Design of a novel multi-epitope vaccine candidate against Chlamydia trachomatis using structural and nonstructural proteins: an immunoinformatics study

Abstract Chlamydia trachomatis (C. trachomatis) is an obligate intracellular bacterium which causes eye and sexually transmitted infections. During pregnancy, the bacterium is associated with preterm complications, low weight of neonates, fetal demise and endometritis leading to infertility. The aim of our study was design of a multi-epitope vaccine (MEV) candidate against C. trachomatis. After protein sequence adoption from the NCBI, potential epitopes toxicity, antigenicity, allergenicity, MHC-I and MHC-II binding, cytotoxic T lymphocytes (CTLs), Helper T lymphocytes (HTLs) and interferon-γ (IFN-γ)- induction were predicted. The adopted epitopes were fused together using appropriate linkers. In the next step, the MEV structural mapping and characterization, three-dimensional (3D) structure homology modeling and refinement were also performed. The MEV candidate interaction with the toll-like receptor 4 (TLR4) was also docked. The immune responses simulation was assessed using the C-IMMSIM server. Molecular dynamic (MD) simulation verified the structural stability of the TLR4-MEV complex. The Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) approach demonstrated the MEV high affinity of binding to the TLR4, MHC-I and MHC-II. The MEV construct was also stable and water soluble and had enough antigenicity and lacked allergenicity with stimulation of T cells and B cells and INF-γ release. The immune simulation confirmed acceptable responses of both the humoral and cellular arms. It is proposed that in vitro and in vivo studies are needed to evaluate the findings of this study. Communicated by Ramaswamy H. Sarma


Introduction
Chlamydia trachomatis (C.trachomatis) is among major causative agents of sexually-transmitted diseases which also causes eye infections with increasing trend annually (Janssen et al., 2018;Phillips, 2019).C. trachomatis prevalence among pregnant women ranges 3-20% and is associated with preterm complications, low weight of neonates, fetal demise and endometritis leading to neonatal infection, mortality and females' infertility.C. trachomatis causes annual infection of 1.7 million reported cases in the United States (US).The bacterium is mostly isolated from young age and adolescent populations (ranging 15-49 years).The actual incidence of the infection is neglected due to the presence of asymptomatic infections (Mascellino et al., 2008;van den Brule et al., 2002).These conditions lead to higher morbidity rates, un-treatment and recurrence of infection.More importantly, nearly 17% (1/6) of infected women develop ascending infection of pelvic inflammatory disease (PID) leading to the impairment of fetus and maternal illness.Notably, C. trachomatis-infected patients are predisposed to other sexuallytransmitted infections such as gonorrhea and human immunodeficiency virus (HIV) (Dzakah et al., 2022;Peters et al., 2021).Infants born from infected mothers also may develop pneumonia, conjunctivitis, cervicitis and urethritis.Moreover, C. trachomatis causes epididymitis, orchitis and urethritis in men.On the other hand, due to failure in exact diagnosis or treatment and lower costs of vaccination, development of preventive strategies is helpful for combat against the disease (Honkila et al., 2018;Nash et al., 2020).C. trachomatis is an intracellular pathogen and efforts to develop vaccines should mainly target elicit of T cells and additional B cells responses.Development of vaccines is far less expensive than antibiotic treatment or screening for infection control worldwide.Major outer membrane proteins (MOMPs)-based vaccine candidates have exhibited both cellular and humoral responses in various conditions.Since the genital mucosa has low immuno-stimulatory potential, other routes of immunization such as intranasal route have been explored.Despite efforts to develop C. trachomatis protective vaccine, there has been no achievement with this regard until today at least in part due to low immune elicit capacity of vaccine candidates (Murray & McKay, 2021;Witkin et al., 2017).Thereby, safe and efficient vaccines may be achieved using subunit vaccines.Polymorphic Membrane Protein family (Pmp A-I) also have the potential to stimulate cellular immune responses.These proteins act as the surface autotransporter adhesins delivering virulence factors to initiate C. trachomatis cell invasion and attachment to epithelium and endothelium.C. trachomatis virulence plasmid gene carries eight protein-encoding genes (pgp1-pgp8) which are essential for the plasmid maintenance (Pgp1,2,6,8), positive regulation (Pgp4 and Pgp5) and enzyme activity (Pgp7,8) (Byrne, 2010;Patton et al., 2018;Versteeg et al., 2018;Yang et al., 2020).Virulence plasmid-deficient C. trachomatis strains have exhibited attenuated virulence (Collar et al., 2020;Jones et al., 2020).The outer membrane complex (OMC) protein exists in the bacterial EB form consisting of various proteins (Schott et al., 2020).Chlamydial protease-like activity factor (CPAF) is an essential factor for the CXCL10 downregulation during the C. trachomatis infection (Murthy et al., 2009;Patton et al., 2016).Intranasal immunization using an rCPAF protein has resulted in the induction of antibodies, cytokines, INF-c and TNF-a, robustly (Dudiak et al., 2019).Several vaccines such as whole bacterial cell, DNA vaccines and subunit vaccines have shown various efficiencies.Even inactivated vaccines could not exert protective immunity.Regarding adjuvants used for the C. trachomatis vaccination, subcutaneous delivery of rPmpD protein was coupled with a TLR4 agonist adjuvant (Russi et al., 2018).Notably, TLR2 and TLR4 have been applied in multi-epitope vaccines (MEVs) targeting chlamydial protein antigens (A candidate MEV against SARS-CoV-2) (Srivastava et al., 2019).TLR4 is expressed in various immune cell types such as granulocytes, immature dendritic cells (DCs), monocytes and macrophages (Demirci et al., 2020).The advantages of using an MEV over whole bacterial proteins candidate mostly include lower costs of production, lack of allergenicity, lack of non-specific host receptor binding and thereby providing safety in terms of cross-reactivity (Behmard et al., 2022;Peres et al., 2015;Yoon et al., 2020).Additionally, despite single-epitope vaccine candidates, MEV candidates induce highly targeted responses preventing recurrence of the infection (Behmard et al., 2022).The aim of this study was efficacy assessment of a novel MEV candidate against C. trachomatis using virulence proteins in silico.

