Identification of Circulating Burkholderia pseudomallei Antigens and Development of Chimeric Antibodies

Abstract

Burkholderia pseudomallei is the causative agent of melioidosis, an emerging tropical disease. Thousands contract this disease every year, mainly in Southeast Asia and northern Australia. Disease manifestations range from fatal acute septicemia to a chronic latent infection. Diagnosis is challenging because melioidosis shares symptoms commonly produced by other pathogens. The diagnostic standard remains culturing B. pseudomallei from patient samples. This can be challenging since the level of bacteria in patient samples is often very low. Our lab has previously used a novel technique termed In Vivo Microbial Antigen Discovery (InMAD) to identify B. pseudomallei diagnostic antigens secreted during a murine model of melioidosis. We have modified this technique by immunizing mice with filtered urine or sera from melioidosis patients. Mice generate an antibody response against the B. pseudomallei antigens present in the patient samples. The mouse serum is used to probe a B. pseudomallei lysate by 2D immunoblot; reactive proteins are identified by mass spectrometry. Proteins identified by mass spectrometry are then targeted by monoclonal antibodies for the rapid diagnosis of melioidosis.Melioidosis is difficult to treat, due to its inherent resistance to common antibiotics. Therefore, novel methods of treatment are vital to increase patient survival from this disease. Immunotherapy is one option that could be used as an adjunct to antibiotics. Our laboratory and others have shown that monoclonal antibodies (mAb) specific to the B. pseudomallei capsular polysaccharide (CPS) can prevent disease in mouse models of melioidosis. Our goal is to produce a chimeric mouse/human mAb specific to CPS that may be useful in treating human disease. The variable region of a capsule specific (mAb) was cloned and sequenced. This variable region was then cloned into a human IgG1 expression vector.