A proteoliposome formulation derived from Bordetella pertussis induces protection in two murine challenge models
© Fernández et al; licensee BioMed Central Ltd. 2013
Published: 25 February 2013
Whooping cough remains a health problem despite high vaccination coverage. It has been recommended that development of new strategies provide long-lasting immunity. The aim of this work was to evaluate the potential of proteoliposomes (PL) extracted from Bordetella pertussis as a vaccine candidate against whooping cough. The size of the B. pertussis PL was estimated to be 96.7±50.9 nm by Scanning Correlation Spectroscopy and the polydispersity index was 0.268. Western blots using monoclonal antibodies revealed the presence of pertussis toxin, pertactin, and fimbriae 3. The Limulus Amebocyte Lisate (LAL) assay showed endotoxin levels lower than those reported for whole cell pertussis licensed vaccines, while the Pyrogen Test indicated 75 ng/mL/Kg. The PL showed high protection capacity in mouse challenge models. There was 89.7% survival in the intracerebral challenge and total reduction of the number of CFU in the intranasal challenge. No significant differences (p>0.05) were observed between mice immunized with B. pertussis PL and the Cuban DTwP vaccine, whichever challenge model used. These results encouraged us to continue the development of the B. pertussis PL as a component of a new combined vaccine formulated with tetanus and diphtheria toxoids or as a booster dose for adolescents and adults.
Whooping cough remains an important cause of public health concern worldwide. Data from WHO revealed that more than 16 million cases and about 195 000 deaths were reported in 2008 . Despite high vaccination coverage with whole-cell (wP) or acellular (aP) vaccines, pertussis is still prevalent, probably in part, as a result of waning immunity and for that reason new vaccine strategies, able to provide long-lasting immunity have been recommended . Bacterial-derived proteoliposomes (PL) are nanoparticulate vesicular structures that contain proteins, lipids and native lipopolysaccharide (LPS). To date, only a few licensed PL-based vaccines are available. One of them is VA-MENGOC-BC®, a vaccine composed of PL obtained from the outer membrane of Neisseria meningitidis serogroup B. This PL has been shown to have high efficacy in controlling meningococcal disease  and has also exhibited potent adjuvant activity when used with other vaccine antigens not only in its native form, but in cochleate form too . Thus, a PL derived from B. pertussis could be a good approach for a new candidate vaccine against whooping cough.
Materials and methods
Cellular pellets of B. pertussis strain 165 grown at industrial scale, inactivated with formalin 0.1% through 48-56 hours, were homogenized with 30 mM Tris buffer, containing 2mM EDTA, pH 8.5 at a ratio of 100-200mg/mL. Sodium deoxycholate (Fluka, Switzerland) was added at a ratio of 0.1-1 mL/g of biomass. The mixture was incubated for 1 hour and centrifuged at 33000g for 15 min. All supernatants were collected, subjected to a sequence of diafiltration processes and filtered using a Sartorius Minisart-plus unit of 0.2 µm. Particle size PL was determined by Photon Correlation Spectroscopy (PCS) (Beckman Coulter, Hialeach, FL, US). SDS-PAGE (polyacrylamide 13%) followed by a R250 Blue Coomassie or silver stain for LPS [5, 6] was carried out using a low molecular weight marker (Amersham, UK). Western blot assays  were carried out using monoclonal antibodies (Mabs) against pertussis toxin (PT), pertactin (PRN) and fimbriae 3 (FIM3) (NIBSC, UK). The biological activity of the endotoxin present in the PL was determined by Limulus Amebocyte Lisate (LAL)  and by a Total Pyrogen Test in rabbits  at 10, 25, 50, 75 ng/mL concentrations of protein. Intracerebral challenge assays were carried out in 4-6 weeks old, 16-18 g, female OF1 mice (CENPALAB, Cuba) injected intraperitoneally with one dose of 0.5 mL of 60 µg of PL and aluminum hydroxide (1mg/mL), DTwP vaccine (Finlay Institute) prepared at 8 Ul/mL or placebo. Intracerebral challenge with B. pertussis 18323 (100-1000 DL50) was performed 2 weeks post immunization. All mice were observed for 14 days after the challenge and daily mortality was registered. For the intranasal challenge assay, BALB/c mice (female, 3-4 weeks, 12-14 g, CENPALAB, Cuba) were immunized subcutaneously with 2 doses, 125 µL separated by a three-week interval. Each dose consisted of 20 µg of PL and 0.25 mg of aluminum hydroxide. A group of mice was immunized with 125 µL of DTwP vaccine (Finlay Institute) while another group received 125 µL of PBS and aluminum hydroxide as placebo. The intranasal challenge and lung extraction was performed following the procedure described by Guiso et al. . Results were expressed as the average Log 10 of the CFU/g of lung for each group of mice at each extraction time after challenge.
The Wilson approximate method and software R 2.10.0  were used to calculate the confidence intervals. The arithmetic means ± standard deviation for Log 10 CFU/ lung were also calculated. The comparison of arithmetic means of the groups was carried out by an analysis of simple variance (Statistic kit, Statgraphics Plus 5.0).
Results and discussion
Intranasal challenge experiments in BALB/c mice using B. pertussis WHO reference strain 18323
Treatments (s.c. immunization)
Bacteria recovered post challenge (Log CFU/g lung ± S.D.)
7.85 ± 0.39
B. pertussis PL
6.68 ± 0.65
8.02 ± 0.2
6.85 ± 0.44
6.48 ± 0.11
The authors want to give thanks to MSc Vladimir Peña for the technical support provided in the particle size analysis of PL. We also acknowledge Dr. Reinaldo Acevedo for useful suggestions.
This article has been published as part of BMC Immunology Volume 14 Supplement 1, 2013: Proceedings from Delivery Systems and Current strategies to drug design. The full contents of the supplement are available online at http://www.biomedcentral.com/bmcimmunol/supplements/14/S1.
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