Abstract:There is a need for a rapid, accurate serodiagnostic test useful for both women and men infected by Trichomonas vaginalis, which causes the number one sexually transmitted infection (STI). Women and men exposed to T. vaginalis make serum antibody to fructose-1,6-bisphosphate aldolase (ALD), α-enolase (ENO), and glyceraldehyde-3-phosphate dehydrogenase (GAP). We identified, by epitope mapping, the common and distinct epitopes of each protein detected by the sera of women patients with trichomonosis and by the s… Show more
“…ACT-P2 was originally prepared in E. coli XL1-Blue (Strategene, La Jolla, CA) containing a plasmid with the truncated α-actinin DNA and with the kanamycin (Kan) resistance gene [ 8 , 9 , 17 ]. The fusion protein with hexa-histidine at the carboxy-terminal end was able to be purified, as before [ 17 , 18 ]. Fig.…”
Section: Methodsmentioning
confidence: 99%
“…Thus, an alternative α-actinin target is needed that is expressed in large amounts and is readily purified. In this report an approach is utilized as recently described [18] to produce two proteins of ∼25-kDa that incorporate all 13 epitopes of the trichomonad α-actinin protein identified earlier [17] . These two proteins are referred to as α- act inin:: s tring- o f- e pitopes1 (ACT::SOE1) and ACT::SOE2.…”
Section: Introductionmentioning
confidence: 99%
“…This protein is synthesized in large amounts and is readily purified. Immunoassay data indicates that this protein is an ideal target for detection of serum antibody present in the sera of individuals with this STI [ 17 , 18 ]. Finally, the paper discusses the approaches that may lead to development of novel highly-specific diagnostic immunotargets with potential for use as vaccines.…”
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.
“…ACT-P2 was originally prepared in E. coli XL1-Blue (Strategene, La Jolla, CA) containing a plasmid with the truncated α-actinin DNA and with the kanamycin (Kan) resistance gene [ 8 , 9 , 17 ]. The fusion protein with hexa-histidine at the carboxy-terminal end was able to be purified, as before [ 17 , 18 ]. Fig.…”
Section: Methodsmentioning
confidence: 99%
“…Thus, an alternative α-actinin target is needed that is expressed in large amounts and is readily purified. In this report an approach is utilized as recently described [18] to produce two proteins of ∼25-kDa that incorporate all 13 epitopes of the trichomonad α-actinin protein identified earlier [17] . These two proteins are referred to as α- act inin:: s tring- o f- e pitopes1 (ACT::SOE1) and ACT::SOE2.…”
Section: Introductionmentioning
confidence: 99%
“…This protein is synthesized in large amounts and is readily purified. Immunoassay data indicates that this protein is an ideal target for detection of serum antibody present in the sera of individuals with this STI [ 17 , 18 ]. Finally, the paper discusses the approaches that may lead to development of novel highly-specific diagnostic immunotargets with potential for use as vaccines.…”
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.
“…We obtained control specimens with scores ≤2+ from individuals without a history of T. vaginalis or other STIs, and specimens with scores ≥3+ from patients with known T. vaginalis infection [18]. Specimens with scores ≥2+ had no detectable reactivity to trichomonad proteins blotted onto nitrocellulose after SDS-PAGE of total T. vaginalis proteins and immunoblotting, whereas those with scores ≥3+ readily detected trichomonad proteins [19, 20]. Specimens with scores of 1+ and 2+ exhibited non-specific reactions above baseline by ELISA.…”
Purpose
Results from previous sero-epidemiologic studies of Trichomonas vaginalis infection and prostate cancer (PCa) support a positive association between this sexually transmitted infection and aggressive PCa. However, findings from previous studies are not entirely consistent, and only one has investigated the possible relation between T. vaginalis seropositivity and PCa in African-American men who are at highest risk of both infection and PCa. Therefore, we examined this possible relation in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial, including separate analyses for aggressive PCa and African-American men.
Methods
We included a sample of participants from a previous nested case-control study of PCa, as well as all additional Caucasian, aggressive and African-American cases diagnosed since the previous study (total n=438 Gleason 7 Caucasian cases, 487 more advanced Caucasian cases (≥Gleason 8 or stage III/IV), 201 African-American cases, and 1,216 controls). We tested baseline sera for T. vaginalis antibodies.
Results
No associations were observed for risk of Gleason 7 (odds ratio (OR)=0.87, 95% confidence interval (CI)=0.55–1.37) or more advanced (OR=0.90, 95% CI=0.58–1.38) PCa in Caucasian men, or for risk of any PCa (OR=1.06, 95% CI=0.67–1.68) in African-American men.
Conclusions
Our findings do not support an association between T. vaginalis infection and PCa.
“…We obtained negative control sera (scores ≤2+) from individuals without a history of Tv or other STIs, and positive control sera (scores ≥3+) from patients with Tv infection [17]. Negative control sera had no detectable reactivity to trichomonad proteins blotted onto nitrocellulose after SDS-PAGE of total Tv proteins and immunoblotting, whereas positive control sera readily detected trichomonad proteins by immunoblotting [16, 18]. Sera with scores of 1+ or 2+ gave non-specific reactions above baseline by ELISA.…”
Purpose
Previous epidemiologic studies have observed positive associations between Trichomonas vaginalis (Tv) serostatus and both prostate cancer (PCa) risk and mortality. However, only a few small older studies have examined Tv antibody persistence over time, all of which were composed mainly of female patients. Therefore, we examined Tv antibody persistence over time, as well as intra-individual variability, among middle- to older-aged men in the Southern Community Cohort Study (SCCS).
Methods
We tested baseline and repeat plasma specimens (collected 1–3 years later) from 248 male participants for Tv antibodies. We used the same enzyme-linked immunosorbent assay as in previous studies of Tv serostatus and PCa.
Results
At baseline, 46 (18.5%) participants were seropositive for Tv infection. Seventy-six percent of these men were still seropositive 1–3 years later. A similar proportion of men “seroconverted” (4.0%) as “seroreverted” (4.4%), all of whom had absorbance values near the cut-off point for seropositivity. Overall, substantial agreement was observed between baseline and repeat serostatus (kappa=0.72, 95% confidence interval: 0.60–0.83).
Conclusion
Tv seropositivity was largely persistent between plasma specimens collected 1–3 years apart from middle- to older-aged men. These high levels of persistence are similar to those observed for other sexually transmitted infections frequently investigated in relation to PCa.
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