Defining biotechnological parameters for a new pertussis acellular vaccine

GAILLARD, M. EMILIA; BARTEL, ERIKA; ZURITA, MARIA EUGENIA; RUMBO, MARTIN; HOZBOR, DANIELA

Congreso; VIII CONGRESO ARGENTINO DE MICROBIOLOGIA GENERAL -SAMIGE-; 2012

Pertussis or whooping cough is an acute respiratory tract infection caused by Bordetella pertussis which causes 300,000 children death mainly in developing countries and afflicts up to 16 million children worldwide per year. Vaccination is the most economical and effective strategy for preventing and controlling pertussis. The introduction of the first generation of pertussis vaccines (whole cell vaccines, WCV) in the 1950s dramatically reduced the incidence of the disease. Now, WCV and acellular vaccines (AV) are the two main types of globally used pertussis vaccines. Despite the high vaccination coverage worldwide, pertussis is still a serious contagious disease, especially in infants less than 6 months old. The current epidemiological situation of pertussis points out the need to review the current control strategies against the disease and the design of new vaccines. We have recently developed an acellular vaccine, which has been shown to be safe and to induce strong protective immunity against B. pertussis infection in mice. The aim of the present work is to adjust culture condition to increase yields keeping the adequate protective capacity of vaccine immunogen. Since our acellular vaccine is a nanoparticle consisting in several different proteins, we used one of them, the well known protective immunogen, the pertussis toxin, as a quantitative marker to assess the protection capacity in an in vitro assay. Here we showed the results obtained with a quantitative western blot assay (Near-Infrared Fluorescence, Odyssey). Using this technique we determined that the minimal level of the protein marker in a protective vaccine dose is 0,04 µg. To improve the yield of the vaccine immunogens we evaluated the biomass production in three different media that are commonly used in pertussis vaccine production: Stainer-Scholte chemically defined medium (SS); a modification of SS by addition of casamino acids (SSC) and THIJS medium. In each media the bacterial growth was estimated by optical density at 650 nm (OD650nm) and dry weight (DW) measures. The results obtained in four independent assays show that bacterial growth in SS achieves maximum OD650nm=0.8-1 in 20 hours (DW= 0.621 ± 0.001 g/l), in THIJS maximum OD650nm=0.9-1.1 is reached in 23 hours (DW= 0.677 ±0.078 g/l) while in SSC the highest OD650nm=3.5-4 is achieved after 45 hours (DW= 2.809±0.32 g/l). To evaluate the quality of biomass from which we obtain the acellular vaccine, the pertussis toxin protein marker was measured using the quantitative immunoblot assay. We observed that though the bacterial yield was different among the media analyzed, the PT yield per biomass were similar, 0.15-0.20 µg/108 cells. Based on these the SSC medium appeared as the best option to obtain higher quantities of biomass and improve the B. pertussis immunogen yield.