Intranasal administration of synthetic recombinant peptide-based vaccine protects mice from infection by Schistosoma mansoni

Abstract

Schistosomiasis is the cause of a chronic debilitating disease which accounts for significant mortality and morbidity every year, especially in tropical and subtropical areas. An epitope derived from the protective surface protein 9B-Ag of Schistosoma mansoni, designated 9B peptide-1, was previously showed to be protective in mice when conjugated to bovine serum albumin and administered subcutaneously in complete Freund's adjuvant. In this work, this protective peptide was expressed in the flagellin of a Salmonella vaccine strain, and the isolated recombinant flagella were used for immunization of mice. Since during the invasion of the parasite into the host the schistosomula migrate first to the lungs, the intranasal route of administration was employed in order to halt the parasite at an early stage of the infection. Such intranasal immunization with this peptide expressed in flagellin, without the addition of adjuvants, resulted in a significant humoral response and also led to protection against challenge infection, manifested as a reduction of the worm burden by an average of 42%.

FIG. 1

Expression of recombinant 9B peptide-1 flagellin protein and its reaction with anti-9B peptide-1 antibodies. Lanes 1 to 3, Coomassie blue staining of purified proteins on sodium dodecyl sulfate–10% polyacrylamide gels. A shift in molecular weight of the recombinant flagellin (lane 3) compared to the native flagellin (lane 2) is seen. Molecular weight markers (in thousands) are shown in lane 1. Lanes 4 and 5, Western blot developed with anti-9B peptide-1–BSA antibodies. No reaction is observed with native flagella (lane 4), while a strong specific reaction is observed with the recombinant 9B-expressing flagella (lane 5).

FIG. 2

Pharmacokinetics of ¹²⁵I-labeled recombinant 9B peptide-1-expressing flagella in mice. Mice were immunized i.n. (A) or f.p. (B) with the labeled protein. The distribution of radioactive protein in the lungs (■), intestine (┌), liver (○), kidney (▭), spleen (Y=), gut (×), heart (+), and leg (▵) was determined at the specified times. A high percentage of the total radioactivity was found at the site of immunization up to 24 h postimmunization.

FIG. 3

Recognition of 9B peptide-1 by serum antibodies. C57BL/6J mice were immunized twice with recombinant 9B peptide-1-expressing flagella i.n. (□) or f.p. (○), native flagella i.n. (⧫) or f.p. (▴), or serum from a CFA-injected mouse f.p. (■) and NMS (●) as controls. The plate was coated with 5 μg of 9B peptide-1 conjugated to BSA per ml. O.D., optical density.

FIG. 4

Recognition of 2-h schistosomula and cercariae by serum antibodies of immunized mice. C57BL/6J mice were immunized i.n. twice with recombinant 9B peptide-1-expressing flagella (empty symbols) or native flagella (filled symbols). ELISA plates were coated with a 10-μg/ml concentration of lysate of either 2-h schistosomula (squares) or cercariae (circles). O.D., optical density.

FIG. 5

Complement-mediated lysis of 3.5-h schistosomula by serum antibodies elicited against recombinant 9B peptide-1-expressing flagella. Lysis of schistosomula in the presence of sera from normal C57BL/6J mice (bars A), sera from mice immunized i.n. with recombinant (bars B) or native (bars C) flagella, and sera from infected animals (positive control) (bars D) is shown. Guinea pig serum was used as a source of complement (■), and lysis was compared to that in the presence of heat-inactivated complement (□). Results are means and standard errors from three experiments.

FIG. 6

Local humoral response against 9B peptide-1 in the lungs of immunized mice. C57BL/6J mice were immunized twice i.n. with recombinant 9B-expressing flagella (▴) or with native flagella (▵). Their lungs were removed, homogenized in PBS, and tested by ELISA for the IgA response against 9B peptide-1 conjugated to BSA. O.D., optical density.

FIG. 7

Isotype profile of serum antibodies following i.n. or f.p. immunization. Serum samples were obtained 2 to 3 weeks after the last immunization of mice. The mice were immunized i.n. either with the native flagellin (squares) or with recombinant flagellin (diamonds) or f.p. with recombinant flagellin (circles). Results with preimmune serum are indicated by triangles. The coating antigen for the ELISA microplate was either the recombinant 9B peptide-1 flagellin (empty symbols) or 9B peptide-1 (filled symbols). The second antibody reacts specifically with different isotypes of the serum antibodies (IgG1, IgG2a, IgG2b, and IgG3) as indicated.