MHC-I binding epitopes prediction
Prediction of epitopes which have the potential of provoking CTLs responses is crucial for designing an MEV candidate.As a source database, the Immuno Epitope Database and Analyzing Resource (IEDB-AR) (www.iedb.org)contains T cells epitopes, MHC and binding ligands for human and animals which predicts MHC-I and peptides binding potential.Hence, the IEDB web server was applied to predict epitopes with 9-11 mer length having the potential of binding to the MHC-I using the ANN 4.0 prediction method.Herein, the MOMP, PmpD, OmcB, CPAF and pGP2-D proteins potential epitopes were employed to predict CTLs stimulation.In this method, the MHC source species was selected as human; the IC 50 �50 nM and the percentile rank < 1 were selected as the cut off values for screening of high affinity epitopes.

Helper T lymphocyte (HTL) epitopes prediction
Prediction of epitopes potentially provoking helper T lymphocytes (HTLs) responses is indispensable in the design of an MEV.The IEDB web server was applied to predict epitopes with 15 mer length of having the potential of binding to the MHC-II using the NN-align 2.3 prediction method.The selected aforementioned protein epitopes were employed to predict HTL epitopes.In this method, the MHC source species was selected as human HLA-DR and the IC 50 �50 nM and the adjusted rank < 1 were selected as the cut off values for screening of high affinity epitopes.

IFN-c-inducing epitopes prediction
The IFN-c plays a pivotal role in the innate and humoral immune responses against intracellular pathogens and also cancer cells.Considering this, epitopes with the potential of INF-c releasing enhancement are crucial for MEV candidate design.The INFepitope server (http://crdd.osdd.net/raghava/ifnepitope) can accurately (81.39%) distinguish the potential of epitopes in the induction of INF-c secretion.This server predicts epitopes by the employment of various strategies such as motive-based analysis, machine learning strategy and accuracy hybrid approach.

