Conformational Nature of the Borrelia burgdorferi Decorin Binding Protein A Epitopes That Elicit Protective Antibodies

Decorin binding protein A (DbpA) has been shown by several laboratories to be a protective antigen for the prevention of experimental Borrelia burgdorferi infection in the mouse model of Lyme borreliosis. However, different recombinant forms of the antigen having either lipidated amino termini, approximating the natural secretion and posttranslational processing, or nonprocessed cytosolic forms have elicited disparate levels of protection in the mouse model. We have now used the unique functional properties of this molecule to investigate the structural requirements needed to elicit a protective immune response. Genetic and physicochemical alterations to DbpA showed that the ability to bind to the ligand decorin is indicative of a potent immunogen but is not conclusive. By mutating the two carboxy-terminal nonconserved cysteines of DbpA from B. burgdorferi strain N40, we have determined that the stability afforded by the putative disulfide bond is essential for the generation of protective antibodies. This mutated protein was more sensitive to thermal denaturation and proteolysis, suggesting that it is in a less ordered state. Immunization with DbpA that was thermally denatured and functionally inactivated stimulated an immune response that was not protective and lacked bactericidal antibodies. Antibodies against conformationally altered forms of DbpA also failed to kill heterologous B. garinii and B. afzelii strains. Additionally, nonsecreted recombinant forms of DbpAN40 were found to be inferior to secreted lipoprotein DbpAN40 in terms of functional activity and antigenic potency. These data suggest that elicitation of a bactericidal and protective immune response to DbpA requires a properly folded conformation for the production of functional antibodies. Lyme disease (41) or Lyme borreliosis, is caused by a group of related tick-borne spirochetes classified as Borrelia burgdorferi sensu lato (including B. burgdorferi sensu stricto, B. afzelii, and B. garinii). Recent clinical trials have shown that monovalent recombinant subunit vaccines composed of the Borrelia outer surface protein A (OspA) lipoprotein were efficacious through two Lyme disease transmission seasons (40, 42). The mechanism of this protective effect differs from that of other vaccines. The OspA protein is expressed by spirochetes in the tick midgut, but this protein is down regulated during tick engorgement (13) and in the mammalian host (7). Protection by immunization with OspA therefore involves prevention of transmission of the spirochetes from the tick to the mammalian host and is dependent on having a critical threshold level of antibodies at the time of the tick bite (12). The addition of mammalian host stage antigens to the OspA vaccines may extend the duration or enhance the level of protective efficacy of such transmission-blocking vaccines (28). Alternatively, vaccines composed of one or more mammalian-stage antigens may be effective without OspA. Several B. burgdorferi proteins expressed in the mammalian stage have been shown to be effective vaccines for preventing infection in laboratory animals challenged by experimental or natural routes. These protective antigens include OspC, P35/ BBK32, P66/Oms66, and decorin binding protein (14, 17, 19, 20, 25, 27, 34). Decorin binding proteins A and B (DbpA and DbpB) are B. burgdorferi lipoproteins (23, 27) that are surface exposed and may act as spirochetal adhesins (24). We have demonstrated that immunization of mice with DbpA protected them from challenge with cultured spirochetes (27), and others (17, 25) have confirmed this protection. DbpA is expressed in vivo during spirochetemia in the mouse model (7) and is recognized by human Lyme disease patient sera (8, 29). These data suggest a potential role for DbpA in an improved Lyme vaccine. Studies of DbpA vaccine effectiveness in other laboratories have relied on Escherichia coli vectors expressing cytosolic products as fusions to affinity tag sequences, a commonly used strategy for generating recombinant immunogens. However, recombinant cytosolic DbpA expressed as amino-terminal fusions to either polyhistidine (25) or glutathione S-transferase (GST) (17) was less than completely effective in these other studies. In the present study we sought to determine which form of the DbpA antigen would be most effective as a vaccine antigen and whether the protective efficacy of the protein was conformationally dependent. Due to the ability to measure ligand binding activity of the DbpA forms, we took advantage of the opportunity of being able to correlate function and therefore correct folding with the ability to elicit a protective response.