Oral immunization with a Salmonella typhimurium vaccine vector expressing recombinant enterotoxigenic Escherichia coli K99 fimbriae elicits elevated antibody titers for protective immunity

Abstract

Bovine enterotoxigenic Escherichia coli (ETEC) continues to cause mortality in piglets and newborn calves. In an effort to develop a safe and effective vaccine for the prevention of F5(+) ETEC infections, a balanced lethal asd+ plasmid carrying the complete K99 operon was constructed and designated pMAK99-asd+. Introduction of this plasmid into an attenuated Salmonella typhimurium Deltaaro Deltaasd strain, H683, resulted in strain AP112, which stably expresses E. coli K99 fimbriae. A single oral immunization of BALB/c and CD-1 mice with strain AP112 elicited significant mucosal immunoglobulin A (IgA) titers that remained elevated for >11 weeks. IgA and IgG responses in serum specific for K99 fimbriae were also induced, with a prominent IgG1, as well as IgG2a and IgG2b, titer. To assess the derivation of these antibodies, a K99 isotype-specific B-cell ELISPOT analysis was conducted by using mononuclear cells from the lamina propria of the small intestines (LP), Peyer's patches (PP), and spleens of vaccinated and control BALB/c mice. This analysis revealed elevated numbers of K99 fimbria-specific IgA-producing cells in the LP, PP, and spleen, whereas elevated K99 fimbria-specific IgG-producing cells were detected only in the PP and spleen. These antibodies were important for protective immunity. One-day-old neonates from dams orally immunized with AP112 were provided passive protection against oral challenge with wild-type ETEC, in contrast to challenged neonates from unvaccinated dams or from dams vaccinated with a control Salmonella vector. These results confirm that oral Salmonella vaccine vectors effectively deliver K99 fimbriae to mucosal inductive sites for sustained elevation of IgA and IgG antibodies and for eliciting protective immunity.

FIG. 1

Construction of pMAK99-asd⁺. A BamHI fragment containing the fanABCDEFGH operon from plasmid pFK99 was subcloned into the BamHI site of pYA292. The derived plasmid, pMAK99-asd⁺, stably expresses K99 fimbria antigen in the asd-negative strains S. typhimurium H683 and E. coli H681.

FIG. 2

SDS-PAGE (A) and Western blot analysis (B) of isolated K99 fimbriae derived from S. typhimurium and E. coli K99 fimbria-expressing strains. Lanes (left to right): isogenic S. typhimurium H647 without K99 operon; S. typhimurium-K99 fimbria construct, strain AP112; E. coli-K99 fimbria construct, strain AP111; E. coli NADC 2323 with plasmid pFK99; the wild-type E. coli K99⁺ strain, B41; molecular size (MW) markers in kilodaltons (Kdal) (ovalbumin, 46.7; soybean trypsin inhibitor, 28.8; lysozyme, 20.5; and aprotinin, 7.4). To demonstrate that the isolated protein obtained from the various strains was K99 fimbria, the nitrocellulose-transferred proteins were reacted with K99 fimbria-specific rabbit antiserum. The AP111 and AP112 constructs were shown to express the K99 fimbriae when compared to the K99⁺ E. coli strains producing the fimbriae of the expected 17.5 kDa, whereas no fimbriae were obtained from the K99⁻ asd⁺ S. typhimurium strain, H647.

FIG. 3

K99 fimbria-specific IgA responses in fecal pellets were induced after oral immunization of mice with a single dose of S. typhimurium AP112 and not with the Salmonella vector control, strain H647. Depicted are the means plus standard deviations of the K99 fimbria-specific IgA log₂ end point titers in fecal pellets that developed after 2, 4, 6, and 11 weeks of oral immunization of mice with 5 × 10⁹ CFU. Mucosal IgA antibody titers to K99 fimbriae were determined by ELISA. ∗, significantly different between 2 and 6 weeks (P = 0.003); ∗∗, significantly different between 2 and 11 weeks (P = 0.036). S-IgA, secretory IgA.

FIG. 4

K99 fimbria-specific IgA and IgG responses after oral immunization of BALB/c mice and CD-1 mice with a single dose of strain AP112. Depicted are the means plus standard deviations of the K99 fimbria-specific IgA and IgG subclass log₂ end point titers in serum that developed at 4 weeks after oral immunization of mice with 5 × 10⁹ CFU. IgA, IgG, and IgG subclass antibody titers in serum and fecal pellets were determined by standard ELISA methods. Both strains of mice produced elevated antibody titers, but the CD-1 mice exhibited higher IgA (P < 0.001) and IgG (P = 0.046) titers in serum than BALB/c mice did. For BALB/c mouse results, an asterisk indicates that K99 fimbria-specific IgG2a titers were significantly different from K99 fimbria-specific IgG1 and IgG2b titers (P < 0.001). IgG1 and IgG2b titers were not significantly different from each other. For CD-1 mice, the IgG1, IgG2a, and IgG2b antibody titers were not statistically different from each other; however, the IgG1 (P = 0.001), IgG2a (P = 0.046), and IgG2b (P = 0.015) titers were statistically different between mouse strains.

FIG. 5

K99 fimbria-specific IgA and IgG SFC responses after oral immunization of mice with strain AP112. K99 fimbria-specific and total IgA and IgG SFC responses in LP, PP, and splenic mononuclear cells were measured by ELISPOT at 4 weeks after oral immunization. Depicted are the means plus standard deviations of K99 fimbria-specific IgA and IgG SFC per 10⁴ IgA⁺ and IgG⁺ SFC, respectively, that developed.

FIG. 6

Oral immunization of CD-1 female mice with AP112 provides passive immunity to pups orally challenged with B41, the wild-type ETEC strain. AP112- and H647-vaccinated as well as unvaccinated dams were mated. Pups from each group were allowed to receive colostrum and milk for the first 24 h after birth, after which the pups were orally challenged with 10³ K99 fimbria-positive cells of wild-type ETEC, strain B41, and monitored for survival for 10 days. Oral immunization with AP112 provided passive immunity to wild-type ETEC-challenged mice, as demonstrated by the survival of 25 of 27 pups. In contrast, pups from dams vaccinated with Salmonella vector only (n = 24) or unvaccinated (n = 23) died.