Engineering N-glycosylation mutations in IL-12 enhances sustained cytotoxic T lymphocyte responses for DNA immunization

Abstract

Interleukin-12 (IL-12), consisting of p40 and p35 subunits, produces both p70 heterodimer and free p40. p70 is essential for the induction of T-helper 1 (Th1) and cytotoxic T-cell (CTL) immunity, whereas p40 inhibits p70-mediated function. Here, we found that mutations introduced into N-glycosylation sites (N220 of murine p40 and N222 of human p40) reduced secretion of p40 but not p70. Co-immunization of N220 mutant mIL-12 gene with hepatitis C virus (HCV) E2 DNA significantly enhanced long-term E2-specific CD8+ T-cell response and protection against tumor challenge compared with that of wild type. Our results indicate that the ratio of p70 to p40 is important for generating sustained long-term cell-mediated immunity. Thus, the mutant IL-12 could be utilized for the development of DNA vaccines as an adjuvant for the generation of long-term memory T-cell responses.

Therapeutic effect of DNA vaccines combined with chemotherapy in a latent infection model after aerosol infection of mice with Mycobacterium tuberculosis

“…We previously demonstrated that IL-12N220L DNA, as a prophylactic vaccine, can significantly protect immunized mice against tumor challenge after DNA immunization, compared with wild-type IL-12 DNA immunization. 26 Based on these two studies, we suggest that IL-12N220L DNA could be used as a more effective vaccine than wild-type IL-12 DNA in both therapeutic and prophylactic modes.…”

“…26 Briefly, pGX10, DNA vaccine vector, was generated by replacing the ampicillin resistance gene of pTV2 46 with the kanamycin resistance gene. pGX10 is composed of a CMV promoter, TPL, simian virus 40 (SV40) late polyA tail, and an SV40 enhancer, linked in tandem.…”

“…For the generation of mp40-N220L construct, asparagines residue at Asn220 of mp40, Nglycosylation site of mp40, was substituted with leucine residue by site-directed mutagenesis using PCR, as described previously. 26 The region of mp40 in pGX10-IL-12 was replaced with mp40-N220L to generate the murine IL-12 mutant construct, pGX10-mp35/IRES/ mp40-N220L (pGX10-IL-12N220L).…”

“…IL-12N220L is N-glycosylation mutant at Asn220 of mouse IL-12p40, and leads to a reduced secretion of the p40 subunit, a natural antagonist of IL-12. 26 Coimmunization of IL-12N220L DNA with hepatitis C virus (HCV) E2 DNA was reported to significantly enhance long-term E2-specific CTL response and IFN-g-producing CD8 + T-cell response compared to wild-type IL-12 DNA. 26 Here, we found that the Ag85A or IL-12N220L DNA vaccination when combined with chemotherapy reduced M. tuberculosis reactivation, compared with chemotherapy alone.…”

“…26 Coimmunization of IL-12N220L DNA with hepatitis C virus (HCV) E2 DNA was reported to significantly enhance long-term E2-specific CTL response and IFN-g-producing CD8 + T-cell response compared to wild-type IL-12 DNA. 26 Here, we found that the Ag85A or IL-12N220L DNA vaccination when combined with chemotherapy reduced M. tuberculosis reactivation, compared with chemotherapy alone. In addition, Ag85A DNA vaccine showed a better therapeutic effect than IL-12N220L DNA vaccine, suggesting the potential use of Ag85A DNA vaccine in combination with chemotherapy as a therapeutic agent against TB for the prevention of TB reactivation as well as for the effective cure of TB in humans.…”