Three dimensional (3-D) structure homology modeling and refinement
The online I-TASSER web server was employed for prediction of the 3D structure of the MEV construct.Afterwards, the 3D model structure was relaxed by the Galaxy Refine web server using repacking and the MD simulation.The Molprobity sites (http://molprobity.biochem.duke.edu/),ProSA web server and ERRAT (http://services.mbi.ucla.edu/ERRAT/)servers were then utilized for the quality assessment of the MEV candidate.The prediction of MEV tertiary structure discontinuous B-cell epitopes was performed using the Ellipro in the IEDB database (http://tools.iedb.org/ellipro/).

Multi-epitope vaccine-Toll-like receptor 4 interaction using molecular docking
The interaction of the MEV-TLR4 is pivotal to elicit efficient immune responses.Thereby, in this study, the extraction of the TLR4 3D structure (PDB ID: 4G8A) from the protein data bank (www.rcsb.org)was performed and the molecular docking was conducted using CLUSPRO 2.0 online server (cluspro.bu.edu/login.php) to analyze the binding pattern of the TLR4 and the MEV candidate.

Molecular dynamics simulation of the vccine-TLR4 complex
The all atoms MD simulation was applied to study the MEV-TLR4 docked complex dynamical behavior and stability of the structure.The MD simulation was implemented by GROMACS package 2021 (Van Der Spoel et al., 2005).In summary, the OPLS-AA was utilized as the force field in the current survey (Linse & Hub, 2021).Additionally, the TIP3P water molecule was employed as an explicit solvent for making a solvation of the complex system (Robertson & Skiniotis, 2022).Next, in order to mimic natural or physiological conditions, sufficient amounts of chloride and sodium ions were added to the simulation box.Periodic bounding conditions were considered in the simulation box (Van Der Spoel et al., 2005).To determine the long-range electrostatics, we used the particle mesh Ewald (PME) (Cerutti et al., 2009).The bonds length in the complex system was restrained using the LINCS algorithm in which the van der Waals connections and Coulombic bonds cut-off lengths were set up to be 1.2 nm (Van Der Spoel et al., 2005).The steepest descent algorithm was used for energy minimization to refinement of the unsuitable contact of the geometry.Then, system's temperature was gradually increased to reach 310 K during 0.5 ns in the NVT ensemble.Afterwards, to adjust the density and pressure of the system, 0.5 ns of simulation was performed at 1 atm pressure in the NPT ensemble.Eventually, the main simulation without any restrain on protein molecules was carried out for 100 ns.The eventual MD binding profile energy analysis was performed using the MMPBSA approach and the structure was visualized through the open-source molecular visualization (PyMOL) software (DeLano, 2002).

Immune simulations
The simulation of immune responses against the MEV construct was implemented using Server C-ImmSim version 10.1.The bone marrow, thymus and lymph nodes were simulation using this server and the incorporated parameters included HLA (B0702, A0101, and DRB1_0101 each in pair) for 100 steps and one injection, 12,345 random seeds and 10 volumes.Other parameters were set as the default (Rapin et al., 2011).

The road map
The whole flowchart of the designed MEV candidate road map has been represented in Figure 1.

T cell epitopes prediction
An efficient vaccine candidate provokes durable and multifunctional native and acquired immunity against infection, in which the CTL and HTL epitopes participate in responses.The CTL epitopes play a crucial role in durable immunity against intracellular pathogens via provocation of the CD8 þ cells.Furthermore, HTL epitopes are responsible for the inducing responses by the both arms including humoral and cellular immunity mainly via induction of CD4 þ cells.Thereby, an efficient proposed vaccine must contain the potential specific epitopes for receptors of the CTL and HTL subtypes.
We predicted the HTL and CTL epitopes using IEDB web server through the NN-align 2.3 and the ANN 4.0 methods, respectively (Tables 1 and 2).
We selected high potential epitopes for affinity of binding to MHC-I and MHC-II alleles.Priorities for selection included antigenicity and immunogenicity.The antigenicity, allergenicity, non-toxicity and immunogenicity features were predicted using VaxiJen v2.0, AllerTOP v.2.0, ToxinPred2 and IEDB class I servers, respectively.Moreover, the 3D structure modeling and refinement were performed using I-TASSER and Galaxy Refine web servers, respectively.

