The protein expression system impacts the efficacy of Phlebotomus papatasi SP32 as a marker of exposure in humans, dogs, and mice

Background Phlebotomus papatasi (Diptera: Phlebotominae) females are bloodfeeding insects that transmit sand fly-borne pathogens of medical and veterinary importance. Control measures include serological assays that evaluate anti- P. papatasi saliva antibodies elicited in repeatedly bitten hosts as a marker of exposure. Recombinant P. papatasi salivary protein SP32, produced in mammalian HEK cells, has recently been validated for use in assessing human exposure to this sand fly species. Methods To improve assay performance, we compared the reactivity of recombinant SP32 produced in three different expression systems: (1) HEK cells, as used in previous studies, (2) Escherichia coli , a widely used and cost-effective option, and (3) Sf9 insect cells, which should provide the most natural option for producing recombinant sand fly proteins. We assessed the reactivity of these three proteins using sera from three different host species—humans and dogs naturally exposed to sand flies in endemic areas, and mice experimentally exposed to a single insect species. Results Using salivary gland homogenate as the reference antigen, SP32 produced in Sf9 cells provided comparable results to, or even outperformed the other two recombinant proteins in terms of assay sensitivity and specificity, correlation, and insect species specificity. The SP32-Sf9 protein was the most effective marker of exposure with human sera; however, the assay parameters were better with canine sera. The presence of O-glycosylation in HEK expressed SP32 protein and its absence in SP32 proteins produced in E. coli and Sf9 cells may contribute to the observed antigenic differences. Conclusions The expression system and its features should be carefully selected and tailored for the purpose of recombinant protein expression, balancing well its advantages and disadvantages.