Borrelia burgdorferi sensu lato (s.l.) is a group of gram-negative bacteria that are the etiological factor of Lyme disease. Currently, this group includes 21 genospecies, six of them have been found to be pathogenic to humans i.e. Borrelia afzelii, Borrelia garinii, B. burgdorferi sensu stricto (s.s.), Borrelia spielmanii, Borrelia bavariensis, and Borrelia mayonii (Lohr et al., 2018).

Since the symptoms of Lyme disease, apart from erythema migrans, are not specific enough for a definite diagnosis, serodiagnostic methods that allow the detection of specific antibodies are the basis for diagnosis. Currently, a two-stage serological test is recommended. A highly sensitive ELISA (enzyme-linked immunosorbent assay) is performed as a first step test. When a positive or doubtful result is obtained, more specific Western blot (WB) is used as a confirmatory test (Lohr et al., 2018).

Despite many years of development, immunodiagnosis of Lyme disease faces numerous problems. This is primarily due to the relatively low conservation of the amino acid sequence of proteins between different representatives of B. burgdorferi s.l. Most serological tests use crude whole-cell lysates (WCL) obtained from single genospecies as antigens, leading to variable sensitivity of the immunoassay depending on which genospecies caused the infection. Additionally, B. burgdorferi s.l. shares common amino acid sequences with other pathogens and even with sequences found in the human body, which may cause false positive serodiagnostic test results (Bruckbauer et al., 1992; Markowicz et al., 2021). For all the above mentioned reasons, new cheap diagnostic tests to screen a large number of samples with a high sensitivity and specificity are needed to enable early detection and treatment of Lyme disease (Kodym et al., 2018; Webber et al., 2019).

Recombinant proteins, produced mainly in prokaryotic expression systems based on E. coli, are an alternative source of antigens. However, in order to ensure the highest efficiency of serodiagnostic tests, carefully selected recombinant proteins should be used (Branda et al., 2017; Grąźlewska et al., 2021; Kodym et al., 2018; Panelius et al., 2003). Preliminary selection of proteins can be carried out by B-cell epitope mapping, to estimate their immunoreactivity and/or identify immunodominant fragments. This approach is predominantly used for the design of vaccines (Batool et al., 2018; Contreras et al., 2022; de la Fuente et al., 2022; Soltan et al., 2020), but it also has applications in the design of new diagnostic tools (Arnaboldi et al., 2022; Maksimov et al., 2012).

Peptide microarrays are most commonly used to identify linear B-cell epitopes. Microarrays consist of many short peptides (15–20 aa) printed on a solid surface with overlapping amino acids. The complete antigen sequence is examined for fragments that are recognized by specific antibodies (Heiss et al., 2020). X-ray crystallography of antigen-antibody complexes is believed to be the most accurate method of mapping structural epitopes, as this technique guarantees the precise identification of both continuous and discontinuous epitopes and provides information about the binding strength (Bahrami et al., 2019). However, most of the available epitope mapping methods are expensive and time-consuming (Potocnakova et al., 2016).

Advances in genomics, proteomics and computational methods have significantly contributed to the development of immunoinformatics. Computational methods are used for predicting the structure of antibodies, B-cells, T-cells and allergens, as well as predicting MHC binding, and identifying epitopes. Therefore, employing specialized software for epitope mapping facilitates the rapid identification of antigenic regions that may be useful in diagnostics or therapeutics (Fathollahi et al., 2021; Maksimov et al., 2012; Potocnakova et al., 2016; Zheng et al., 2017). The search for potential antigens that may indicate diagnostic utility should begin with surface proteins as they are the first molecules to interact with the host organism's cells and tissues. These proteins are involved in the infection process and/or pathogen-host interactions. In addition, in the case of B. burgdorferi s.l., greater attention should be paid to antigens that are increasingly produced after transmission to the mammalian organism and those that are conserved among genospecies.

In this study, the epitopes of 9 B. burgdorferi s.l. (BB0108, BB0126, BB0298, BB0689, BB0323, FliL, PstS, SecD, EF-Tu) proteins were mapped using computational methods to pre-select those that are worth experimentally evaluating for diagnostic utility.