Specificities and Sensitivities of Four Monoclonal Antibodies for Typing of Borrelia burgdorferi Sensu Lato Isolates

Abstract

Borrelia burgdorferi, the agent of Lyme borreliosis, is genetically more heterogeneous than previously thought. In Europe five genospecies have been described from the original B. burgdorferi sensu lato (sl): B. burgdorferi sensu stricto (ss), B. garinii, B. afzelii, B. lusitaniae, and B. valaisiana. In the United States, B. burgdorferi ss as well as B. bissettii in California and B. andersonii on the East Coast were differentiated. In Asia, B. japonica has been identified along, with B. garinii, B. afzelii, and B. valaisiana. In order to evaluate sensitivity and specificity of four species-specific monoclonal antibodies, we analyzed 210 B. burgdorferi sl isolates belonging to eight genospecies by immunoblot and confirmed genospecies by restriction fragment length polymorphism (RFLP) of rrf (5S)-rrl (23S) intergenic spacer amplicon. Monoclonal antibody H3TS had 100% sensitivity for 55 B. burgdorferi ss isolates but showed reactivity with all four isolates belonging to B. bissetii. Monoclonal antibody I 17.3 showed 100% specificity and sensitivity for 45 B. afzelii isolates. Monoclonal antibody D6 was 100% specific for B. garinii but missed 1 of 64 isolates (98.5% sensitivity). Monoclonal antibody A116k was 100% specific for B. valaisiana but was unreactive with 4 of 24 isolates (83.5% sensitivity). Genetic analysis correlated well with results of reactivity and confirmed efficacy of the phenotypic typing of these antibodies. Some isolates showed atypical RFLP. Therefore, both phenotypic and genotypic analyses are needed to characterize new Borrelia isolates.Borrelia burgdorferi, the agent of Lyme borreliosis, has been found to be genetically more heterogeneous than previously thought. In Europe, five genospecies have been described from the original B. burgdorferi, now called B. burgdorferi sensu lato: B. burgdorferi sensu stricto, B. garinii, B. afzelii, B. lusitaniae, and B. valaisiana (3, 9, 19, 32 (1,2,24,29). We have previously reported serological evidence for a pathogenic potential of B. valaisiana in humans (29). Sera from three patients with neuroborreliosis and from one patient with Lyme arthritis showed higher reactivity with this Borrelia species.Genetic analysis based on 16S rDNA, restriction fragment length polymorphism (RFLP), arbitrarily primed PCR, and other methods for phylogenetic study of bacterial population, such as multilocus enzyme electrophoresis, all confirmed the subdivision of B. burgdorferi sensu lato into different species worldwide.The serotyping method developed by Wilske et al. (34) and classification based on protein profiles provided similar data. Monoclonal antibodies specific to some of these species have been described (3,9,22), and a new monoclonal antibody to B. valaisiana has been produced in our laboratory. In the present study, we evaluated the specificity and sensitivity of four species-specific monoclonal antibodies based on the analysis of 210 isolates of B. burgdorferi sensu lato. MATERIALS AND METHODSCulture of Borrelia isolates. All isolates (Table 1) were cult...

OspA heterogeneity of Borrelia valaisianaconfirmed by phenotypic and genotypic analyses

“…Phenotypic [16,21,22] and genetic [23] analyses of European isolates of B. burgdorferi sl. have demonstrated antigenic and structural heterogeneities of the OspA protein.…”

“…isolates were all characterized by RFLP of amplicons digested by Mse I (Boehringer, Mannheim, Germany) and Dra I (Life technology, CH) as described by Postic [18]. All the Borrelia isolates were submitted to reactivity of mAb H3TS, I 17.3, D6 and A116k [16]…”

“…Electrophoretic mobility of the major proteins allows us to distinguish most Borrelia genospecies, but monoclonal antibodies (mAb) are used to formally identify species on immunoblots. Up to now, only B. burgdorferi ss., B. garinii and B. afzelii among European isolates could be characterized by mAbs, H3TS was directed against OspA of B burgdorferi ss., D6 directed against a 12 kDa protein of B. garinii and I17.3 reacted with OspB of B. afzelii [16]; specific primers GI, GII and GIII were directed against ospA gene of Borrelia burgdorferi ss, B. garinii and B. afzelii , respectively [17]. In this study, the major outer surface protein OspA of the reference strain VS116 was selected as antigen for mAbs production, because of its particular protein profile in SDS-page and its potential discriminative utility.…”

Development of a sequence-basedin silicoOspA typing method forBorrelia burgdorferisensu lato

“…Moreover, some of the OspA-specific mAb reagents have been prone to cross-reactivity due to high sequence homology. As an example, cross-reactivity between B. burgdorferi and B. bissettii has been reported using H3TS, an OspA-specific mAb used for the identification of OspA ST1 (42). By comparison, the IST pipeline described herein unambiguously differentiates B. burgdorferi as IST1 based on OspA sequence.…”