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In-Silico Proteomic Exploratory Quest: Crafting T-Cell Epitope Vaccine Against Whipple’s Disease

Whipple’s disease is one of the rare maladies in terms of spread but very fatal one as it is linked with many disorders (like Gastroenteritis, Endocarditis etc.). Also, current regimens include less effective drugs which require long duration follows up. This exploratory study was conducted to commence the investigation for crafting multi target epitope vaccine against its bacterial pathogen Tropheryma whipplei. The modern bioinformatics tools like VaxiJen, NETMHCII PAN 3.2, ALLERGEN-FP, PATCH-DOCK, TOXIC-PRED, MHCPRED and IEDB were deployed, which makes the study more intensive in analyzing proteome of T. whipplei as these methods are based on robust result generating statistical algorithms ANN, HMM, and ML. This Immuno-Informatics approach leads us in the prediction of two epitopes: VLMVSAFPL and IRYLAALHL interacting with 4 and 6 HLA DRB1 alleles of MHC Class II respectively. VLMVSAFPL epitope is a part of DNA-directed RNA polymerase subunit beta, and IRYLAALHL epitope is a part of membranous protein insertase YidC of this bacterium. Molecular-Docking and Molecular-Simulation analysis yields the perfect interaction based on Atomic contact energy, binding scores along with RMSD values (0 to 1.5 Ǻ) in selection zone. The IEDB (Immune epitope database) population coverage analysis exhibits satisfactory relevance with respect to world population.


Introduction
George Hoyt Whipple in 1907 explains Whipple's disease, as a multisystemic chronic infectious disease. He identified silver stained rod shaped bacterium in vacuoles associated with macrophages of patients, he initially did not think of them as the cause for the disease rather he think that intestinal lipodystrophy (Whipple's disease) was caused due to some novel disturbances in fat metabolic schemes (Whipple 1907). When the first successful treatment started by using antibiotics in 1952, determined that this bacterium might be the major causative agent of this disease (Paulley 1952). An electron microscopic study in 1960′s provided additional support for this hypothesis (Cohen et al. 1960;Yardley and Hendrix 1961). Whipple's disease occurs uncommonly, as a multisystemic disorder (inexact annual frequency less than 1 per 1,000,000 populace) that specially affects middle-aged Caucasian men (Fenollar et al. 2007;Ramharter et al. 2014, Dobbins et al. 1981). This bacterium was found to mostly affect small children (Keita et al. 2015) and sewage workers (Schöniger-Hekele et al. 2007). Since, its first portrayal by Whipple in very beginning of first decade in twentieth century (Whipple 1907), a limited progresses with in pathogenesis, prognosis, and treatment of the malady have been made. The bacterium gets internalized in to lamina propria of intestine and then make its way to mucosal macrophages, as this bacterium induces the decreased expression of CD11b in such macrophages (CD11b on macrophages frequently mediates the intracellular degradation of bacteria) causes flip in the scenario (inappropriate antigen presentation by such macrophages and dendritic cells). This specially reasons the boom in IL-10, TGF-β and CCL-18 expression and decrease in IFN-γ, which in turn causes destroy in maturation of phagosomes and decrease in thioredoxin expression, lead them unable to kill bacterium and antigen presentation (Moss et al. 2006(Moss et al. , 2010.

