Production and characterization of recombinant pertactin, fimbriae 2 and fimbriae 3 from Bordetella pertussis

Abstract

Background: Bordetella pertussis is a causative agent of pertussis or whooping cough in humans. Pertactin (Prn), fimbriae 2 (Fim2) and fimbriae 3 (Fim3) of B. pertussis are important virulence factors and immunogens which have been included in some acellular pertussis vaccines. In this present study, we cloned, expressed and purified Prn, Fim2 and Fim3, respectively. The immunogenicity and protective efficacy of the three recombinant proteins (rPrn, rFim2 and rFim3) were investigated in mouse model.

Results: Three recombinant proteins with amount of 12 to 25 mg/L were produced. Compared to the control mice only immunized with adjuvant, serum IgG antibody responses were significantly induced in the mice immunized with rPrn, rFim2 or rFim3 (P < 0.001 for all three proteins). Furthermore, T cell responses characteristic of increased production of IL-2 and TNF-alpha (only for rPrn) were elicited in the mice immunized with the three proteins (P < 0.05 for all three proteins). Immunization with rPrn, but not with rFim2 or rFim3, significantly enhanced clearance of bacteria in the lungs of mice after intranasal challenge with B. pertussis (P < 0.05). When tested in a lethal intracerebral infection model, certain protection was observed in mice immunized with rPrn.

Conclusions: We have developed an efficient method to produce large amounts of rPrn, rFim2, and rFim3 from B. pertussis. The three recombinant proteins induced both humoral and cellular immune responses in mice. Immunization with rPrn also conferred protection against pertussis in mouse infection models. Our results indicated that the recombinant proteins still retain their immunological properties and highlighted the potential of the recombinant proteins for the future development of the B. pertussis vaccines.

Figure 1

SDS-PAGE and Western blotting analysis. (A) SDS-PAGE of the purified recombinant proteins. The proteins were electrophoresed on a 10% SDS-PAGE gel under reducing condition and stained by Coomassie blue. Lane 1: Molecular mass marker, the molecular mass standards are indicated in kDa on left side; lane 2: rPrn (10 μg); lane3: rFim2 (10 μg); lane 4: rFim3 (10 μg). (B) Western blotting of the recombinant proteins. Lane 1: Pre-stained molecular mass marker (170 kDa, 130, 100, 70, 55, 40, 35, 25, 15, 10, Fermentas), the molecular mass standards are indicated in kDa on left side; lane 2: rFim2 was detected with mouse anti-Fim2 monoclonal antibodies; lane 3: rFim3 was detected with mouse anti-Fim3 monoclonal antibodies; lane 4: Pre-stained molecular mass marker, the molecular mass standards are indicated in kDa on right side; lane 5: rPrn was detected with mouse anti-Prn monoclonal antibodies; lane 6: Pre-stained molecular mass marker, the molecular mass standards are indicated in kDa on right side.

Figure 2

Antibody responses in immunized and control mice. Two weeks after the second immunization, sera were collected, and IgG antibody titres were determined by ELISA. Results represent the mean antibody titres for five mice per group. An asterisk symbol (*) indicates a statistically significant difference (P < 0.001) between immunized and control group.

Figure 3

Cytokine responses in immunized and control mice. Two weeks after the second immunization, blood samples were collected from five mice from each group. The cytokines were determined by ELISA and are expressed as pg/mL sera. Results are the mean responses for five mice per group. An asterisk symbol (*) indicates a statistically significant difference (P < 0.05) between immunized and control group.

Figure 4

Protection against intranasal challenge with B. pertussis. Two weeks after the second immunization, the mice were challenged intranasally with B. pertussis 18323, and CFU counts were performed on individual lung homogenate. Results are mean viable B. pertussis counts from five mice per group. An asterisk symbol (*) indicates a statistically significant difference (P < 0.05) between immunized and control group.

Figure 5

Protection against intracerebral challenge with B. pertussis. Three weeks after immunization, all mice (sixteen mice per group) were challenged intracerebrally with a lethal dose of B. pertussis 18323, and survival of challenge mice was monitored. For recombinant proteins immunized groups, A, B, and C indicated 100 μg, 20 μg, and 4 μg dose of immunization. The reference vaccine is used as national reference standard in the intracerebral challenge assay in China and this standard have an assigned activity of 14 IU/ampoule. A, B and C indicated 0.5 IU, 0.1 IU and 0.02 IU dose of immunization. All mice of control group were immunized adjuvant alone. An asterisk symbol (*) indicates a significant difference (P < 0.05) between immunized and control group.