Recombinant Protein of Immunogenic Metabolic Enzyme Epitopes of Trichomonas vaginalis are Common to Humans and Microorganisms

Protein Immunogenic Metabolic Enzyme Epitopes vaginalis are Common to Abstract The number one, non-viral sexually transmitted infection (STI) worldwide is caused by the ancient protist . Recently, I reported on an ~40-kDa String-Of-Epitopes chimeric protein (AEG::SOE2) consisting of immunogenic epitopes unique to the vaginalis fructose-1,6bisphosphate aldolase (A), α -enolase (E), and glyceraldehyde-3-phosphate dehydrogenase (G). This report is on another construct of a 49.41-kDa AEG::SOE3 chimeric protein comprised of 8, 9 and 12 non-unique, immunogenic epitopes of A, E and G, respectively. These non-unique epitopes were detected by sera of women and men patients but not control, seronegative sera of women and men. The epitopes were found to have ≥50% to 100% sequence amino acid sequence identity with the human homolog and homologs of Candida albicans, Escherichia coli, Saccharomyces cerevisiae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes as well as the homologs of the STI agents Chlamydia trachoma- tis, Neisseria gonorrhoeae and Treponema pallidum . The AEG::SOE3 protein was not detected by mouse anti AEG::SOE2 serum and by monoclonal antibodies (MAbs) to AEG::SOE2, showing the distinctness of these two chimeric proteins. AEG::SOE3 was detected by ELISA and immunoblot with sera of mice immunized with fixed T. vaginalis organisms, with sera of women and men patients with trichomonosis, and with MAbs to AEG::SOE3. The patient sera reactive to these immunogenic metabolic enzyme epitopes of AEG::SOE3 may be indicative of immune-crossreactive antibodies to human proteins during this STI. The role of these possible immune-crossreactive antibodies produced during this STI and those conceivably gener- ated by other STIs and microbial pathogens to heretofore unappreciated disease pathogenesis is discussed.

that are unique to the T. vaginalis proteins [4]. A second recombinant protein target for a serodiagnostic test, referred to as ACT::SOE3, is a 72.4-kDa truncated version of the 106.2-kDa highly immunogenic α actinin protein to which women and men patients make serum IgG antibodies [7]. This protein is also unique to T. vaginalis in that it has no sequence identity with other proteins in databanks.
This protein target has seven epitopes detected by women, within which are five epitopes reactive with men patients [5,7]. Both the AEG:: SOE2 and ACT::SOE3 serodiagnostics have utility for largescale screening for this STI, which is preparatory to advancing the reproductive health of at-risk humans.
In the earlier paper on AEG::SOE2 [4] it became evident that A, E, and G also had immunogenic epitopes that were not unique to T. vaginalis. In this report another novel recombinant chimeric protein called AEG:: SOE3 is presented. This epitope chain protein is comprised of the non-unique epitopes of A, E and G that have ≥50% to 100% identity with homologous proteins of humans and of Candida albicans, Escherichia coli, Saccharomyces cerevisiae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, including Chlamydia trachomatis, Neisseria gonorrhoeae, and Treponema pallidum, which also cause STIs [8]. Based on numerous published papers [9][10][11][12][13][14][15][16][17][18], I hypothesize that these non-unique, immunogenic epitopes with possible immunocrossreactivity with human proteins may play a role in disease pathogenesis during this STI. I further discuss and hypothesize that T. vaginalis, other STIs and infectious diseases may produce immune-crossreactive antibodies during natural infections, and these antibodies may cause previously unreported and unappreciated host cytopathology.

Non-unique epitopes to the A, E and G proteins of T. vaginalis
The identification of the immunogenic epitopes of A, E and G reactive with sera of women and men patients was performed using the SPOTS system (Sigma-Genosys, The Woodlands, TX, USA) as detailed in several earlier reports [4,5,7,8]. Briefly, oligopeptides were derived from GenBank® accession numbers AAW78361, AAK73099 and AAA30325 for sequences of T. vaginalis of A, E and G proteins, respectively. Commercially available oligopeptides (Custom Peptide Arrays) were synthesized on activated membranes on the SPOTS system for screening with patient sera of women and men using established [4,5,7,8] and the manufacturer's protocols. The membranes were also screened with monoclonal antibodies (MAbs) generated to the A, E and G proteins, as described below and previously [4,8]. The BLAST sequence identity analysis permitted identification of the epitopes (Table 1) with ≥50% to 100% amino acid sequence identity to other enzyme homologs, as before [8].
Finally, and as recently reported [4], the Immune Epitope Database and Analysis Resource (www.iedb.org) was used to show the linear nature of the epitope sequences.