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An unseemly development of proficient antigen-presenting cells caused by the presence of interleukin 10 and interleukin 16, and the non appearance of interferon γ and interleukin 12 might lead to inadequate antigen-presentation and hinder the incitement of antigen-specific T-helper 1 cells enhancing growth and systemic spread of Tropheryma whipple. The nearby generation of provocative cytokines through macrophages and endothelial cells within the fringe might actuate lymphocyte invasion through a defective endothelial obstruction taken after by central aggravation, indeed in immunologically ensured tissues such as joints or the neuronal domain (Schneider et al. 2008).Currently hydroxychloroquine (600 mg/day) and doxycycline (200 mg/day) used for treatment of whipple's disease for 12-18 months, but life time follow up is required (Lagier et al. 2014), so it is time consuming treatment process and only few handful trials were conducted in earlier studies (Feurle et al. 2013). Nowadays epitope based vaccines provide better options in search of good treatment strategy for such type of harmful and rare malady, even if the individuals are genetically predisposed as in case of classical Whipple's disease (Trotta et al. 2017). This modern approach of putative vaccine determination which involves the use of proteomic databases is very handy and easy to use method not only for rare bacterial pathogens, but also very effective in case of harmful viruses like Nipah (Kaushik 2019). Tropheryma whipplei was found to be associated with major ailments like gastroenteritis and endocarditis (Fenollar et al. 2013).
In this research work, five proteins from proteomic data of T. whipplei were analyzed for allergenicity. Non-allergenic proteins were deployed for predicting epitopes. Predicted epitopes were subjected for immunogenic properties, structural modeling and the docking with corresponding MHC II alleles to investigate the strong binding affinity. Method is more economic, time efficient, and harmless when compared to the vaccine designing and testing in wet lab and animal testing strategies (Kumar et al. 2015). Reverse vaccinology is the suitable approach as well as novel science method that use the genomic data with the utilization of computer for the arrangement of antibodies without culturing bacterium species (Kanampalliwar et al. 2013;Tang et al. 2012). It allow the choice in hands of human interface for selecting antigens from pathogenic set of DNA and most antigenic areas could be used to synthesize potential immunization to initiate defensive responses against such pathogenic species (Ada et al. 2018). Epitopes based antibodies selection and production is explicitly less time consuming, economical and considered safest approach in vaccine designing. Earlier computational methods were found to be successful in analyzing genome and prediction of putative drugs for T. whipplei (Palanisamy 2018), such studies provide motivation to craft vaccine targets by deploying in-silico approach. T-cell epitopes were screened out in this study may effectively elicit immune responses against this bacterium, and also similar type of recent study was found to be successful in determining epitope based vaccine agents for SARS-Cov2 (Joshi et al. 2020). Brief flow chart of the study used to determine putative epitope based vaccine candidates against T. whipplei is presented in Fig. 1.

Retrieval of Proteins for T. whipplei
Proteomes were retrieved in fasta format from NCBI-Genbank and UniProtKB databases. Five proteins of different functionality were selected with following accession no's: WP_042507409.1 DNA-directed RNA polymerase subunit beta (RPO-B), WP_033800049.1 co-chaperone GroES, WP_038104819.1 TerC/Alx family metal homeostasis membrane protein, WP_042505650.1 membrane protein

Allergenicity Prediction for Proteins
The protein sequences were then deployed for further analysis based on Allergen FP V 1.0 for predicting allergenicity (Dimitrov et al. 2014).

T-Cell Epitope Prediction
Net MHCII PAN 3.2 server is used to find and screen out HLA alleles which have good interaction with selected non-allergens of pathogenic origin (Jensen et al. 2018). To bring higher confidence in selecting epitope, VaxiJen server is deployed to determine antigenicity with threshold ≥ 0.7 for selected rare bacterium (Doytchinova et al. 2007). By subjecting proteomic sequences to Net MHCII PAN 3.2 server we obtained 1147 epitopes for WP_042507409.1, 90 epitopes for WP_033800049.1, 309 epitopes for WP_038104819.1, 302 epitopes for WP_042505650.1, and 510 epitopes for WP_011096746.1,this server was used because of its neural networking algorithm based approaches for fine predictions. 1-log50k (affinity score) ≤ 0.6 is used to screen out possible epitopes presented in Table 2. These epitopes were further subjected for antigenicity analysis based on VaxiJen scores.

Prediction of Epitope's Toxic Behavior
Toxicity for putative peptides was designated by using SVM scores from Toxin Pred web server (Gupta et al. 2013). Non toxic peptides were finalized for further analysis.

Molecular Structural Modeling for Epitopes and Probable HLA Alleles
The tertiary structure or 3D structure for epitope is determined by using PEP-FOLD 3 web server (Lamiable et al. 2016;Shen et al. 2014;Thévenet et al. 2012). And predicted Human leukocyte antigen alleles 3D structure was obtained from RCSB PDB database (Berman et al. 2000). Also Ramachandran plot analysis was conducted for verification of results by using Molprobity server (Williams et al. 2018).

