A comparison of immunogenicity and protective immunity against experimental plague by intranasal and/or combined with oral immunization of mice with attenuated Salmonella serovar Typhimurium expressing secreted Yersinia pestis F1 and V antigen

Abstract

We investigated the relative immunogenicity and protective efficacy of recombinant X85MF1 and X85V strains of DeltacyaDeltacrpDeltaasd-attenuated Salmonella Typhimurium expressing, respectively, secreted Yersinia pestis F1 and V antigens, following intranasal (i.n.) or i.n. combined with oral immunization for a mouse model. A single i.n. dose of 10(8) CFU of X85MF1 or X85V induced appreciable serum F1- or V-specific IgG titres, although oral immunization did not. Mice i.n. immunized three times (i.n. x 3) with Salmonella achieved the most substantial F1/V-specific IgG titres, as compared with corresponding titres for an oral-primed, i.n.-boosted (twice; oral-i.n. x 2) immunization regimen. The level of V-specific IgG was significantly greater than that of F1-specific IgG (P<0.001). Analysis of the IgG antibodies subclasses revealed comparable levels of V-specific Th-2-type IgG1 and Th-1-type IgG2a, and a predominance of F1-specific Th-1-type IgG2a antibodies. In mice immunized intranasally, X85V stimulated a greater IL-10-secreting-cell response in the lungs than did X85MF1, but impaired the induction of gamma-interferon-secreting cells. A program of i.n. x 3 and/or oral-i.n. x 2 immunization with X85V provided levels of protection against a subsequent lethal challenge with Y. pestis, of, respectively, 60% and 20%, whereas 80% protection was provided following the same immunization but with X85MF1.

Fig. 2

Comparison of Yersinia pestis F1 and V antigen expression in vitro in a Salmonella strain. The overnight cultures of (a) X85MF1 and (b) X85V bacterial strains were fractionated, separated by SDS-PAGE, and then probed, separately, with mAb against F1 (4B5-3) and V (4H-10). Samples equivalent to 5 × 10⁷ CFU of X85MF1 and 10⁷ CFU of X85V from overnight culture of bacteria lysate were loaded into SDS-PAGE. Lanes codes: M, the supernatant fraction collected from centrifugation following overnight culture; CL, total cell lysate. Various amounts of purified rF1 and V protein (in ng) are shown at the top of the figure, and molecular-weight markers (in kDa) are indicated on the right-hand side of the figure.

Fig. 1

Plasmids used for the expression of Yersinia pestis F1 or V antigen for an attenuated Salmonella typhimurium strain. The Y. pestis CafMF1- or LcrV-encoding gene was cloned into the expression vector pYA3495 (see ‘Materials and methods’ section) downstream of the Bla-secretion system. This Bla-secretion system consisted of the β-lactamase signal sequence and 12 amino-acid residues of the N-terminus of the mature β-lactamase signal sequence, which derived from plasmid pBR322, and which was expressed under the control of the Ptac promoter. 5ST1T2 is a transcriptional terminator.

Fig. 3

Confocal immunofluorescent microscopy images of Yersinia pestis F1- and V-antigen expression for X85MF1 and X85V bacterial strains within mouse macrophage-like J774 A1 cells. The J774 A.1 cells were infected with the Salmonella vaccine strain, and the intracellular bacteria were probed with anti-F1 or anti-V mAb and then detected with Alex-Red conjugated anti-mouse IgG (red). The cell morphology, as characterized by the cytoskeleton, was observed by Phllodin-FITC-conjugated antitubulin antibody (green).

Fig. 4

Total F1/V-specific IgG responses in pooled sera collected on days 14, 28 and 42 following i.n. × 3 or oral-i.n. × 2 immunization regimens with the Salmonella (a) X85MF1 and (b) X85V vaccine strains. The arrow indicates the time point, on, respectively, days 15 and 29 subsequent to primary immunization, at which each i.n. boost was administered for the specific vaccination regimen. Error bars indicate SD.

Fig. 5

F1 and/or V antigen-specific IgG1 and IgG2a responses in sera collected on day 14 after the delivery of a dose of Salmonella (a) X85MF1 and (b) X85V, following i.n. × 3 and/or oral-i.n. × 2 prime–boost immunization regimens. Error bars indicate SD.

Fig. 6

IgG-, A- and cytokine-producing cells in the spleen and lung of immunized mice. Mice were i.n. × 3-immunized with Salmonella X85MF1 (F1) and/or X85V (V), following which spleen (S) and lung (L) cells were examined for the production of F1/V antigen-specific antibody (a) and cytokine-secreting cells (b) by ELISPOT assay. Error bars indicate SD.

Fig. 7

Protection against Yersinia pestis challenge following mucosal prime–boost immunization with X85MF1 and X85V. Two weeks after the last immunization following i.n. or oral − i.n. × 2 administration with Salmonella vaccine, mice were i.p. challenged with 2 × 10³ CFU of Y. pestis, and individual test-mouse survival was recorded daily for a period of 2 weeks.