Epitopes within recombinant α-actinin protein is serodiagnostic target for Trichomonas vaginalis sexually transmitted infections

Abstract

To date there is no commercially-available serodiagnostic for women and men infected with Trichomonas vaginalis. Thirteen epitopes of the immunogenic T. vaginalis α-actinin (106.2-kDa) are detected by sera of women patients, and 5 epitopes, a subset of the 13, are detected by sera of men. A truncated recombinant protein called ACT-P2 (63.5-kDa) encoding the 5 epitopes is used for screening by ELISA for antibody in sera of women and men. Because ACT-P2 is poorly expressed in E. coli, we wanted alternative recombinant α-actinin proteins as serodiagnostic targets. We, therefore, constructed plasmids encoding two novel, small recombinant proteins of ∼25-kDa comprised of 15-mer peptides, each peptide of which contains one of the 13 epitopes. We refer to these novel proteins as α-actinin::string-of-epitopes1 (ACT::SOE1) and ACT::SOE2. We found the proteins to be unrecoverable from insoluble inclusion bodies without denaturing conditions, which rendered the proteins unsuitable for antibody detection. Thus, we synthesized a third ACT::SOE3 protein (72.4-kDa) with 7 epitopes that was synthesized in high amounts and was readily purified. Monoclonal antibodies to α-actinin detected ACT::SOE3 and ACT-P2 by ELISA. Further, we show that ACT::SOE3 is equal to ACT-P2 as a target protein for detection of serum IgG in positive sera of women and men. Data indicate that ACT::SOE3 is a target for screening of populations at-risk for this STI. Finally, the paper discusses the findings with regard to Point-of-Care diagnostic targets and vaccine candidates.

Advancing Prevention of STIs by Developing Specific Serodiagnostic Targets: Trichomonas vginalis as a Model

“…The description of ACT-P2 in E. coli has been described [24]. Both recombinant proteins have hexa-histidine at the carboxy terminus for purification [21,25].…”

“…The α-actinin protein is one of the most immunogenic proteins of T. vaginalis [26,27] and has been shown to be a serodiagnostic target because it has no sequence identity to other known proteins in databases [24]. The truncated α-actinin protein called ACT-P2 of 558-amino acids (64.1-kDa) has been previously described [25]. ACT-P2 has been used to screen both male and female patient sera [24] and more recently to examine the relation of serum antibody in men and prostate cancer [8][9][10].…”

“…This earlier report showed a proof-of-principle for the construction of a novel recombinant chimeric protein, called AEG::SOE, with two each of the A, E, and G epitopes of the three enzymes. This earlier construct, however, failed to detect some positive sera when compared with the gold standard immunogenic truncated version of α-actinin called ACTP2 [8][9][10]24,25]. In this report I test this hypothesis and develop a stepwise approach to show that a new recombinant protein, two epitopes of A, ten epitopes of E, and seven epitopes of G (AEG::SOE2), is a serodiagnostic target equal to ACTP2.…”

Humoral and T cell–mediated immune response against trichomoniasis

“…In addition, the α‐actinin protein and its epitopes are unique and invariant . A truncated recombinant form of α‐actinin called ACT‐P2 was utilized as a valuable target for screening of the specific antibodies in serum samples . The protective potency of the recombinant α‐actinin fragments against T. vaginalis infection was evaluated in a mouse model.…”

“…64 A truncated recombinant form of α-actinin called ACT-P2 was utilized as a valuable target for screening of the specific antibodies in serum samples. 65 The…”