Project description:Epitope mapping studies aim to identify the binding sites of antibody-antigen interactions to enhance the development of vaccines, diagnostics and immunotherapeutic compounds. However, mapping is a laborious process employing time- and resource-consuming M-bM-^@M-^Xwet benchM-bM-^@M-^Y techniques or epitope prediction software that are still in their infancy. For polymorphic antigens, another challenge is characterizing cross-reactivity between epitopes, teasing out distinctions between broadly cross-reactive responses, limited cross-reactions among variants and the truly type-specific responses. A refined understanding of cross-reactive antibody binding could guide the selection of the most informative subsets of variants for diagnostics and multivalent subunit vaccines. We explored the antibody binding reactivity of sera from human patients and Peromyscus leucopus rodents infected with Borrelia burgdorferi to the polymorphic outer surface protein C (OspC), an attractive candidate antigen for vaccine and improved diagnostics for Lyme disease. We constructed a protein microarray displaying 23 natural variants of OspC and quantified the degree of cross-reactive antibody binding between all pairs of variants, using Pearson correlation calculated on the reactivity values using three independent transforms of the raw data: (1) logarithmic, (2) rank, and (3) binary indicators. We observed that the global amino acid sequence identity between OspC pairs was a poor predictor of cross-reactive antibody binding. Then we asked if specific regions of the protein would better explain the observed cross-reactive binding and performed in silico screening of the linear sequence and 3-dimensional structure of OspC. This analysis pointed to the C-terminal helix of the structure as a major determinant of type-specific cross-reactive antibody binding. We developed bioinformatics methods to systematically analyze the relationship between local sequence/structure variation and cross-reactive antibody binding patterns among variants of a polymorphic antigen, and this method can be applied to other polymorphic antigens for which immune response data is available for multiple variants. Antibody profiling was performed on sera from Borrelia burgdorferi infected and non-infected humans and Peromyscus leucopus rodents against 23 variants of the surface protein OspC . For infected human serum samples, the OspC type of the infecting B. burgdorferi strain is unknown; for experimentally-infected P. leucopus serum samples, it is known. Of human serum samples, 55 were from infected individuals and 25 from naive controls. Of P. leucopus serum samples, 23 were from infected individuals and 7 were from naive controls.

Project description:The white-footed deermouse Peromyscus leucopus is a primary reservoir for the agents of Lyme disease and other zoonoses in North America. These and other species of Peromyscus are tolerant of the infection by the bacteria, protozoa, and viruses they host. This is mainly by mitigating the damaging effects of the innate immune response. In previous studies we demonstrated differences between P. leucopus and either inbred or outbred M. musculus in the degree of sickness and profiles of biomarkers after exposure to bacterial lipopolysaccharide, a TLR4 agonist. With the objective of developing a method for broadly assessing of innate immunity of capture-release mammals in nature, we evaluated using bulk and single cell RNA-seq primary dermal fibroblast cultures of P. leucopus and M. musculus in their short-term responses to a lipopeptide that is a TLR2 agonist. As we had observed for experimental animals, the fibroblast cultures of the two species displayed both similarities and differences in their responses to the agonist. The notable differences included the greater magnitude of an anti-viral profile of cytokines and other effectors in the deermouse fibroblasts and the occurrence of an interleukin-11 response in the mouse cultures but not deermouse. We also observed in both species' cultures an increased transcription of several types of endogenous retrovirus (ERV) elements after exposure of the cells to the agonist. The P. leucopus cells were distinguished from M. musculus cells in the generally shorter retroviral open reading frames among the differentially expressed sequences. This was consistent with previous findings about ERV transcription in P. leucopus and M. musculus and suggests a greater suppression of ERV activity in P. leuocopus. The results affirm the feasibility of this in vitro model for both laboratory- and field-based studies without need for euthanasia and that inherent differences between deermice and mice in innate immune responses can be demonstrated in primary fibroblasts as well as the animals themselves.

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