Borrelial C6 peptides as antigens for serological diagnosis of Lyme borreliosis
Diagnosis of Lyme borreliosis depends on clinical signs supported by serological findings—enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Various B. burgdorferi sensu lato protein antigens are used for ELISA tests (OspA, OspC, FlaB, VlsE).
VlsE is the most promising diagnostic antigen among them due to the conserved immunogenic epitopes.VlsE gene consists of expression site and 15 silent cassettes with a high degree of homology and high rate of reassortments. Each cassette consists of six variable and six invariable regions. The 26-amino-acid-long sixth invariable region (IR6) is immunodominant and much conserved among B. burgdorferi sensu lato species. It was shown that IgG antibodies to IR6 (C6) are often detected in early and late Lyme borreliosis [1, 2]. These two statements, i.e. that C6: (1) possess high immunogenicity and (2) is highly conserved among species of the complex B. burgdorferi sensu lato, we decided to test in practice with serum samples from Bulgarian patients with Lyme disease.
Four 26-amino-acid-long peptides were synthesized (ProteoGenix SAS, France), which corresponded to IR6 regions of VlsE proteins from B. burgdorferi sensu stricto, B. garinii, and B. afzelii. Because of the previously described difference in reactivity [1], two peptides were synthesized from two strains of B. burgdorferi sensu stricto—B31, isolated from a tick and 297, isolated from cerebrospinal fluid of a patient with neuroborreliosis.
Four different peptide ELISA tests were developed based on IR6 regions of two B. burgdorferi sensu stricto strains (B31 and 297), of one B. afzelii (PT7) and one B. garinii (IP90) strain. Two different serum panels were tested. The first one consisted of serum samples from Bulgarian patients with Lyme borreliosis—50 sera from patients with erythema migrans (clinical hallmark of early Lyme borreliosis), 20 sera from patients with neuroborreliosis, and 10 sera from patients with Lyme arthritis.
This serum panel was used to analyze sensitivity of the tests. It contained 40 serum samples from patients with known cross-reactive serological results— patients with syphilis (n = 10), leptospirosis (n = 10), rheumatoid arthritis (n = 10), and sclerodermia (n = 10). The second serum panel was applied to test specificity of the developed peptide ELISA tests.Test results showed that the two C6 peptides from B. burgdorferi sensu stricto had higher reactivity than the corresponding C6 peptides from B. afzelii and B. garinii with sera from patients with erythema migrans and those with Lyme arthritis. On the contrary, the C6 peptide from B. garinii was more reactive with sera from patients with neuroborreliosis [3]. The two peptides from B. burgdorferi sensu stricto showed different reactivity with sera from patients with erythema migrans (Table 1).
Concerning non-specific reactivity of the peptide antigens with sera from patients with syphilis, leptospirosis, rheumatoid arthritis, and sclerodermia, the lowest level of specificity (87.5%) was found for the C6 peptide from B. afzelii; specificity was higher (90% and 92.5%) with the C6 peptides from B. burgdorferi s.s. and highest (100%) with the C6 from B. garinii. Overall, specificity of the four peptides was high [3].
In order to test applicability of the C6 peptide for serological diagnosis, we used peptide ELISA tests for detection of antibodies in Lyme borreliosis. Four peptide antigens from the C6 regions of VlsE proteins from the three Borrelia species that mainly cause Lyme disease in Europe were tested. The findings were very promising since up to 80% of the patients with early Lyme borreliosis and neuroborreliosis and all patients with Lyme arthritis can be diagnosed by the peptide ELISA tests. In addition, overall specificity of the C6 tests was high (87.5-100%). Notably, the tests are easy to perform and cheap as the peptide synthesis is much more easy to implement than the production of recombinant protein antigens.
The C6 peptides from B. burgdorferi sensu stricto showed the highest sensitivity in detection of specific anti-borrelia antibodies in patients with early Lyme disease
| Serum panel | C6 B31 (B. burgdorferi s.s.) | C6 297 (B. burgdorferi s.s.) | C6IP90 (B.garinii) | C6 PT7 (B. afzelii) |
| Erythema migrans (n = 50) | 36 (72%) | 39 (78%) | 26 (52%) | 29 (58%) |
| Neuroborreliosis (n = 20) | 11 (55%) | 11 (55%) | 16 (80%) | 9 (45%) |
| Lyme arthritis (n = 10) | 10 (100%) | 10 (100%) | 8 (80%) | 8 (80%) |
| Total number of reacted samples (% sensitivity) | 57 (71.3%) | 60 (75%) | 50 (62.5%) | 46 (57.5%) |
| Number of reactive sera from | 3/40 (92.5%) | 4/40 (90%) | 0 (100%) | 5/40 (87.5%) |
patients with other diseases (% specificity)
Table 1.
Reactivity of peptide C6 ELISA with serum panels of patients with Lyme disease in Bulgaria.
from Bulgaria. Our previous studies on borrelia infections of Bulgarian ticks have shown that the ticks are mostly infected with B. afzelii, followed by B. burgdorferi sensu stricto and B. garinii [4]. The discrepancy between the abundance of B. afzelii in our ticks and predominant B. burgdorferi sensu stricto reactivity of Lyme borreliosis patients could be explained by different pathogenic potential of the Borrelia species.
It is well known that different Borrelia species cause predominantly certain clinical manifestations: neuroborreliosis is associated with B. garinii and Lyme arthritis with B. burgdorferi sensu stricto [5]. This finding may explain the higher reactivity of sera from patients with neuroborreliosis with the C6 peptide from B. garinii as well as the predominant reactivity of the sera from Lyme arthritis with the C6 from B. burgdorferi sensu stricto.
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