Epitopes 3D Interaction or Molecular Docking with HLA Alleles
The docking experiments was conducted by using Patch-Dock tool (Schneidman-Duhovny et al. 2005), The predicted docked models of putative epitope and HLA alleles was selected on the basis of score, which relies on highest geometric shape complementarities and Atomic contact energy (Zhang et al. 1997). This allows the best selection of epitope and HLA allele interaction. This tool is easy to deploy for all life science domains.

Analysis of Population Coverage
Immune Epitope Database (IEDB) analysis Resource tool of population coverage was used to predict population coverage of the putative epitopes that are exhibiting interaction to HLA alleles and based on MHC-II restriction data (Bui et al. 2006). MHCPred tool was deployed for quantitative prediction of selected epitopes interacting to major Histocompatibility complexes (Guan et al. 2003).

Molecular Docking Simulations
Epitope-HLA allele docked sets were then used for simulation and dynamics analysis by deploying NAMD (Phillips et al. 2005) associated with VMD (Visual Molecular Dynamics) tool (Humphrey et al. 1996).

Non-allergen Determination
Total 5 protein sequences were analyzed for allergenicity and depicted as non-allergen in Table 1 by using AllergenFP tool.

T-Cell Epitope Prediction
Net MHCII PAN 3.2 server is deployed to identify promiscuous epitopes and probable HLA alleles of MHC Class II

Molecular 3D Modeling of Epitopes and HLA Alleles
3D structural models of selected epitopes were designed by using PEP-FOLD 3 web server and than most common HLA DRB1 proteins structural models were derived by using RCSB-PDB database. In Table 3 PDB Id along with HLA alleles is exhibited. Molprobity results of Ramachandran plot analysis results shows satisfactory structural prediction (> 85% residues in favorable region) of epitopes that were finalized at last in Fig. 8.

Molecular Docking of Epitopes and HLA Alleles
PatchDock tool was deployed for interaction between selected structures of epitopes and HLA DRB1 proteins. Then interaction data produced by docked molecules include ACE (Atomic contact energy) and best model score that leads to the final selection in the way of prediction for each pair. In Table 4 the selected models and rejected models both were included to enhance the comparative analysis. The two selected epitopes were VLMVSAFPL and IRYLAALHL interacting with 4 and 6 HLA DRB1 alleles respectively. VLMVSAFPL epitope is a part of DNA-directed RNA polymerase subunit beta and IRYLAALHL epitope is a part of murein biosynthesis integral membrane protein of T. whipplei and are major identifiers of this bacterium. Figure 2 clearly depicts the good interaction between epitopes and HLA Alleles in docked results. In Fig. 2a Docked result of IRYLAALHL with HLA-DRB1* 01:01 exhibits perfect hydrogen bond due to presence of tyrosine residue in epitope at 3rd position, while most of the other non polar amino acids of this epitope are depicts vander waals interactions with in the HLA model and in Fig. 2c Docked result of VLMVSAFPL with HLA-DRB1* 04:04 exhibits perfect   (Schlundt et al. 2012;Chen et al. 2013). The reference docked peptides have great difference in amino acid sequence in comparison to our screened epitopes but exhibits some resemblance alike of our epitopes in interaction towards antigen binding pocket. Figure 3a, b represents the free undocked HLA-DRB1 receptors (4AH2, 4IS6 respectively), while Fig. 3c, d represents free unbound putative epitopes (IRYLAALHL, VLMVSAFPL respectively) and their side chains. Figure 4 graphically represents the selected epitopes and HLA alleles of MHC II on the basis of ACE values.

Toxicity, Half Life, and Stability Analysis of Putative Epitopes
Predicted epitopes VLMVSAFPL and IRYLAALHL have VaxiJen scores 0.9461 and 1.2114 respectively, they are also of non toxic nature as per the study of Toxin Pred tool and its toxicity scores (SVM score) represented in Table 5. In Table 6 quantitative estimation of best interaction of epitope with HLADRB1 alleles were achieved with upright IC 50 values by using MHCPred tool, this allows confidence of prediction. Table 7 shows Half-life and instability index for putative epitopes by deploying ProtParam expasy tool.

