Phage display techniques have been widely employed to map the
epitope structures which served as the basis for developing
molecular vaccines. In the present study, we applied this technique to map the
epitopes of Mycoplasma hyopneumoniae, the etiologic agent causing
swine enzootic pneumonia, and evaluated directly the immune responses in mice of the selected phage-displayed
epitopes (phagotopes). Two phage-displayed
random peptide libraries were biopanned with the
protein A-purified
IgG of the rabbit anti-M. hyopneumoniae hyperimmune serum and the selected phage clones were sequenced and analyzed. Some of the inserts of the selected phagotopes showed a good match with the known
proteins of M. hyopneumoniae. Others, which did not match with any known
proteins, but shared extensive homology with each other, were clustered and classified as the conformational
epitopes of M. hyopneumoniae. To evaluate the potential of using these phagotopes as effective
vaccines, several phage clones were chosen to immunize mice.
IgA coproantibody,
IgA in bronchoalveolar lavage fluid and serum
IgG responses were assayed. The serum raised by the phage clones clearly recognized several major mycoplasmal
proteins indicating that the phagotope-induced immune responses were
antigen-specific. The stronger
IgG1 response revealed that the immune responses of the
epitope-displaying phage were mainly through Th2 activation. The growth inhibition assay showed that the selected phage clones CS4 and varphi58 are potential
vaccine candidates and suggested that the mycoplasmal 97 kDa, 56 kDa, 30 kDa and 23 kDa
proteins may play important roles in the immune responses. The present work demonstrates that the whole
epitope profile of a microorganism can be obtained through screening the phage displayed
peptide libraries with the hyperimmune serum and reveals the potential of using
epitope-displaying phages as
peptide vaccines.