Masters Theses

Date of Award

8-1992

Degree Type

Thesis

Degree Name

Master of Science

Major

Microbiology

Major Professor

Robert N. Moore

Committee Members

Thomas C. Montie, Michael A. Breider

Abstract

Pasteurella haemolytica A1 is commonly isolated from cattle exhibiting the shipping fever complex. Because pneumonic pasteurellosis is responsible for economic losses in the cattle industry, a vaccine to combat the disease is desired. Recently, leukotoxin culture supernatant was shown to confer some protection against intrabronchial challenge (53). Furthermore, recombinant leukotoxin enriched with culture supernatant of Pasteurella haemolytica A1 resulted in fewer clinical signs and pneumonic lesions in calves (16). Therefore, the leukotoxin appears to be an important virulence factor, and could be a potential target in vaccine development. Because anti-idiotypic antibodies have been shown to confer protective immunity against the hepatitis virus (34), Streptococcus pneumoniae (40) and other microorganisms, an anti-idiotypic antibody could be a potential vaccine component for pneumonic pasteurellosis. Monoclonal and polyclonal approaches were taken to develop anti-idiotypic antibodies to Mab-2N, a neutralizing monoclonal antibody (Mab-2N) directed against an epitope of the leukotoxin. Twelve clones produced by the fusion of a splenocytes from a mouse injected with Mab-2N with SP2/O cells secreted Mabs that inhibited binding of Mab-2N to the leukotoxin and bound directly with Mab-2N as shown by ELISA. Three of these Mabs abolished protection conferred by Mab-2N against the leukotoxin in a neutral red cytotoxicity assay. Eight of these Mabs exhibited binding to glutaraldehyde fixed BL-3 cells, target cells for P. haemolytica leukotoxin. When injected into rabbits, none of the clones injected produced neutralizing antibodies, indicating the possibility that none of the Mabs were internal image anti-idiotypic antibodies (Ab2β) or that the immunogens were not optimally presented for response. Three rabbits that were injected with Mab-2N produced polyclonal sera giving high ELISA titers of anti-2N antibodies. The rabbit sera also showed inhibition of 2N-Horseradish Peroxidase (HRP) binding to affixed leukotoxin. The polyclonal rabbit sera was purified by immunoaffinity chromatography. An affinity column containing non-neutralizing IgG Mabs, 35, 58 and 81, and another affinity column containing non-neutralizing IgG Mabs 4 and 37 were used to deplete the anti-idiotypic polyclonal sera of anti-isotypic antibodies. Finally, an affinity column containing Mab-2N was used to obtain polyclonal anti-idiotypic antibodies. The purified polyclonal anti-idiotypic antibodies abolished Mab-2N protection of BL-3 cells from the leukotoxin. In addition these antibodies inhibited binding of Mab2N-HRP to affixed leukotoxin. In order to confirm the presence of Ab2β, mice were injected with the purified polyclonal antibodies. Neutralizing antibodies were produced, as measured by a neutral red cytotoxicity assay. It appears that the monoclonal antibodies were Ab2γ antibodies or perhaps Ab2β antibodies which were not optimally presented to the rabbits so that they could respond. However, it appears that Ab2β antibodies were present in the purified polyclonal sera.

Download
Files over 3MB may be slow to open. For best results, right-click and select "save as..."

Share

COinS