Mucosal and cellular immune responses elicited by recombinant Lactococcus lactis strains expressing tetanus toxin fragment C

Abstract

The mucosal and cellular responses of mice were studied, following mucosal-route administration of recombinant Lactococcus lactis expressing tetanus toxin fragment C (TTFC), which is a known immunogen protective against tetanus. A TTFC-specific T-cell response with a mixed profile of T-helper (Th) subset-associated cytokines was elicited in the intestine, with a Th2 bias characteristic of a mucosal response. These results correlated with the humoral response, where equivalent titers of anti-TTFC immunoglobulin G1 (IgG1) and IgG2a in serum were accompanied by an elevated IgA-specific response at more than one mucosal site. The route of vaccination had an important role in determining the immune response phenotype, as evidenced by the fact that an IgG1-biased subclass profile was obtained when lactococci were administered parenterally. Stimulation of splenic or mesenteric lymph node cells with lactococci resulted in their proliferation and the secretion of gamma interferon via antigen-specific and innate immune mechanisms. The data therefore provide further evidence of the potential of recombinant lactococcal vaccines for inducing systemic and mucosal immune responses.

FIG. 1.

Serum anti-TTFC IgG, IgA, and IgM titers elicited by recombinant lactococci or purified TT. Groups of six mice were immunized, either i.g., i.n., or by i.p. injection, with lactococci expressing TTFC (pTREX1-TTFC) or a control strain (pTREX1). For comparison, a group of mice were injected with TT in FCA. On day 35 post-initial treatment, blood samples were collected and TTFC-specific serum IgG (solid bars), IgA (striped bars), and IgM (open bars) titers were measured by ELISA. Bars, mean titers; error bars, standard deviations. Asterisks indicate values significantly different from those for unvaccinated controls (P < 0.01).

FIG. 2.

Serum anti-TTFC IgG1 and IgG2a titers elicited by recombinant lactococci or purified TT. Groups of six mice were immunized, either i.g., i.n., or by i.p. injection, with lactococci expressing TTFC (pTREX1-TTFC) or a control strain (pTREX1). For comparison, a group of mice was injected with TT in FCA, while another group received no treatment. On day 35 post-initial treatment, blood samples were collected, and serum TTFC-specific IgG1 (open bars) and IgG2a (solid bars) titers were measured by ELISA. Bars, mean titers; error bars, standard deviations. Asterisks indicate values significantly different from those for nonvaccinated controls (P < 0.05).

FIG. 3.

Ratios of specific to total IgA in intestinal lavage fluids of mice immunized i.g. with lactococci expressing TTFC (pTREX1-TTFC) or a control strain (pTREX1). Intestinal lavage fluids were collected from groups of six mice on days 6 (open bars), 20(striped bars), and 46 (shaded bars) post-initial treatment. The samples were tested by ELISA for TTFC-specific and total IgA. Bars, mean ratios; error bars, standard deviations. Asterisks indicate values significantly different from those for nonvaccinated controls (P < 0.05).

FIG. 4.

Ratios of specific to total IgA in lung lavage fluids of mice immunized i.n. with lactococci expressing TTFC (pTREX1-TTFC) or a control strain (pTREX1). Lung lavage fluids were collected from groups of six mice on days 7 (open bars), 20(striped bars), and 45 (shaded bars) post-initial treatment. The samples were tested by ELISA for TTFC-specific and total IgA. Bars, mean ratios; error bars, standard deviations. Asterisks indicate values significantly different from those for nonvaccinated controls (P < 0.05).

FIG. 5.

Quantification of TTFC-specific IgA-secreting cells in lymphoid and mucosal tissues over 42 days after lactococcal immunization. Groups of six mice were immunized i.g. (i, ii, and iii) or i.n. (iv, v, and vi) with lactococci expressing TTFC (pTREX1-TTFC) (solid bars) or a control strain (pTREX1) (striped bars). A control group remained untreated (open bars). On days 7, 18, 35, and 42 post-initial treatment, MLN (i and iv), PP (ii and v), and PBS-perfused lung (iii and vi) cell suspensions were cultured and tested by ELISPOT for the presence of TTFC-specific IgA-secreting cells. Bars, mean frequencies of SFCPM; error bars, standard deviations.

FIG. 6.

Proliferative responses of SPLC from groups of six mice immunized i.p. with lactococci expressing TTFC (pTREX1-TTFC), a control strain (pTREX1), or TT in FCA (a) and of MLNC from mice immunized i.g. with recombinant lactococci (b). Control nonvaccinated groups were also included. SPLC and MLNC were cultured with LP-TT (open bars), LP-U (striped bars), or formalin-fixed pTREX1-TTFC (solid bars) or pTREX1 (speckled bars) lactococci. Unstimulated cells were also cultured. Proliferation was determined by [³H]thymidine incorporation, and results were expressed as SI (ratios of counts from stimulated cells to counts from unstimulated cells). Bars, mean SI. Error bars, standard deviations.

FIG. 7.

Percentages of cytokine-positive events among CD4-positive LPL. Mice were immunized i.g. with lactococci expressing TTFC (pTREX1-TTFC) (solid bars) or a control strain (pTREX1) (striped bars). Control unvaccinated mice were also included (open bars). LPL were isolated from the small intestine on days 7 and 42, cultured, fixed, and stained for CD4 and the intracellular cytokines IL-4, IL-5, IL-10, and IFN-γ.