Structure-guided design of Anaplasma P44 protein-based chimeric antigens for serodiagnosis of anaplasmosis in dogs

ABSTRACT Canine and human anaplasmosis are tick-borne diseases caused by the obligate intracellular bacteria, Anaplasma phagocytophilum and Anaplasma platys , respectively. In veterinary medicine, peptides derived from the immunodominant P44 protein are used to detect antibodies in serological point-of-care tests for anaplasmosis. To inform efforts to develop improved chimeric diagnostic proteins for anaplasmosis, the structure of the predicted surface-exposed domain (residues 151–308) of P44 was determined at a resolution of 1.65 Å. This domain comprises six α-helices and six short β-strands connected by loop regions. A structural similarity search of the P44 151–308 domain using the Distance Matrix Alignment server against the AlphaFold Database revealed that the domain represents a previously uncharacterized protein fold. The determined structure is consistent with AlphaFold v2 predictions, which further predict the core of P44 to be an eight-stranded β-barrel. Using P44 fragments, immunodominant epitopes were mapped within residues 151–308. Four chimeric genes (ADC1, ADC2, ADC3, and ADC4) were designed that incorporate the analogous immunodominant P44 domains from A. phagocytophilum and A. platys . The recombinant ADC chimerics were assessed for sensitivity and specificity by screening with serum from dogs diagnosed with anaplasmosis, Lyme disease, or ehrlichiosis, with sera from specific pathogen-free dogs serving as negative controls. Accuracy was further demonstrated by screening characterized serum samples from coyotes ( Canis latrans ; wild canids). The ADC1 chimeric provided 100% sensitivity and 98.04% specificity. The sensitivity and specificity of ADC1 support its potential use as a diagnostic Ag for anaplasmosis.