SCRCADIFHRLEKYL
a. AEG::SOE3 refers to the chimeric protein derived from non-unique immunogenic epitopes to the T. vaginalis A, E, and G proteins as described in Methods and Figure 1. The epitopes with ≥50% to 100% identity to other proteins of the human homologue; C. albicans, C. trachomatis, E. coli, N. gonorrhoeae, S. cerevisiae, S. aureus, S. pneumoniae, S. pyogenes and T. pallidum were reported in an earlier published paper [8]. The first epitope of E (underlined) had no identity with other proteins in databanks and is included because it is recognized by a MAb ALD13 as shown in Figure 1B.

Plasmid encoding AEG::SOE3 and purification of the protein
GenWay Biotech, Inc (San Diego, CA) synthesized the DNA coding sequence for AEG::SOE3 in the pET23a(+) plasmid with ampicillin and chloramphenicol genes. The protein as shown in Figure 1 was synthesized by induced E. coli BL21DE3 cells, as before [4,5,7,8]. AEG::SOE3 has hexa-histidine at the carboxy terminus for purification by Ni-NTA superflow affinity column chromatography (Qiagen Inc., Valencia, CA USA) [4]. The amino acid sequence of the protein was confirmed by sequencing of the plasmid. Because the protein was found within inclusion bodies, GenWay provided the purified protein for this study. Alternatively, the protein was purified in the laboratory by growing E. coli on Luria Broth with the antibiotics as described recently [4,5,7]. Briefly, a starter culture of 100ml of E. coli was grown at 37°C to OD600. The E. coli was then transferred to a 2-liter Erlenmeyer flask with 600ml Luria Broth and placed in a shaking incubator at 18°C prior for 2-hours prior to induction with isopropyl-β-Dthiogalactopyranoside and incubated for 4-hours. The protein was purified in the Ni-NTA column as shown in (Figure 2). The peptide-epitope sequences are linked by EE amino acids (underlined). Epitopes within the peptide sequences are red. B. Linear sequence with A, E and G refer to amino acid sequences from the T. vaginalis proteins, respectively, as described for Table 1. The epitopes were detected by pooled positive women (W) or men (M) sera, as shown and as indicated previously [8], and the number next to W and M are the order the epitope was identified within the protein during epitope mapping on the SPOTs system (Methods). The MAbs labeled ALD11, ALD12, ALD13, ALD25, B44 and B43 are reactive with the epitopes, as before [8].

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Enzyme-linked immunosorbent assay (ELISA)
The preparation of the microtiter plates with AEG::SOE3 for ELISA is as before [4,5,7,8]. For all assays 2% ELISA-grade bovine serum albumin (eBSA) (Sigma Chemical Co., St. Louis, MO, USA) prepared in PBS (eBSA-PBS) was used for blocking the wells of microtiter plates and for dilution of sera. The eBSA-PBS also served as a negative control during ELISAs ( Table 2). As shown in Figure   2,3 in addition to negative controls, ELISA was performed using recently generated mouse anti-AEG::SOE2 and MAbs to AEG:: SOE2 [4], mouse anti-T. vaginalis (Tv) serum [19], and three different MAb cocktails to AEG::SOE3 (described below), and pooled sera of women and men patients, as before [4,5,7,8].
a. The normal mouse serum served as a negative control for the use of mouse anti-T. vaginalis (Tv) serum and mouse anti-AEGSOE2 serum and gave low values equivalent to other negative controls. The AEG::SOE3 MAb cocktail mixes 1, 2 and 3 are as described above for Figure 3. The MAbs for AEG::SOE2 were described recently [4] and are unreactive with AEG::SOE3. The MAbs cocktails were all equal volumes of 25µl of hybridoma supernatants prior to addition to the protein-coated microtiter wells. As with figure 3 HA423 and L64 are irrelevant negative control antibodies that react with a α-actinin and a cytoplasmic protein of T. vaginalis, respectively, and were unreactive with either AEG::SOE3 or AEG::SOE2 (data not shown). All monoclonal antibodies are of the IgG 1 isotype.
b. eBSA-PBS is ELISA-grade bovine serum albumin prepared in PBS (Methods).
c. Absorbance values were obtained at 405nm. The standard deviation was calculated for the average of all ELISA performed in quadruplicate samples. All experiments were performed using quadruplicate wells and were performed on four different occasions with similar data.
*Asterick indicates the values are statistically significant with p<0.005. Independent t-tests were used to compare ELISA values for each sera and the MAb cocktail mixes for AEG::SOE3 and AEG::SOE2.

