A multiepitope subunit vaccine conveys protection against extraintestinal pathogenic Escherichia coli in mice

Abstract

Infections due to extraintestinal pathogenic Escherichia coli (ExPEC) are common in humans and animals and include urinary tract infections (from uropathogenic E. coli [UPEC]), septicemia, and wound infections. These infections result in significant morbidity and mortality and in high health care costs. In view of the increasing number of ExPEC infections and the ever-growing antibiotic resistance capability of ExPEC isolates, preventive measures such as an effective vaccine against ExPEC are desirable. An ExPEC vaccine may be cost-effective for select patient groups. Previous vaccine candidates consisted of single target proteins or whole ExPEC cells. Here we describe a subunit vaccine against ExPEC which is based on immunodominant epitopes of the virulence-associated ExPEC proteins FyuA, IroN, ChuA, IreA, Iha, and Usp. Using a novel approach of computer-aided design, two completely artificial genes were created, both encoding eight peptide domains derived from these ExPEC proteins. The recombinant expression of these two genes resulted in a protein vaccine directed against ExPEC but not against commensal E. coli of the gut flora. In mice, the vaccine was highly immunogenic, eliciting both strong humoral and cellular immune responses. Nasal application resulted in high secretory immunoglobulin A (sIgA) production, which was detectable on the mucosal surface of the urogenital tract. Finally, it conveyed protection, as shown by a significant reduction of bacterial load in a mouse model of ExPEC peritonitis. This study provides evidence that a novel vaccine design encompassing distinct epitopes of virulence-associated ExPEC proteins may represent a means for providing a protective and pathogen-specific vaccine.

FIG. 1.

(A) Example of structure prediction of the FyuA protein, using PROFtmb software. The protein is drawn in linear form from left to right. The start codon is numbered 1. Schemes of the vaccine proteins Vol1 (B) and Vol2 (C) are shown, including peptide subunits (red), spacers (blue), the m45 tag (green), and His tag sequences (black).

FIG. 2.

Titer increases of vaginal wash IgA antibodies specific for the vaccine proteins Vol1 and Vol2 after intranasal vaccination. (A) ELISA plates were coated with the Vol1 and Vol2 vaccine proteins. As a control, His-DHFR-m45 protein was used to exclude an antibody response against the N- or C-terminal tag sequences. (B) Western blot of the Vol1 and Vol2 vaccine proteins with the vaginal washes of mice immunized with Vol1 and Vol2, respectively. Vaginal washes were diluted 1:250, and the detection antibody, anti-mouse IgA-horseradish peroxidase, was diluted 1:1,000. **, P < 0.01; n.s., not significant.

FIG. 3.

(A) Titer increases of serum IgG/IgM antibodies specific for the Vol1 and Vol2 vaccine proteins after intranasal vaccination. As a control, His-DHFR-m45 protein was used to exclude an antibody response against the N- or C-terminal tag sequences. (B) Western blot of the Vol1 and Vol2 vaccine proteins with sera of Vol1- and Vol2-immunized animals, respectively. Sera were diluted 1:2,500, and the detection antibody, anti-mouse IgG/IgM-horseradish peroxidase, was diluted 1:10,000. **, P < 0.01; n.s., not significant.

FIG. 4.

IgA immunoblots of sera obtained from Vol1- and Vol2-immunized mice. (A) IgA binding to full-length target proteins for Vol1-immunized serum (upper part) and Vol2-immunized serum (lower part). (B) IgA binding to peptides derived from IutA and IreA. The peptides included in the vaccine proteins are numbered 1, 2, and 3. (C) Controls comprised full-length Vol1 and Vol2 proteins as well as the murine DHFR protein containing an N-terminal His tag and a C-terminal m45 tag to exclude an antibody response against the tag sequences.

FIG. 5.

IgG immunoblots of sera obtained from Vol1- and Vol2-immunized mice. (A) IgG binding to full-length target proteins for Vol1-immunized serum (upper part) and Vol2-immunized serum (lower part). (B) IgG binding to peptides derived from IutA and IreA. The peptides included in the vaccine proteins are numbered 1, 2, and 3. (C) Controls comprised full-length Vol1 and Vol2 proteins as well as the murine DHFR protein containing an N-terminal His tag and a C-terminal m45 tag to exclude an antibody response against the tag sequences.

FIG. 6.

IFN-γ-producing splenocytes in ELISPOT assay after intranasal immunization. In vitro restimulation was performed with the whole vaccine protein Vol1 or Vol2, as indicated, with protein buffer, or with protein buffer with BSA. Mock-immunized mice received CT with protein buffer intranasally. *, P < 0.05; **, P < 0.01.

FIG. 7.

Bacterial load per organ weight for livers (A) and spleens (B) of mice 48 h after challenge infection. The PBS group was immunized with PBS alone, the mock-immunized group received CT with protein buffer, and the Vol1 and Vol2 groups received vaccine protein in protein buffer, with CT as adjuvant. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant.