The broad, long-term goal that is envisaged is the development of a """"""""second generation"""""""" of surface-antigen-based vaccines for the prevention of Lyme disease in humans. Towards this aim, the objective of this proposal is the identification and cloning of surface proteins of Borrelia burgdroferi that are antigenically conserved and whose conserved epitopes are targeted by antibody-dependent complement-mediated lysis (ADCL) in primate hosts. An initial assessment of whether the cloned antigens are subjected to antigenic variation will be made as well. In making this the proposal's objective, it is recognized that, although very significant progress has been made towards providing the develop a second generation of vaccines based on more conserved and non-variant surface antigens. This necessity arises because OspA is antigenically polymorphic, such that recombinant OspA derived from one strain of B. burgodorferi may not protect against a challenge infection with spirochetes of a different strain and, moreover, OspA is the subject of an as yet unidentified mechanism of antigenic variation. The strategy to be employed unfolds from the following results: using serum from rhesus monkeys infected with B. burgdorferi strain JD-1, an antibody reagent that specifically recognizes 4 surface antigens of this spirochete was generated. These antigens are neither OspA nor OspB. They were named P1-4, and it was demonstrated that they are the target of (ADCL) of B. burgdorferi, i.e. the expressed by strain B-31 and, moreover, that at least one isomorph of these molecules appears to be present also on the taxonomically distant strain IP-90, considered by some authors to belong to a different species altogether (Borreli garinii). It is planned to extend this survey of strains killed by anti- P1-4 antibodies to a larger number of isolates, encompassing 10 isolates from 3 different genospecies, and thus generate an order of priority for the cloning of P1-4, such that only those most conserved antigens will be cloned. Cloning of the selected subpopulation of P1-4, will be done with the aid of the anti-P1-4 antibody reagent mentioned above, and whole-DNA libraries made in the bacteriophage expression vector gammagt11. After the antigens are subcloned, overexpressed and purified, likely in the form of fusion proteins with the Escherichia coli maltose binding protein, rhesus monkeys will be immunized with the individual recombinant antigens and monospecific antisera will be generated. These sera will be used initially for two purposes: 1) to confirm that all strains expressing the cloned (conserved) surface antigens can be killed by ADCL in vitro, and 2) to assess whether the chosen antigens undergo antigenic variation, by quantifying the frequency of appearance of escape variants in vitro, under selective pressure induced by the monospecific antibodies. At this point an informed decision will be made on whether it is merited to solicit the large amount of money required to assess two or more surface antigens, individually or in combinations, as a comprehensive vaccine against Lyme disease in nonhuman primates. This is a desired next step to which the research described in this proposal should lead us.
