Selection of peptides from random peptide libraries for a recombinant vaccine against dermatophilosis

Dermatophilosis commonly known as "lumpy wool" in sheep in Australia, is a skin disease of animals and man caused by Dermatophilus congolensis. In sheep dermatophilosis causes significant wool production losses both directly and indirectly due to down grading of wool and hides, low meat production and high mortality. particularly in young sheep. Control of the disease by vaccination is desirable but has only been partially successful and antigens are difficult to produce in sufficient quantity from D. congolensis. Dr T.M. Ellis and colleagues (Agriculture WA) have developed a vaccine that can produce a significant reduction in ovine dermatophilosis against several strains of the bacterium. In pen and field studies, the haemolysin based vaccine did not prevent initial infection but approximately 70% were protected against the development of lumpy wool. Their previous research showed that a vaccine prepared from a serine protease produced by D. congolensis also gave some protection against the disease. This antigen was expensive to prepare by conventional culture. An alternative approach is to use recombinant protein as an antigen. However, this is not a simple task if the protective antigens have not been identified and even if they have been, it can be difficult to prepare a recombinant protein in high yield. Random peptide libraries provide an alternative approach to the identification of peptides which might be useful in a vaccine. While these libraries have been used to identify epitopes and prepare diagnostic tests, their potential to produce antigens which could generate a protective immune response has still to be explored. The main aim of this thesis was to use random peptide libraries to identify new antigens which could be used in the future to immunise sheep and other animals against dermatophilosis. Because of the commercial availability of large random peptide libraries displayed on phage and flagellin there is an opportunity to produce low cost and immunologically potent peptide vaccines. To explore the effectiveness and reliability of random peptide libraries (Ph.D.™ and FliTrxTM libraries) for the selection of peptides, polyclonal antibodies against a recombinant serine protease from D. congolensis were used to pan the libraries. This recombinant serine protease was produced using a pQE expression vector and purified by immobilised metal affinity chromatography. Clones selected from the libraries were sequenced and the peptides aligned with the original amino sequence of serine protease. Many of the peptides aligned with varying homology to the serine protease sequence which demonstrated that the antibodies were selecting specific sequences from the libraries rather than just random peptides. To obtain peptides, which might be associated with a protective immune response to D. congolensis sheep which had been immunised with a crude enzyme preparation form D. congolensis by Dr T.M. Ellis were used as a source of antibodies. Four peptides from Ph.D.TM and three peptides from FliTrx™ libraries were chosen for vaccination of sheep. Sheep were given two doses of vaccine one month apart. Twentyone days after the second vaccination each sheep was challenged with a zoospore suspension of the MB and W14 strains of D. congolensis and the presence of lesions and their severity were measured at 7, 14 and 21 days after challenge. The immune response to D. congolensis antigens was also studied. There was a striking production of antibodies to antigens from D. congolensis induced by the peptides selected from the random peptide libraries. Vaccination with recombinant serine protease and with the peptides selected from the Ph.D.™ library increased the rate of resolution of the lesions caused by one strain of D. congolensis. The results in this thesis provide the first demonstration in large animals that phage displayed peptides can induce a specific immune response against an infectious agent