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PRAME antigen, Vaccines, Breast neoplasms, Computer simulation, What&,rsquo s Known Standard therapies used to treat breast cancer are only effective in approximately half of the patients. Among various immunotherapy methods, vaccination has gained much attention in treating breast cancer. What&,rsquo s New Preferentially expressed antigen in melanoma (PRAME) combined with FliC&,Delta D2D3 is a stable and soluble chimeric protein. PRAME+FliC&,Delta D2D3 stimulated humoral and cellular immunity against breast cancer. IntroductionBreast cancer is considered as the most common women&,rsquo s cancer worldwide, and is the second leading cause of death after lung cancer. 1, The World Health Organization (WHO) has estimated 2.1 million new cases of breast cancer each year, and has reported 627,000 related deaths in 2018. 2,, 3, Such a high number of deaths has been attributed to tumor progression and metastasis to other organs. Breast cancer is a heterogeneous disease, which requires targeted therapy consistent with the tumor&,rsquo s characteristics. 4, Standard therapies used to treat breast cancer (e.g., surgery, radiation therapy, anti-estrogen therapy, and chemotherapy individually or in combination) are only effective in approximately half of the patients. It has been shown that these therapies pose adverse side-effects, since they do not target specific tumor cells. 5,- 7, Immunotherapy is a targeted approach and a promising method to treat cancer patients. Among various immunotherapy methods, vaccination has gained much attention in treating breast cancer. Recently, it has been shown that the use of antigen-specific vaccines are effective in eradicating breast tumor cells. 8,, 9, Preferentially expressed antigen in melanoma (PRAME) is a human tumor antigen found in melanomas. PRAME is also known as melanoma antigen, which is preferentially expressed in tumors (MAPE), OPA-interacting protein 4 (OIP4), and cancer-testis antigen 130 (CT130). The human PRAME gene is located on chromosome 22q11.22 and encodes a protein of 509-amino acids. 10, PRAME induces cell proliferation in melanoma cells by repressing retinoic acid (RA) signaling. 11, It belongs to the cancer-testis antigen (CTA) family based on its chromosomal position, expression, and immunogenicity. Typically, PRAME has no expression in normal tissues. However, low level of expression has been observed in some organs such as testis, ovaries, adrenal glands, and endometrium. 12, Aberrant expression of PRAME has been demonstrated in various cancers such as neuroblastoma, 13, ovarian adenocarcinomas, 14, head and neck cancer, 15, acute and chronic leukemia, 10, and breast cancer. 16, Previous studies have reported the overexpression of PRAME in breast cancer, making it a suitable prognostic and predictive biomarker. It is viewed as a potential candidate for immunotherapeutic strategies. 4,, 16, The goal of vaccination is to provide a strong immune response for long-term protection against antigens. To achieve this goal, a vaccine requires an adjuvant. Adjuvants are used to enhance the immune response against the vaccine and are classified in various groups. Toll-like receptors (TLR) ligands are known as a popular group of adjuvants that improve the immunogenicity of vaccines. 17,, 18, Bacterial flagellins (also termed FliC) is considered as an effective adjuvant candidate. Salmonella typhimurium FliC, a principal structural protein of flagella, is identified as a TLR5 ligand. FliC stimulates several innate immune cells to release specific cytokines and chemokines, which in turn stimulate an adaptive immune response. FliC is a 494-amino acid protein consisting of two separate domains, namely D0/D1 and D2/D3. The domain D0/D1 (with 170 and 90 amino acids) plays a role in TLR5 agonist activity, and the domain D2/D3 (with 170 and 400 amino acids) is essential for flagellin antigenicity. 19,- 21, Various studies have shown the high antigenicity and inflammatory effects of FliC. As a result, truncated flagellin (FliC&,Delta D2D3) has been designed by removing the hypervariable D2/D3 domain. In comparison to FliC, FliC&,Delta D2D3 exhibits remarkably lower inflammatory responses, TLR5 agonist potency, more efficient immunoreactivity, and least allergenicity. 22,, 23, The design and development of a new vaccine are still time-consuming and requires extensive experimental investigations. Among various approaches, bioinformatics methods have been utilized to design more effective vaccines. 