Receptor-dependent immune responses in pigs after oral immunization with F4 fimbriae

Abstract

F4 receptor-positive (F4R+) and F4 receptor-negative (F4R-) pigs were orally vaccinated with purified F4 fimbriae of enterotoxigenic Escherichia coli (ETEC). Serum immunoglobulin G (IgG) and IgA responses were readily detected in F4R+ animals, whereas immune responses were not detected in F4R- animals. Even after a subsequent oral infection with virulent F4(+) ETEC and a booster immunization with F4, the F4R- animals remained F4 seronegative whereas the unvaccinated F4R+ pigs exhibited clear IgA and IgG responses. These results clearly demonstrate that F4Rs are a prerequisite for an immune response following oral immunization. Furthermore, indications that oral F4 vaccination can induce mucosal protection were obtained, since the experimental ETEC infection did not induce a systemic booster response or fecal ETEC excretion in orally vaccinated F4R+ pigs, in contrast to the clear immune response and ETEC excretion of unvaccinated F4R+ animals. F4-specific IgA antibodies could be found in the feces of the vaccinated F4R+ pigs. They are secreted at the intestinal mucosal surface and appear to prevent ETEC infection. The F4R-dependent induction of a mucosal immune response can be used as a model to better understand mucosal immunization and mucosal immune responses and can contribute to the development of oral vaccines in veterinary as well as in human medicine.

FIG. 1

Small intestinal villous brush borders after the in vitro adhesion assay with F4ac⁺ E. coli. (A) F4R⁺ brush border with strong adhesion; (B) F4R⁻ brush border without adhesion. Bars, 10 μm.

FIG. 2

Adhesion of F4⁺ ETEC to F4R⁺ intestinal villi (of two pigs) with and without addition of 10 μg of F4ac- and swine IgG-specific MAb per ml (A) and adhesion of ETEC grown at 37 or 18°C to F4R⁺ intestinal villi (of two pigs) (B). Bars and T bars represent mean numbers of adhering bacteria per 250-μm length of villous brush border ± SEM (n = 2).

FIG. 3

Adhesion of F4⁺ ETEC to intestinal villi of control pigs (n = 5) and vaccinated pigs (n = 10). The adhesion assay was repeated twice for each pig. Bars and T bars represent mean numbers of adhering bacteria per 250-μm length of villous brush border for individual pigs ± SEM (n = 3).

FIG. 4

Adhesion of F4⁺ ETEC to intestinal villi of pigs in the CF4R⁺ group (n = 5), the VF4R⁺ group (n = 4), and the VF4R⁻ group (n = 6). Bars and T bars represent mean numbers of adhering bacteria per 250-μm length of villous brush border ± SEM.

FIG. 5

Evolution of the F4-specific IgG, IgM, and IgA antibody titers in serum (±SEM) in CF4R⁺ animals (n = 5), in VF4R⁺ animals (n = 4), and in VF4R⁻ animals (n = 6) following vaccination on days 0 and 16; all animals were challenged on day 36, and F4-specific IgG, IgM, and IgA antibodies in serum were quantified. prim. vacc., primary vaccination; sec. vacc., secondary vaccination.

FIG. 6

Mean F4-specific IgA titers in feces of VF4R⁺ pigs (n = 4), VF4R⁻ pigs (n = 6), and CF4R⁺ pigs (n = 5) 35 days ppv (1 day before oral ETEC challenge). Bars and T bars represent mean titers ± SEM.

FIG. 7

Evolution of the F4-specific IgA titers (±SEM) in feces of VF4R⁺ pigs (n = 4), VF4R⁻ pigs (n = 6), and CF4R⁺ pigs (n = 5) following oral ETEC challenge.