Evaluation of antibody response to group 2 outer membrane proteins of Brucella abortus S99 in animal model

Abstract number: P652

Siadat S.D., Karami S., Tabaraiee B., Norouzian D., Nejati M., Aghasadeghi M., Sadat S.M., Kheirandish M., Zangeneh M., Sharifat Salmani A., Moshiri A., Ahmadi H., Izadi Mobarakeh J.

Objective: Outer membrane proteins of Brucella are considered as potential immunogens to develop a Brucellosis subunit vaccine. Although the antigen that dominates the humoral response in brucellosis is the lipopolysaccharide, outer membrane proteins would be characterised and evaluated since they are T-dependent antigens. In this study, Group 2 of OMPs or Porins of Brucella abortus S99 was initially extracted through an optimised method. Following the immunisation of animal models with extracted Porins, antibody response against these OMPs analyzed by ELISA method to determine anti-Brucella IgG titer in the sera of immunised animals.

Methods: Cells were suspended at 10 mM tris buffer, and 1 mg each of DNase and was added per 100 ml. Samples were centrifuged twice to remove unbroken cells. Supernatants were submitted to ultracentrifugation to pellet the crude membranes. Detergent extraction of cytoplasmic membranes was performed by using sodium N-lauroyl sarcosinate. Resultant insoluble material was dialyzed against tris buffer. In order to isolate the peptidoglycan from outer membrane proteins, lysozyme (1 mg/50 mg of membrane protein) was added. Samples were finally ultracentrifuged and supernatants were kept at 4°C. After biochemical evaluations of the extracted sample, animal models were immunised intramuscularly with B. abortus Porins and hyper immune sera of immunised animals collected. Animal models were immunised in three different patterns: (1) B. abortus Porins, (2) B. abortus Porins in combination with B. abortus LPS, (3) B. abortus Porins in combination with Complete Freund's adjuvant. Finally, titer of the elicited specific anti-Brucella IgG assayed by ELISA method in the sera of immunised animals and titers expressed in OD units.

Results: Among the three different compounds applied to promote humoral immune responses in the animal models, B. abortus Porins in combination with B. abortus S99 LPS have been the most potent immunogen that significantly elicited the highest IgG titer against B. abortus (P < 0.05). The difference between IgG titer following the immunisation with B. abortus Porins and B. abortus Porins + Complete Freund's adjuvant has not been significant (P > 0.05).

Conclusion: The application of both B. abortus Porins and B. abortus LPS would be a promising aspect to design a human brucellosis subunit vaccine since Porins are able to promote T-dependent and Cell mediated responses while the highest titers of anti-Brucella IgG antibodies are directed against LPS.