Population Coverage Analysis of Epitopes
VLMVSAFPL and IRYLAALHL manifest 28.82% and 37.06% elicitation of immune responsiveness by world population by availing IEDB tool. The epitopes VLMVS-AFPL and IRYLAALHL shows greater effect in European population by 29.63% and 42.68% respectively, and correspondingly similar results with North American population coverage analysis. This indicates its greater relevance in treatment of Whipple's disease as it is mostly seen in Caucasoid population. In Figs. 5 and 6 it is clearly represented in a graphical representation.

Molecular Dynamic Simulation Studies
NAMD was deployed for simulation studies on docked Epitope-HLA allele sets to obtain RMSD values. Maximum value of RMSD for VLMVSAFPL and IRYLAALHL epitopes were analyzed, this gives more confidentiality in selection of vaccine candidate against Tropheryma whipplei. Figures 7 and 8 shows RMSD plots that indicates clear picture of selection of these two epitopes.

Discussion
Immuno-informatics is the suitable approach as well as novel science method that use the proteomic data with the utilization of computer systems for predicting epitopes without culturing bacterium species (Kanampalliwar et al. 2013;Tang et al. 2012). It allow the choice in hands of human interface for selecting antigens from pathogenic set of DNA and most antigenic areas could be used to synthesize potential immunization to initiate defensive responses against harmful pathogenic species (Ada et al. 2018). Insilico approach was earlier successful in case of Staphylococcus aureus (Delfani et al. 2015), Mycobacterium  -367.28-321.11-353.51-353.51-353.51-349.18-443.31-443.31-226.35-297.99   tuberculei (Mustafa 2013) and numerous bacterial species, but T. whipplei is still not fully explored in this domain. Current regimens include hydroxychloroquine and doxycycline for treatment of Whipple's disease for 12-18 months, but life time follow up is required (Lagier et al. 2014), so it is time consuming treatment process and  (Feurle et al. 2013).
In present study we identified two possible epitopes that can interact with MHC-II alleles to elicit immune response on individuals namely VLMVSAFPL epitope (part of DNAdirected RNA polymerase subunit beta), and IRYLAALHL epitope (part of murein biosynthesis integral membrane protein). These epitopes exhibit better interaction with HLA DRB1 alleles, as confirmed by deploying Molecular-Docking and Molecular-Simulation studies (Adhikari et al. 2018). Population coverage analysis was found to be satisfactory and in earlier studies it was used in strengthening vaccine prediction aspects (Misra et al. 2011). Very similar studies were also conducted successfully for related bacterium Mycobacterium avium and found to be successful in predicting epitopes (Gurung et al. 2012 − 321.1, − 353.5, − 353.5, − 353.5, − 349.1 respectively) in docking results similar type of methodology was seen in recent studies in screening epitopes for SARS-Cov-2 (Joshi et al. 2020). Both selected epitopes exhibit structural integrity as possess less than 35% instability index score, and half life greater than 20 h for mammalian reticulocytes, this makes the screening criteria more reliable. Also, more than 85% residues of selected epitopes come under favorable region in Ramachandran plot analysis (Fig. 9). Still no one has used vaccine based treatments for Whipple's disease, as it is thought to be rare and possess reduced genome but considered one of the harmful pathogen of human ( Raoult et al. 2003;La Scola et al. 2001;Marth et al. 2016).The effectiveness of epitope based vaccines for treatment of endocarditis has already been claimed (Priyadarshini et al. 2014). But in our study we found the short peptides that can easily be synthesized and deployed in developing immunity in Caucasian populations against Whipple's disease.

Conclusion
In this study we obtained VLMVSAFPL and IRYLAALHL as predicted epitopes for vaccine crafting. This novel approach in crafting vaccine based treatment of T. whipplei will open new doors in research for creating regimens to treat such harmful bacterium by developing adaptive immune response and eradicating it globally before any future escalations takes place. The predicted epitopes can be deployed in crafting vaccines against T. whipplei bacterium after Molecular-wet lab corroboration.