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Antibodies for detection of proteins and trichomonads
Numerous reports describe the sera from women and men used for detection of proteins to T. vaginalis [4,5,7,8]. These sera permitted determining levels of serum IgG antibodies to trichomonad proteins and particularly α-actinin [5]. Individual seropositive sera of women and men had identical reactivities to parasite proteins, and the limitation of amounts of individual sera was overcome by pooling of the seropositive sera of both women and men to obtain sufficient volumes for doing epitope mapping analysis. Likewise, pooled negative control sera from both women and men were used in ELISA experiments. Institutional Review Board (IRB) approvals for using the sera were obtained. A 1:25 (vol/vol) dilution of sera with eBSA-PBS was previously found to be optimal for detection of purified proteins immobilized onto wells of microtiter plates [4,5,7,8]. When doing ELISAs using MAbs to AEG::SOE3 ( Figure 1B), a 50µl volume of undiluted hybridoma supernatant was added to wells. Alternatively, a cocktail of 25µl of hybridoma supernatant for each of MAb cocktails was used as The mouse anti-AEG::SOE2 serum was described recently [4] and was unreactive with AEG::SOE3. Mouse anti-T. vaginalis (Tv) serum was generated to chemically stabilized T. vaginalis isolate NYH 286 [19]. The AEG::SOE3 cocktail mix 1 was comprised of the MAbs ALD12, ALD11, ALD25. Two other MAb cocktails included mix 2 with ALD12, B44, B43 and mix 3 with ALD12, ALD11, ALD25, B44, B43 and gave results as mix 1. The MAbs were to epitopes as per Figure 1B. The MAb cocktails included 25μl of each MAb. The AEG::SOE2 MAb cocktail as recently reported [4] consisted of MAbs ALD13, ALD55, ALD30 and ALD32 and did not detect AEG::SOE3. The individual MAbs ALD11 and ALD12 detected AEG::SOE3. As expected, negative control irrelevant MAbs HA423 to α-actinin and L64 to a cytoplasmic protein [4,5,7,8] of T. vaginalis were unreactive to AEG::SOE3. All MAbs were of the IgG1 isotype. Women and men patient sera have been described before, and the optimal pooled sera dilution was 1:25 (vol/vol) in eBSA-PBS [4,5,7,8]. The ELISA was repeated on four different occasions using quadruplicate samples and gave similar data.

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Mouse anti-AEG::SOE2 serum was used and described before [4]. Further, mouse anti-T. vaginalis serum generated in mice immunized with glutaraldehyde-fixed trichomonads has been reported [19]. The MAbs to A, E and G proteins were generated at the MAB Core Facility of the College of Veterinary Medicine at Washington State University using established protocols described before [7]. All animals were handled humanely as per guidelines studies [8,20,21] were analyzed further with AEG::SOE3.

Statistical analysis
All statistical analyses were conducted in R (Version 4.0.4).
Independent t-tests were used to examine differences in ELISA results between AEG::SOE3 and AEG::SOE2 for each sera and MAbs examined. Statistical significance was defined as a two-sided p-value less than 0.05.

Reproducibility
Experiments were performed on at least four occasions using quadruplicate samples under identical conditions and gave similar data. Averages and standard deviations were calculated.

Results
The T. vaginalis non-unique, immunogenic epitopes of A, E, and G and the AEG::SOE3 protein An earlier report [8] on epitope mapping revealed that pooled sera of women and men patients recognized 12 epitopes of fructose-1,6-bisphosphate aldolase (A), 18 epitopes of α-enolase (E), and 19 epitopes of glyceraldehyde-3-phosphate dehydrogenase (G) for a total of 49 epitopes for the three proteins [18]. Eighteen of these 49 epitopes were found to be unique to the A, E and G T. vaginalis proteins [4,8]. In this report Table 1 shows the non-unique epitopes (colored red) identified by IgG antibody in the SPOTS system (Methods). There were 8 epitopes for A, 8 epitopes for E, and 12 epitopes for G for a total of 28 non-unique epitopes to these proteins. Except for the first epitope for E, these epitope amino acid sequences had ≥50% to 100% identity with other bacterial, fungal, and human sequences [8]. The first sequence for E (underlined) had no identity with other proteins in databanks and is included as an internal control because the epitope is recognized by MAb ALD13 (Figure 1). Figure 1A presents the recombinant protein amino acid sequence of the peptides containing the 15mer sequences shown in Table 1. Each peptide was linked with two glutamic acid residues (underlined). The protein with six histidine residues at the carboxy terminus is 436 amino acids in length and is 49.41kDa with a pI of 4.83. Figure 1B shows the reactivity with women (W) and/or men (M) positive sera indicated above the red epitope amino acid sequences. The epitopes for A(M1) and A(W2) overlapped. Overall, there are 17 and 14 epitopes detected by women and men sera, respectively. The six monoclonal antibodies (MAbs) that detect epitopes are labeled ALD11, ALD12, ALD13, ALD25, B44 and B43, as indicated. These MAbs have been previously characterized [8].