24, The present study aimed to employ bioinformatics methods to design a chimeric protein as a novel recombinant construct against breast cancer, composed of PRAME as the antigenic fragment and FliC&,Delta D2D3 as the adjuvant part. Materials and MethodsThe present study was conducted in 2019 at the School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran. A complete amino acid sequence of PRAME was obtained from the UniProt Knowledgebase (http,//www.uniprot.org,, UniProt ID, P78395). FliC&,Delta D2D3 (D0/D1+D1/D0) sequence was in accordance with a previous study. 22, The construct included PRAME at N-terminus connected to FliC&,Delta D2D3. Physicochemical Properties and SolubilityDifferent physicochemical characteristics, such as theoretical isoelectric point (pI), the total number of positive and negative residues, molecular weight (MW), instability index, aliphatic index (AI), and grand average hydropathy (GRAVY) of the chimeric protein were computed using the ProtParam web server (https,//web.expasy.org/protparam,). Protein solubility was evaluated using the Protein-sol web server (http,//scratch.proteomics.ics.uci.edu/,).Secondary Structure PredictionThe secondary structure of PRAME, FliC&,Delta D2D3 (D0/D1+D1/D0), and PRAME+FliC&,Delta D2D3 (D0/D1+D1/D0) proteins was predicted using the GOR IV web server with an accuracy of 73.5% (https,//npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_gor4.html,). 3D Model Building, Refinement, and DockingThe I-TASSER web server (https,//zhanglab.ccmb.med.umich.edu/I-TASSER/,) was used to build a 3D model of the chimeric protein. The server is a combined platform for computerized protein structure and function prediction. The optimal 3D model from I-TASSER was used for refinement using the GalaxyRefine web server (http,//galaxy.seoklab.org/cgi-bin/submit.cgi?type=REFINE,). Validation of the 3D model of the chimeric structure was performed using the PROCHECK and RAMPAGE web servers. The PROCHECK (http,//servicesn.mbi.ucla.edu/PROCHECK/,) was used to check the stereochemical quality of the protein structure, and its results were verified using the RAMPAGE web server (http,//mordred.bioc.cam.ac.uk/~rapper/rampage.php,). The interaction between the 3D model of the chimeric protein and TLR5 was predicted using the ZDOCK web server with &,amp gt 70% accuracy.Antigenicity and Allergenicity EvaluationThe ANTIGENpro and VaxiJen web servers were used to evaluate the antigenicity of PRAME, FliC&,Delta D2D3, and the chimeric protein. ANTIGENpro (http,//scratch.proteomics.ics.uci.edu/,) is a sequence-based alignment-free and pathogen-independent predictor, which uses protein antigenicity microarray data for predicting protein antigenicity. VaxiJen (http,//www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html,) was used for the anticipation of conserved regions of antigens, and candidate vaccines according to physiochemical properties of proteins, neglecting alignment of sequences. The allergenicity of the chimeric protein was analyzed using the AlgPred and Allergen FP v.1.0 web servers. Allergen FP v.1.0 (http,//ddg-pharmfac.net/Allergen_FP,) is a descriptor-based fingerprint bioinformatics tool. AlgPred (http,//crdd.osdd.net/raghava/algpred/,) predicts allergens based on similarity to known epitopes.Prediction of MHC Binding PeptidesMajor histocompatibility complex (MHC) class I and class II binding peptides were identified using the HLApred web server (http,//crdd.osdd.net/raghava/hlapred/,). The server predicts HLA binder peptides that can be potential vaccine candidates. Prediction of B-Cell EpitopesThe ABCpred web server (http,//crdd.osdd.net/raghava/abcpred/,) was used to predict linear (continuous) epitopes with 65.93% accuracy. This server has been established through a machine-based technique, which considered a recurrent neural network via fixed-length patterns. ElliPro (http,//tools.iedb.org/ellipro/,) is an online bioinformatics web server that has been used for the prediction of conformational (discontinuous) antigens, based on the 3D structure of antigenic proteins. This structure-based server uses three algorithms, namely approximation of a protein surface patch by an ellipsoid, computation of the residue protrusion index (PI), and cluster neighboring residues based on their PI values. ResultsPhysicochemical Analysis of PRAME+FliC&,Delta D2D3 Protein ProtParam analysis of PRAME+FliC&,Delta D2D3 protein showed that the molecular weight and theoretical isoelectric point (pI) of the chimeric protein were approximately 87 kDa and 5.89 units, respectively. We found 79 negatively charged aspartate and glutamate amino acids, as well as 70 positively charged arginine and lysine in the protein sequence (i.e., the net charge of the protein was -9). Based on the computed instability index of 42.44, the chimeric protein was considered an unstable protein. The AI and GRAVY indices were 101.63 and -0.107, respectively. The solubility of the protein was determined using the SOLpro web server. The result showed that this protein could be soluble with a probability of 0.585946 (cut-off, 0.5).Secondary and Tertiary Structure The results of GOR IV for the prediction of the secondary structure of the chimeric protein indicated that the PRAME consisted of 46.95% alpha-helix, 36.94% random coil, and 16.11% extended strand. Whereas the chimeric protein consisted of 50% alpha-helix, 37.34% random coil, and 12.66% extended strand. Therefore, the protein was mainly composed of regular structures. The 3D models of PRAME+FliC&,Delta D2D3 protein were built using the I-TASSER web server. A model with the most positive c-score was chosen (data not shown). The putative tertiary structure is shown in figure 1,.Figure 1. Tertiary structure of PRAME-FliC&,Delta D2D3 is illustrated. The structure was modeled and built using I-TASSER homology-based modeling.3D Structure Refinement, Validation, and DockingAn optimal I-TASSER model was used as an input to the GalaxyRefine web server. This server suggested five refined protein models (data not shown) of which the model with the highest Rama favored score was chosen to carry out more analysis. Based on the Ramachandran plot, the results of PROCHECK and RAMPAGE web servers showed that residues in the favored and most favored regions were increased after model refinement (figure 2,). The results of PROCHECK showed that residues in most favored regions before and after 3D model refinement were 80.6% and 88.5%, respectively. In addition, the results of RAMPAGE showed that residues in the favored region before and after 3D model refinement were 83.0% and 94.4%, respectively. PRAME+FliC&,Delta D2D3 tertiary refined model and the TLR5 interaction showed that the chimeric vaccine could bind to TLR5 receptor (figure 3,).Figure 2. Ramachandran plot for the 3D structure before (left) and after (right) refinement using the PROCHECK web server is illustrated.Figure 3. Docking of PRAME-FliC&,Delta D2D3 and toll-like receptor (TLR5) shows suitable interaction between them.Antigenicity and AllergenicityThe results of ANTIGENpro showed that the antigenicity of the chimeric protein was increased compared to that of the PRAME alone (from 0.161 to 0.485, threshold, 0.4). In addition, using the VaxiJen web server, the predicted antigenicity of PRAME and the chimeric protein was 0.527 and 0.611 (threshold, 0.5), respectively. This implied that FliC&,Delta D2D3 might increase the antigenicity of PRAME. The AlgPred and Allergen FP v.1.0 web servers predicted the non-allergenic potential of the chimeric protein. MHC-I and MHC-II Binding Peptides The HLApred web server predicted possible MHC-I binding peptides in the PRAME+FliC&,Delta D2D3 chimeric protein. The position, sequence, and score of the 10 best-ranked peptides with the potential to bind to MHC-I are listed in table 1,. Moreover, the predicted binding peptides, which had an affinity to the DRB1_1101, DRB1_0301, and DRB1_1001 alleles of MHC-II are listed in table 2,. AlleleRankPositionSequenceScorePredictionHLA-A*0201 (threshold, 7.470)1371ALLERASAT12.900Binder2194YLIEKVKRK12.770Binder3190ELFSYLIEK12.390Binder428ELAGQSLLK12.290Binder5237DLEVTCTWK12.230Binder6429HLIGLSNLT11.030Binder7118KLQVLDLRK10.780Binder8401SLSHCSQLT9.940Binder9469ELLCELGRP8.970Binder1068TLKAMVQAW8.710BinderTable 1. Major histocompatibility complex I binding peptides of preferentially expressed antigen in melanomaAlleleRankPositionSequenceScorePredictionHLA-DRB1*0301 (threshold, 2.960)1223IKMILKMVQ5.200Binder2230VQLDSIEDL4.960Binder3383LVFDECGIT4.800Binder4301LYVDSLFFL4.360BinderHLA-DRB1*0401 (threshold, 1.480)1194YLIEKVKRK4.300Binder2738YATEVSNMS3.800Binder3321MNPLETLSI3.200Binder4430LIGLSNLTH3.180BinderHLA-DRB1*0701 (threshold, 4.100)1513VINTNSLSL8.200Binder27WGSIQSRYI6.500Binder3669LKQINSQTL6.300Binder4238LEVTCTWKL5.720BinderHLA-DRB1*1501 (3% threshold=3.250)1259MINLRRLLL5.500Binder2262LRRLLLSHI4.600Binder3513VINTNSLSL4.300Binder4321MNPLETLSI4.160Binder PRAME, Preferentially expressed antigen in melanoma |