Recombinant BBK32 Protein in Serodiagnosis of Early and Late Lyme Borreliosis

Abstract

Borrelial protein BBK32 was evaluated as an antigen in the serodiagnosis of early and disseminated Lyme borreliosis (LB). bbk32 was cloned and sequenced from eight isolates of the three pathogenic Borrelia species. The identities between the amino acid sequences of the BBK32 proteins from Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii isolates were 71 to 100%. By immunoglobulin G (IgG) Western blotting (WB) or enzyme-linked immunosorbent assay (ELISA), up to 74 and 100% of acute-and convalescent-phase samples, respectively, from 23 patients with erythema migrans (EM) were positive for recombinant BBK32 protein from B. afzelii. In the serology of disseminated LB, the three variant BBK32 antigens cross-reacted. In total, 14 of 14 samples from patients with neuroborreliosis and 15 of 15 samples from patients with Lyme arthritis were positive. The specificities of the IgG ELISA with the variant BBK32 antigens for EM and disseminated borreliosis were 81 to 92% and 89 to 95%, respectively. Our findings indicate that the BBK32 proteins are promising serodiagnostic antigens for the detection of early and disseminated LB but that variant BBK32 proteins may be needed either in parallel or in combination with an immunoassay for LB to cover all the relevant borrelial species that cause the disease.Lyme borreliosis (LB) is a tick-transmitted spirochetal infectious disease, caused by Borrelia burgdorferi, which is characterized by multistage skin, joint, neurologic, and cardiac manifestations (29). The diagnosis of LB is based on clinical evaluation of the patients, but serologic assays, most frequently, enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB), are often used to provide supporting evidence of infection with B. burgdorferi. The antigens predominantly used in the present routine LB serodiagnostic tests are borrelial flagellin protein or whole-cell lysate (WCL) of the microbes cultured in vitro. In different laboratories, the performances of serologic assays with these antigens in terms of their specificities and sensitivities are highly variable (26). Especially in LB patients with early manifestations, e.g., erythema migrans (EM) and facial palsy, antibody responses to the antigens used at present may be weak or delayed (22). In Europe, evaluation of the serologic responses is further complicated by the existence of three species of B. burgdorferi sensu lato as causes of human LB: B. burgdorferi sensu stricto, B. afzelii, and B. garinii (6). One of the main reasons for these problems is the antigenic diversity due to variations in the sequences and expression of immunogenic proteins in these different borrelial species (26,27).The expression of borrelial proteins also varies at different stages in the life cycle of Borrelia in ticks and in the mammalian hosts. Several genes, e.g., bbk32, bbk50, vls, and ospE and ospF homologs (4,8,10,12,14,25,(30)(31)(32)(33)36), have been shown to be selectively expressed in vivo. Moreover, bbk32 expression is detectable in spirochetes during tick f...

Improved serodiagnosis of erythema migrans using novel recombinant borrelial BBK32 antigens

“…However, our recent Abbreviations: EM, erythema migrans; LB, Lyme borreliosis. study suggested species-specific variations in the immune responses to the variant BBK32 proteins in early LB (Heikkilä et al, 2002). The rBBK32 antigen originating from a local B. afzelii strain appeared to be superior to the other heterologous rBBK32 variants.…”

“…Serum samples were collected from patients with clinically documented or culture-or PCR-confirmed EM from Finland, Germany, Slovenia and the USA. All 23 Finnish sera were from culture (n ¼ 18) and/or from PCR (n ¼ 21) proven patients taken at the time of diagnosis (acute) and 1-3 months after treatment (convalescent) (Heikkilä et al, 2002). In the 23 patients with EM, genotyping by PCR analysis (Junttila et al, 1999) showed B. afzelii in 17 and B. garinii in 4 of the skin biopsies.…”

“…bbk32 genes were cloned and sequenced from Finnish isolates of borrelial species representing B. afzelii, B. garinii and B. burgdorferi sensu stricto, as described previously (Heikkilä et al, 2002). The respective rBBK32 antigens were produced as glutathione-S-transferase-BBK32 fusion proteins, and used as separate antigens in ELISA.…”

“…The respective rBBK32 antigens were produced as glutathione-S-transferase-BBK32 fusion proteins, and used as separate antigens in ELISA. The IgG BBK32 ELISAs were performed as described earlier (Heikkilä et al, 2002). Briefly, serum samples were diluted 1 : 100 in 5 mg BSA ml À1 in 0·155 M NaCl-0·04 % Tween 20 buffer (BSA-NaCl-Tween).…”

“…New recombinant borrelial proteins or synthetic peptides have been tested in LB serology with promising results (Liang et al, 1999;Gomes-Solecki et al, 2001). We recently evaluated recombinant BBK32 (rBBK32) protein antigens in serologic assays for LB (Heikkilä et al, 2002). The rBBK32 protein appeared to be a useful antigen in early and late LB, especially in IgG serology, but not in IgM serology.…”