Ehrlichia chaffeensis and E. canis hypothetical protein immunoanalysis reveals small secreted immunodominant proteins and conformation-dependent antibody epitopes

Immunomolecular characterization of Ehrlichia chaffeensis (E. ch.) and E. canis (E. ca.) has defined protein orthologs, including tandem repeat proteins (TRPs) that have immunodominant linear antibody epitopes. In this study, we combined bioinformatic analysis and cell-free protein expression to identify undiscovered immunoreactive E. ch. and E. ca. hypothetical proteins. Antigenicity of the E. ch. and E. ca. ORFeomes (n=1105 and n=925, respectively) was analyzed by the sequence-based prediction model ANTIGENpro, and we identified similar to 250 ORFs in each respective ORFeome as highly antigenic. The hypothetical proteins (E. ch. n=93 and E. ca. n=98) present in the top 250 antigenic ORFs were further investigated in this study. By ELISA, 46 E. ch. and 30 E. ca. IVTT-expressed hypothetical proteins reacted with antibodies in sera from naturally E. ch.-infected patients or E. ca.-infected dogs. Moreover, 15 E. ch. and 16 E. ca. proteins consistently reacted with a panel of sera from patients or dogs, including many that revealed the immunoreactivity of gold standard TRPs. Antibody epitopes in most (>70%) of these proteins exhibited partial or complete conformation-dependence. The majority (23/31; 74%) of the major immunoreactive proteins identified were small (<= 250 aa), and 20/31 (65%) were predicted to be secreted effectors. Unlike the strong linear antibody epitopes previously identified in TRP and OMP orthologs, there were contrasting differences in the E. ch. and E. ca. antigenic repertoires, epitopes and ortholog immunoreactivity. This study reveals numerous previously undefined immunodominant and subdominant antigens, and illustrates the breadth, complexity, and diversity of immunoreactive proteins/epitopes in Ehrlichia.