Carrier liposome systems of Pasteurella multocida membrane antigenic proteins for the prevention of pasteurellose
Pasteurellosis is a common disease in the respiratory tract of commercial and/or biomedical rearing of research rabbits. The bacterium Pasteurella multocida is the pathogen responsible for a range of clinical syntomes, including chronic rhinitis (snuffles), otitis media, pneumonia, genital infection, pulmonary and cutaneous abscesses, conjunctivitis and hemorrhagic septicemia. However, between 50 and 70 % of the animals can harbour the microorganism asymptomatically. The factors that cause the clinical syntomes include the ammonium accumulation in the air (foul ventilation), pregnancy, another concomitant disease, disorder in the rabbit production environment and experimental manipulation. Outbreaks of this disease occur in Brazil with relative frequency; however diagnosis is generally based on the clinical signals and necropsy. Therefore, it is difficult to estimate the extent of losses caused by pasteurellosis druing cuniculture. However, specific commercial vaccines against pasteurellosis in rabbits are not available and prevention is through the use of antibiotics in drinking water, even though this type of treatment generally does not protect the animals. Initially, pure bacteria colonies were obtained, which were cultivated in specific growing media (BHI). The microorganisms were isolated, lysed and the antigenic proteins were detected by SDS-PAGE and Western Blotting. These results show that most protein bands were recognized by the policlonal antibody against P. multocida. Since this protein pool presented antigenicity, the protein mixture was solubilized by incubating 0,5 mg/ml of the membrane fraction with SDS 1 % (w/v) under constant agitation for 2 hours. This procedure resulted in a 85 % solubilization yield. The proteoliposomes wew formed using a lipid, protein and detergent co-solubilization method. A good yield of protein incorporation in liposomes seems to be related to the methodology used for the removal of the detergent from the lipid:protein:detergent mixture during the co-solubilization process, as well as the nature of the phospholipid used. The results indicated that the Calbiosorb® resin was the most efficient for SDS removal and, among the various phospholipids tested, DPPC best incorporated the proteins, presenting an incorporation yield of 93% and average proteoliposome diameter of 180 nm. In addition, SDS-PAGE of the proteoliposomes has shown that all the proteic species present in the crude solubilized extract were incorporated in the DPPC liposomes. The Western Blotting has shown that the proteins incorporated in the liposomes continue to be recognized by the policlonal antibody against P. multocida. For the immunization assays, three animal groups were separated: (i) rabbits immunized with liposomes; (ii) rabbits immunized with crude solubilized extract (CSE) and (iii) rabbits immunized with the proteoliposomes. After twenty-one days of immunization with the described preparations, the animals were challenged with 105 ufc of bacteria. All animals previously vaccinated with the liposomes or CSE died while the animals vaccinated with the proteoliposomes systems had 95 % survival after the challenge. Moreover, a control group vaccinated with the attenuated bacteria in the presence of aluminum hydroxide as an immunoadjuvant had only 30% survival, indicating that the conventional vaccine does not protect against pasteurellosis. The serum of animals vaccinated with liposome, CSE and proteoliposomes were collected weekly before and after the experimental infection for the detection of IgG, IgM and IgA antibodies production using ELISA. Animals vaccinated with liposomes did not present stimulation of any of the specific antibodies for the P. multocida analyzed. The animals immunized with CSE presented a significant increase in the IgA serum level seven days after the immunization, but these levels were not maintained until the moment of the experimental infection. After the experimental infection, the serum levels of IgG in rabbits immunized with proteoliposomes showed a significant increase, while for those animals immunized with the CSE the levels were maintained. The analysis of IgM antibodies specific for the P. multocida showed a higher production to animals vaccinated with proteoliposomes than for the animals immunized with CSE. Furthermore, after experimental infection, the production of IgM in animals immunized with proteoliposomes continued to be stimulated, which was not observed for those immunized with EBS. The proteoliposome system does not induce IgA systemic antibodies that were specific for the bacterium. However, after the experimental infections it was possible to observe the gradual appearance of IgA in the nasal lavage of the infected animals on the time course of the experiment. Animals previously immunized with the proteoliposomes which survived the first experimental infection were observed during 140 days and re-infected. After the re-infection, the survival of these animals was 100 %, indicating that the proteoliposome system was able to generate a possible immunological memory. The global analysis of the results obtained in the antibody detection indicates that the protection given by the proteoliposome against pasteurellosis is due to the stimulation of antibodies IgG and mainly of IgM. The other delivery system of antigenic proteins developed during this work is of lipidic microspheres. Different protocols were tried, but the one which was more adequate to our experimental conditions was elaborated from joining and adapting two methodologies described in literature. Scanning electron microscopy studies have shown that the lipidic microspheres are formed when 3 % (w/v) of PVA is used in the formulation. Furthermore, we have marked the proteins with fluorescein isothiocyanate and the microscopy revealed the presence of fluorescent spherical structures which indicated the encapsulation of the proteins in the lipophilic region of the microspheres. Systematic studies varying the concentration of oil, phospholipid, proteins and PVA in the microcapsules formulation has given a yield of encapsulation of 99%. We have established methodologies of incorporation of the antigenic proteins in liposomes constituted of DPPC and lipidic microspheres. Moreover, the proteolipossome systems have shown a satisfying property of protection of rabbits against pasteurellosis in face of the experimental challenge with P. multocida indicating that the system proposed here can be used as a vaccine to prevent the pasteurellosis either in commercial or biomedical research rearing of rabbit.
Advisor:Pietro Ciancaglini; Maria Helena Bueno da Costa; Iolanda Midea Cuccovia; Eneida de Paula; Richard John Ward; Pietro Ciancaglini
School:Universidade de São Paulo
Source Type:Master's Thesis
Keywords: antigenic membrane proteins Delivery
Date of Publication:12/07/2004