فهرست مطالب seyed reza banihashemi

Pertussis is a current contagious bacterial disease caused by Bp. Given the prevalence of pertussis, development of new vaccines is important. This study was attempted to evaluate the expression of main virulence factors (PTX, PRN, and FHA) from Bp predominant strains and also compare the expression of these factors in the OMVs obtained from predominant circulating Bp isolate.

The physicochemical features of the prepared OMVs were analyzed by electron microscopy and SDS-PAGE. The presence of the mentioned virulence factors was confirmed by Western blotting. BALB/c mice (n =21) immunized with characterized OMVs were challenged intranasally with sublethal doses of Bp, to examine their protective capacity.

Electron microscopic examination of the OMVs indicated vesicles within the range of 40 to 200 nm. SDS-PAGE and Western blotting demonstrated the expression of all three main protective immunogens (PTX, PRN, and FHA), prevalent in the predominant, challenge, and vaccine strains, and OMVs of the predominant IR37 strain and BP134 vaccine strain. Significant differences were observed in lung bacterial counts between the immunized mice and control group (p < 0.001). In mice immunized with OMVs (3 µg), the number of lungs recovered colonies after five days dropped at least five orders of magnitude compared to the control group.

OMVs obtained from circulating isolates with the predominant profile may constitute a highly promising vaccine quality. They also can be proposed as a potential basic material for the development of new pertussis vaccine candidate.

Pertussis is still one of the major public health problems. The increase of the disease emerged in recent decades due to the replacement of the reactogenic whole cell vaccine with the safer acellular vaccine and the genetic diversity of the bacterium. As outer membrane vesicles (OMVs) obtained from Bordetella pertussis contains surface immunogenic antigen in its structure, it has an acceptable characteristic that could be considered as a good candidate for pertussis vaccine.

Vaccinal strain BP134 strain of B. pertussis was cultured under standard conditions and OMVs were isolated by modifying the method without the use of ultracentrifuge. The isolated vesicles were examined by transmission electron microscopy and protein content was measured by the Bradford method. The expression of virulence factors was confirmed by SDS-PAGE and protein expression was confirmed by Western immunoblot. Pyrogenicity test and abnormal toxicity test were performed on extracted vesicles.

The morphology of the vesicles was confirmed in the range of 40 to 200 nm. The protein concentration of the extracted vesicles was determined as 600 μg. Expression analysis by SDS-PAGE and western blot confirmed the presence of virulence factors, pertussis toxin, filamentous hemagglutinin, and pertactin using monoclonal antibodies in OMVs of the vaccinal strain. Pyrogenicity test and abnormal toxicity test were negative.

The proposed method is a simple and efficient method for isolation of the B. pertussis OMVs. The OMVs extracted from B. pertussis could be a candidate for a new generation of pertussis vaccine alone or in combination with adjuvants to design future acellular vaccines.