRNA (P<0.05 for each). After the virus challenge, the virus titers in the spleens and salivary glands of mice given pcDNA3.1-pp65 and BAK + BAX siRNAs were significantly lower than those in mice immunized with pcDNA3.1-pp65 and control siRNA (P<0.05 for each). Furthermore, mice immunized with pcDNA 3.1-pp65 and control siRNA or BAK + BAX siRNAs survived for longer, and at 21 days after lethal CMV challenge, 66 and 100% of these mice survived, respectively. These mice also experienced less weight loss compared with mice immunized with pcDNA3.1-pp65 and control siRNA (P<0.05). In conclusion, intradermal administration of siRNAs targeting BAK and BAX improved the efficacy of CMV pp65 DNA vaccine.

Keywords: BAK; BAX; DNA vaccine; cytomegalovirus; cytomegalovirus 65 kDa matrix phosphoprotein; small interfering RNA.

Figure 1.

Splenocyte IFN-γ secretion following immunization with CMV pp65 DNA vaccine combined with or without BAK + BAX siRNAs. Mice were immunized with pcDNA 3.1-pp65 and BAK + BAX siRNAs; pcDNA 3.1-pp65 and control siRNA; pcDNA 3.1 with BAK + BAX siRNAs (negative control); or no vaccine (unimmunized group). Fourteen days after the last immunization, splenocytes were collected from mice and stimulated with 4 µg/ml synthesized CMV pp65 peptide. Splenocyte IFN-γ secretion was assessed by ELISPOT. Values are expressed as the mean ± standard deviation (n=4 in each group). *P<0.05 vs. unimmunized group; ^#P<0.05 vs. negative control; ^ΔP<0.05 vs. pcDNA 3.1-pp65 combined with control siRNA. CMV, cytomegalovirus; IFN, interferon; BAK, B-cell lymphoma 2 homologous antagonist killer; BAX, B-cell lymphoma 2-associated X protein; siRNA, small interfering RNA.

Figure 2.

In vivo CTL responses following immunization with CMV pp65 DNA vaccine with or without BAK + BAX siRNAs. In vivo CTL responses were assessed by measurement of the relative proportion of CFSE_high and CFSE_low cells by flow cytometry. Values are expressed as the mean ± standard deviation. *P<0.05 vs. unimmunized group; ^#P<0.05 vs. negative control; ^ΔP<0.05 vs. pcDNA 3.1-pp65 combined with control siRNA. CTL, cytotoxic T lymphocytes; BAK, B-cell lymphoma 2 homologous antagonist killer; BAX, B-cell lymphoma 2-associated X protein; siRNA, small interfering RNA; CFSE, carboxyfluorescein.

Figure 3.

Body weight changes after CMV challenge of mice. Two weeks after the final immunization, 6 mice in each group were challenged with a lethal dose (3xLD₅₀) of CMV and the body weights were determined within 3 weeks after the challenge. Values are expressed as the mean ± standard deviation. CMV, cytomegalovirus; BAK, B-cell lymphoma 2 homologous antagonist killer; BAX, B-cell lymphoma 2-associated X protein; siRNA, small interfering RNA.