ELISA using AEG::SOE3-coated wells probed with different antibodies
ELISA was then performed with 1 µg AEG::SOE3 immobilized onto microtiter wells, as before [4], and representative results of four separate experiments, each with quadruplicate samples, are shown in Figure 3. All experiments yielded similar data. One microgram of protein was found to be optimal for detection by antibody [4,5,7,8]. Not unexpectedly, there was no reaction of AEG::SOE3 by the secondary peroxidaseconjugated goat anti-mouse IgG (negative control) and normal mouse serum (NMS). Both the mouse antiAEG::SOE2 serum and a cocktail of MAbs to AEG::SOE2 previously reported [4,8] were unreactive with AEG::SOE3. The  Figure 2 was detected by immunoblot with these antibody reagents and pooled sera of women and men patients (not shown) as was done for AEG::SOE2 recently [4]. Pooled patient sera from 5 women and 5 men readily detected AEG::SOE3. The α-actinin protein MAb HA423 and a cytoplasmic protein MAb L64 of T. vaginalis [4,5,7,8] were other negative controls and unreactive to AEG::SOE3. All MAbs were of the IgG 1 isotype. Statistical analysis (Methods) was performed to compare ELISA reactions with sera and MAbs to AEG::SOE2 and AEG::SOE3 that the values were statistically significant as shown in Table 2.
Representative ELISA comparing ACT::SOE3 and AEG::SOE2 with different antibodies AEG::SOE3 was then compared with AEG::SOE2 for detection by IgG of different antibodies. Table 2

Discussion
The approach recently published [4] was used to synthesize another epitope chain protein [6] called AEG::SOE3 (Figures 1,2). This AEG::SOE3 is different from AEG::SOE2 because the epitopes of fructose1,6-bisphosphate aldolase (A), α-enolase (E), and glyceraldehyde-3-phosphate dehydrogenase (G) are not unique to T. vaginalis and share ≥50% to 100% amino acid identity with the human homologue as well as homologues of C. albicans, E. coli, S. cerevisiae, S. aureus, S. pneumoniae, and S. pyogenes and the three other STI agents, C. trachomatis, N. gonorrhoeae, and T.
pallidum [8]. The fact that mouse anti-AEG::SOE2 sera and MAbs to AEG::SOE2 [4] were unreactive with AEG::SOE3 and that MAbs to AEG::SOE3 did not detect AEG::SOE2 shows the specific and distinct nature of the different epitopes and the two proteins. Further, data from the earlier work [4] and in this study support the surface location of the epitopes of these metabolic enzymes. For example, mouse antiserum generated to chemically-stabilized T. vaginalis [19] reacts to both AEG::SOE2 [4] and AEG::SOE3 (Figure 2,3).
In addition, it was shown recently that this antiserum and MAbs detects trichomonads in a whole-cell ELISA [4]. These data and earlier reports [20,21] show that the enzymes E and G reside on the surface of T. vaginalis. These surface-associated enzymes are ligands for binding host pathogenicity proteins, like plasminogen and other proteins, such as fibronectin, laminin, and collagen [20,21]. That there is an antibody response to these epitopes during infection by T. vaginalis may indicate that the host immune surveillance recognizes these enzyme proteins not as homologs of human proteins but as virulence factors involved in disease pathogenesis.
It is noteworthy that the host immune response resulted in IgG antibodies to 12 immunogenic epitopes of A, 18 epitopes of E and 19 epitopes of G given the highly conserved nature of the proteins among humans. Of these 49 total epitopes, 8 of 12 epitopes of A, 9 of 18 epitopes of E and 12 of 19 epitopes of G were not unique to T. vaginalis and have ≥50% to 100% sequence identity with homologues of humans and other bacterial pathogens and fungi, including three other organisms that cause STIs. The AEG::SOE3 epitopes with high sequence identity to C. trachomatis, N. gonorrhoeae and T. pallidum enzymes perhaps suggests that IgG antibodies may be made to these enzyme epitopes during infection by the other STI agents as well. If this is true, this fact would be significant and may further suggest that assumptions that the immune response to microbial pathogens may not aggressively be towards protein homologues of humans requires additional attention.
It is important to consider that antibodies to these non-unique trichomonad enzyme epitopes may elicit immune cross-reactions with host proteins and tissues. While it is acknowledged that this requires future examination, at a minimum this work suggests that this is an issue that should be considered within the framework of pathogenesis of trichomonosis, especially as it has been shown that human serum antibody to α-enolase of group A streptococcus cross-reacts with host tissues [22]. Indeed, there is a growing literature showing autoantibody in relation to infectious diseases [9][10][11][12][13][14][15][16][17][18]. For example, bacterial pathogens, viruses, and parasites are now considered triggers of autoimmunity [9,10]. Hepatitis C in patients is considered in cases of autoimmune encephalitis due to West Nile virus and other bacteria [11,18]. The role of viruses and microbial pathogens is related to multiple sclerosis [9].
Among some organisms implicated in diseases are the following: Chlamydia pneumoniae and biliary cirrhosis [15], Helicobacter pylori and autoimmune gastritis [12], Trypanosoma cruzi and Chagas' cardiomyopathy [13], Leishmania and autoimmune hepatitis [17] and Mycoplasma arthritidis and rheumatoid arthritis [16]. Therefore, it is reasonable to hypothesize that the epitopes presented in Table 1 with sequence identities to human and other microbial pathogen proteins may play a role in heretofore unknown and unappreciated disease pathogenesis via eliciting host immunocrossreactive antibodies.

