Evaluation of the Borrelia burgdorferi BBA64 protein as a protective immunogen in mice

Abstract

The Borrelia burgdorferi bba64 gene product is a surface-localized lipoprotein synthesized within mammalian and tick hosts and is involved in vector transmission of disease. These properties suggest that BBA64 may be a vaccine candidate against Lyme borreliosis. In this study, protective immunity against B. burgdorferi challenge was assessed in mice immunized with the BBA64 protein. Mice developed a high-titer antibody response following immunization with soluble recombinant BBA64 but were not protected when challenged by needle inoculation of culture-grown spirochetes. Likewise, mice passively immunized with an anti-BBA64 monoclonal antibody were not protected against needle-inoculated organisms. BBA64-immunized mice were subjected to B. burgdorferi challenge by the natural route of a tick bite, but these trials did not demonstrate significant protective immunity in either outbred or inbred strains of mice. Lipidated recombinant BBA64 produced in Escherichia coli was assessed for possible improved elicitation of a protective immune response. Although inoculation with this antigen produced a high-titer antibody response, the lipidated BBA64 also was unsuccessful in protecting mice from B. burgdorferi challenge by tick bites. Anti-BBA64 antibodies raised in rats eradicated the organisms, as evidenced by in vitro borreliacidal assays, thus demonstrating the potential for BBA64 to be effective as a protective immunogen. However, passive immunization with the same monospecific rat anti-BBA64 polyclonal serum failed to provide protection against tick bite-administered challenge. These results reveal the challenges faced in not only identifying B. burgdorferi proteins with potential protective capability but also in producing recombinant antigens conducive to preventive therapies against Lyme borreliosis.

FIG 1

(A) Amino-terminal coding sequence of bba64 gene. Nucleotides in bold type represent the signal sequence, with the lipidation signal peptidase site underlined. The arrow denotes the start of the protein sequence cloned to express BBA64 minus the signal peptide. (B) SDS-PAGE stained with GelCode Blue (Thermo Fisher, Rockford, IL) (lanes 1 to 3) of soluble rBBA64 protein expression in E. coli. Lane 1, IPTG-induced culture lysate soluble fraction; lane 2, flowthrough fraction from His tag affinity Ni column; lane 3, elution fraction from column; lane 4, immunoblot (IB) of elution fraction with anti-BBA64. (C) SDS-PAGE stained with GelCode Blue of full-length lipidated rBBA64 protein expression in E. coli. (D) Immunoblot of lipidated rBBA64 using anti-BBA64. (E) Click-iT blot for detection of lipidated proteins. (+/+), IPTG-induced E. coli culture expressing BBA64 plus labeled palmitic acid; (−/+), noninduced E. coli culture containing BBA64 expression plasmid plus labeled palmitic acid; (+/−), IPTG-induced E. coli culture expressing BBA64 minus labeled palmitic acid; (ap), affinity-purified lipidated rBBA64 minus labeled palmitic acid. (B to E) Numbers on left denote molecular mass in kilodaltons. MW, molecular weight standards; IB, immunoblot.

FIG 2

(A) ELISA of mouse anti-rBBA64 IgG antibodies 8 to 14 days after final rBBA64 boost and prior to tick challenge. Bars represent the mean (with standard error) OD₄₀₅ of pooled serum samples at 1:25,600. Shown are 3 groups of soluble rBBA64-immunized mice (n = 10, 4, and 5, left to right), lipidated rBBA64-immunized mice (n = 10), and representative PBS-adjuvant-only-immunized mice (n = 5). Mice were boosted twice with soluble rBBA64 or once with lipidated rBBA64. (B) Representative immunoblot showing BBA64-specific reactivity of immunized mouse serum against B. burgdorferi whole-cell lysate (left). The anti-BBA64 monoclonal antibody served as a reference blotted against the B. burgdorferi whole-cell lysate (right).