Linear Peptide Epitopes Derived from ErpP, p35, and FlaB in the Serodiagnosis of Lyme Disease

Abstract

Lyme disease is the most common vector-borne disease in the northern hemisphere. Current serodiagnostics are insensitive in early infection. Sensitivity in these seroassays is compromised by the necessity to preserve specificity in the presence of cross-reactive epitopes in Borrelia burgdorferi target antigens. We evaluated the efficacy of using synthetic peptides containing epitopes unique to B. burgdorferi as antigen targets in a Lyme disease seroassay. We performed linear B cell epitope mapping of the proteins p35 (BBH32) and ErpP to identify unique epitopes. We generated peptides containing these newly identified linear epitope sequences along with previously identified epitopes from the antigens FlaB and VlsE and evaluated their diagnostic capabilities via ELISA using large serum sets. Single-epitope peptides, while specific, demonstrated insufficient sensitivity. However, when epitopes from FlaB, ErpP, or p35 were combined in tandem with an epitope from VlsE, the sensitivity of the assay was significantly increased without compromising specificity. The identification of additional unique epitopes from other B. burgdorferi antigens and the further development of a combined multi-peptide-based assay for the laboratory diagnosis of Lyme disease offers a way to address the poor specificity associated with the use of whole protein antigen targets and thus significantly improve the laboratory diagnosis of Lyme disease.

Lyme borreliosis diagnosis: state of the art of improvements and innovations

“…The purpose is dual: i) solve the specificity issues thanks to peptides—rather than complex protein mixes—able to specifically capture serum Abs, and ii) solve the sensitivity issues thanks to the use of a rationalized variety of such peptides, each representing an immunodominant epitope from a bacterial mix. The final aim is to ensure the development of a multi-peptide-based assay [ 53 ].…”

Antibody Cross-Reactivity in Serodiagnosis of Lyme Disease

“…Even greater possibilities in the selection of fragments for the construction of chimeric proteins are provided by B-cell epitope mapping, which allows for detailed knowledge of the distribution of immunodominant fragments and potential cross-reaction fragments [ 135 , 136 ]. There are several methods for identifying conformational and linear epitopes, including both computational and experimental methods [ 137 , 138 ].…”

“…Experimental B-cell epitope mappings involve methods such as peptide microarray [ 140 ], X-ray crystallography of antigen-antibody complexes [ 141 ] and nuclear magnetic resonance [ 137 ]. Their main advantage is that they allow the identification of cross-reactive epitopes, thanks to which these fragments can be excluded and not used in the construction of chimeric proteins [ 135 , 136 , 142 ]. Another aspect is the possibility of distinguishing fragments of antigens that react with different classes of immunoglobulins [ 143 ] and thus designing chimeric proteins dedicated to the detection of a specific isotype of antibodies.…”

Epitope Mapping of BmpA and BBK32 Borrelia burgdorferi Sensu Stricto Antigens for the Design of Chimeric Proteins with Potential Diagnostic Value

“…Only the addition of a fragment derived from VlsE increased the sensitivity of ELISA. 55 In this study, the tertiary structure of B/32-M and B/32-G recombinant chimeric proteins may not contain any naturally occurring structural epitopes, as noncontiguous protein sequences in the native proteins have been joined together into a single amino acid chain. In addition, sequences -GGGwere introduced, forcing a linear conformation for the exposure of the epitopes to the antibodies, maintaining the plasmid were grown in LB medium supplemented with 100 μg/mL ampicillin and 34 μg/mL chloramphenicol, until the optical density at λ = 600 nm reached 0.4.…”

“…The FlaB fragment provided the highest sensitivity; tests based on it reached a sensitivity of 37.6%, which is insufficient for the effective diagnosis of Lyme disease. Only the addition of a fragment derived from VlsE increased the sensitivity of ELISA …”