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These findings strongly reaffirm an earlier report [4] that argues against the use of whole cells and/or lysates as diagnostics and vaccines [23][24][25]. The ideal serodiagnostic target would be a protein with immunogenic epitopes unique to the pathogen of interest [4]. It is shown here that A, E and G of T. vaginalis leads to IgG to epitopes shared with other infectious diseases, including C. trachomatis, N. gonorrhoeae and T. pallidum. If these STI microbial pathogens likewise make IgG crossreactive with T. vaginalis proteins A, E and G, and if a lysate of trichomonads is used for diagnosis, this may lead to the false identification of trichomonosis as the STI. Likewise, this work shows that a pathogen with immunocrossreactive epitopes to human proteins is unsuitable as a vaccine.
In this scenario, the immune response may lead to antibodies that contribute to autoimmune-like tissue cytopathology. pathogens. Second, it would show that IgG to epitopes common to human and other microorganism proteins is more common than previously appreciated. Third, it may be possible to determine whether infections with certain microbial pathogens, such as those that cause STIs, make serum IgG to a common set of epitopes. This is because the nature of the extent of crossreactivity to AEG::SOE3 among infectious diseases may mean we must reconsider metabolic enzymes as virulence factors and possible mediators of disease pathogenesis.
Finally, this work highlights the possibility of constructing a novel SOE protein with epitopes found to be common among select groupings of microbial pathogens. For example, in the case of T. vaginalis, C. trachomatis, N. gonorrhoeae, and T. pallidum, a chimeric protein comprised of epitopes with 100% sequence identity could represent a broad-spectrum diagnostic that can be used in large at-risk populations. Positive results could then lead to additional specific diagnosis of the STI pathogen or multiple STI agents for treatment and cure. As it is recognized that multiple STIs can exist within patients [26], this type of diagnostic may have merit in screening of at-risk populations.
In the context mentioned above, we need a new paradigm when we find IgG to metabolic enzymes during immune responses to infectious diseases. When considering that trichomonosis is a significant asymptomatic disease and that women and men may be infected for long periods of time, it is reasonable to hypothesize that a positive serum IgG test to AEG::SOE3 indicates the presence of immuno-crossreactive antibodies, which may have consequences heretofore not considered for this STI in disease pathogenesis.

Conclusion
This paper presents a new epitope chain protein (AEG::SOE3) made up of immunogenic epitopes that are common and have sequence identity to humans and other bacteria and fungi, including three organisms that cause STIs. Data show that patients with trichomonosis make serum IgG antibody to A, E and G proteins. The work highlights the possibility of immuno-crossreactive antibody to human proteins, which may lead to disease pathogenesis. The work underscores that, in the future, an SOE protein can be constructed as a diagnostic that identifies multiple, related infectious agents, such as the four microorganisms causing STIs in this study, which then permits more specific diagnosis of single or multiple organisms causing disease.

Conflict of Interest
The author declares no conflicts of interest. I alone designed the study and was responsible in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.