Intranasal immunization with recombinant Lactococcus lactis secreting murine interleukin-12 enhances antigen-specific Th1 cytokine production

Abstract

Interleukin-12 (IL-12), a heterodimeric cytokine, plays an important role in cellular immunity to several bacterial, viral, and parasitic infections and has adjuvant activity when it is codelivered with DNA vaccines. IL-12 has also been used with success in cancer immunotherapy treatments. However, systemic IL-12 therapy has been limited by high levels of toxicity. We describe here inducible expression and secretion of IL-12 in the food-grade lactic acid bacterium Lactococcus lactis. IL-12 was expressed as two separate polypeptides (p35-p40) or as a single recombinant polypeptide (scIL-12). The biological activity of IL-12 produced by the recombinant L. lactis strain was confirmed in vitro by its ability to induce gamma interferon (IFN-gamma) production by mouse splenocytes. Local administration of IL-12-producing strains at the intranasal mucosal surface resulted in IFN-gamma production in mice. The activity was greater with the single polypeptide scIL-12. An antigen-specific cellular response (i.e., secretion of Th1 cytokines, IL-2, and IFN-gamma) elicited by a recombinant L. lactis strain displaying a cell wall-anchored human papillomavirus type 16 E7 antigen was dramatically increased by coadministration with an L. lactis strain secreting IL-12 protein. Our data show that IL-12 is produced and secreted in an active form by L. lactis and that the strategy which we describe can be used to enhance an antigen-specific immune response and to stimulate local mucosal immunity.

FIG. 1.

Site-specific mutagenesis. The p35 intron was eliminated by site-directed mutagenesis. Briefly, we inserted a BamHI restriction site (underlined) in the three codons (encoding Arg, Gly, Ser) to the right of the intron. Primers were designed to PCR amplify two fragments of p35 that were joined at the BamHI site, such that p35 was reconstituted but intron free.

FIG. 2.

Expression cassettes to produce and secrete IL-12 in L. lactis: schematic structures of p35 and p40 subunits and scIL-12 cassette expressed under the lactococcal P_nisA promoter and carried by the plasmids indicated. For details concerning plasmid construction, see the text and Table 1. The arrows indicate the presence of the nisin-inducible promoter (P_nisA); the solid vertical bars indicate the RBS of the usp45 gene (bar 1 for p35) or of the exp4 gene (bar 2 for p40); the dark gray bars indicate the SP of the usp45 gene; the light gray bar indicates the SP of the exp4 gene (48); the cross-hatched bar indicates the p35 mature coding sequence; the dotted bar indicates the p40 mature coding sequence; the open bar indicates the scIL-12 coding sequence; and the stem-loop symbols indicate trpA transcription terminators (not to scale).

FIG. 3.

Production of IL-12 by recombinant L. lactis. IL-12 production was analyzed by immunoblotting by using anti-IL-12 antibodies. Protein samples were prepared from induced recombinant L. lactis cultures. (A) Immunodetection after SDS-PAGE. Lane C, cell fraction of the NZ(pSEC:p35-p40) strain encoding the p35 and p40 subunits (IL-12p35-p40); lane S, supernatant samples. The positions and sizes of molecular weight markers are indicated on the left. (B) Quantification of IL-12p35-p40 by ELISA (R&D Systems). STD, 80 pg of commercial murine rIL-12 per ml; IL-12p35-p40, supernatant sample of NZ(pSEC:p35-p40) culture.

FIG. 4.

Secretion analysis of the two IL-12 forms produced by L. lactis. (A) rIL-12 production compared by SDS-PAGE and Western blot analysis of supernatant samples prepared from induced cultures of NZ(pSEC:p35-p40) and NZ(pSEC:scIL-12) encoding IL-12p35-p40 and scIL-12, respectively. Lane CTRL contained rIL-12 (R&D Systems) as a control. (B) Quantification of IL-12 forms produced by L. lactis by ELISA by using supernatants of induced cultures of NZ(pSEC:p35-p40) and NZ(pSEC:scIL-12) encoding IL-12p35-p40 and scIL-12. STD, 80 pg of commercial rIL-12 per ml.

FIG. 5.

In vitro induction of IFN-γ in mouse splenocytes by recombinant L. lactis. Mouse splenocytes were incubated in the presence of 50 μg of rIL-12 per ml as a control and with cells (C) and supernatant (S) of wild-type L. lactis (L. lactis wt), L. lactis NZ(pSEC:p35-p40) IL-12p35-p40, or L. lactis NZ(pSEC:scIL-12) encoding scIL-12. The concentrations of the induced culture samples were adjusted to ∼50 pg/ml as determined by ELISA. The IFN-γ concentrations are the means and standard deviations determined in three independent experiments. Significant differences compared to the data obtained for recombinant L. lactis cells and supernatant samples are indicated by one asterisk and two asterisks, respectively (P < 0.05).

FIG. 6.

Production of IFN-γ in mouse splenocytes after intranasal inoculation of recombinant L. lactis. Levels of IFN-γ were determined following sacrifice on day 35 for mice that received 5 × 10⁸ CFU of wild-type L. lactis [L. lactis (wt)], NZ(pSEC:p35-p40), or NZ(pSEC:scIL-12) or PBS alone. The data are representative of one of three separate experiments in which similar results were obtained. The values are the means and standard deviations for three mice per treatment group. Statistically significant differences (P < 0.05) compared with the wild-type L. lactis and PBS control groups are indicated by one asterisk for the NZ(pSEC:p35-p40) group and by two asterisks for NZ(pSEC:scIL-12) group.

FIG. 7.

Production of Th1 cytokines by splenocytes of mice immunized with recombinant L. lactis. Levels of Th1 cytokines were determined following sacrifice on day 35 for mice immunized with 5 × 10⁸ CFU of wild-type L. lactis (wt) or recombinant L. lactis displaying E7 antigen (E7) and for mice coimmunized with L. lactis displaying E7 and an L. lactis strain secreting active murine IL-12 (E7/IL-12). Spleen cells were cultured for 24 h with 2 μg of E7 peptide (RAHYNIVTF) (peptide) or PBS, and the levels of the Th1 cytokines IL-2 (A) and IFN-γ (B) in the culture supernatants were determined by ELISA. The values are the means and standard deviations for three mice per treatment group. Statistically significant differences (P < 0.05) compared to the E7/PBS group are indicated by one asterisk and by two asterisks for the E7/peptide and E7/IL-12/peptide groups, respectively.