Multi-epitope vaccine design, structural characterization, refinement and validation
Highly potential epitopes of each protein (immunogenic, antigenic, non-allergen, INF-c-inducer, and T cells epitopes stimulating) sequence was adopted for MEV construct designing (Supplementary Tables S1 and S2) and the overlapping regions containing CTL epitopes were taken from the protein sequences.Accordingly, the designed linear MEV construct included six CTL, five HTL and five INF-c epitopes (Tables 1 and 2, Supplementary Tables S1 and S2) joined by GPGPG and KK linkers facilitating antigen immune processing and junctional epitopes formation.Additionally, the TLR4 agonist (CTB adjuvant) was linked to the N-terminal region of the construct utilizing EAAAK linker to provoke the levels and duration of responses.The molecular weight of the  adopted MEV construct was 64.769 kDa and contained 588 amino acids.The 3D structure was modeled using I-TASSER (Roy et al., 2010) and refined by the Galaxy Refine web servers.Furthermore, the structural quality validation of the model was performed by the Z-score, Ramachandran plot and overall quality factor analyses.The best model was adopted with a Z-score of À 5.28 (Figure 2), being in range of those from comparable size proteins, hence it was reliable (Figure 2).Ramachandran plot demonstrated that 92.7% of residues were in favored region, 5.4% in allowed and 1.9% of them in outlier regions of the 3D-model structure, which validated the overall quality of the MEV construct.A cut-off reliability of �90% should be considered for residues being in the favored region (Krieger et al., 2009).Considering our results in which >90% of MEV residues were located in the favored region, the reliability was confirmed.The ERRAT overall quality factor following analysis was 80.20, indicating the percentage of the sufficient quality and validity (Figure

3(e)
).An ERRAT score higher than 50 exhibits a good quality model and therefore, the overall quality factor of 80.20 verified our MEV candidate structural quality (Mora Lagares et al., 2020).

Immunogenicity, allergenicity and physicochemical characteristics
The antigenicity, allergenicity, solubility, safety and efficacy (using physiochemical characterization) assessment of the MEV candidate validated an appropriate designed construct (Table 3).Its feature scores included theoretical pI (9.83), aliphatic index (79.90)indicating thermo-stability, half-life in mammalian reticulocytes, yeast and E. coli included >30h, >20h and >10h, respectively.The GRAVY score included À 0.287 exhibiting its hydrophilic nature which facilitates its interaction with other proteins.Additionally, predicted solubility upon over-expression included 0.71 in aqueous environment confirming the MEV solubility.The final MEV instability index included 32.40 which exhibited a high stability.

B cell epitope prediction
B cells participate in efficient humoral immunity which can develop memory immunity against various infections particularly via interaction with epithelial cells.Enough B cell receptors are required to this aim and the presence of B cell epitopes in the MEV construct is crucial.Applying the ElliPro server, linear and conformational (continuous and discontinuous, respectively) B cell epitopes were predicted utilizing default parameters.A total of two conformational epitopes having scores of 0.72 and 0.78 and seven linear epitopes with scores ranging 0.71-0.82were adopted as the final B cell epitopes (Table 4).The PI value of 0.78 indicated that 78% of residues reside within the predicted ellipsoid zone of the epitopes which demonstrated high solvent accessibility.
Considering predicted epitopes (two conformational and seven linear epitopes), the MEV probably provokes humoral immunity.

Molecular docking of MEV-TLR4
TLRs recognize the pathogen associated molecular patterns (PAMPs) and initiate the native and acquired immune responses.Bacterial proteins such as those from C. trachomatis are detected by membrane TLR2, TLR4 and TLR6 and induce immunity via pro-inflammatory cytokines release.TLR4 is expressed in various immune cells.A stable immune response is exerted via an efficient interaction of MEV with corresponding immune cell receptors.In order to study these interactions, MEV docking with TLR4 agonist (CTB adjuvant) was performed.Hence, the ClusPro web server was applied and its results were ranked based on the cluster size providing a series of docked structures according to lowest energy structure in the cluster.The top cluster with the lowest energy score is the most reliable cluster.Thereby, the optimal docked structure was selected considering these conditions (Figure 3(a)).We used the optimal docked complex as initial structure for the running MD simulation studies.

Molecular dynamic simulation
The conformational changes of MEV-TLR4 complex were evaluated using the MD simulations.Furthermore, this simulation is implemented for assessment of the complex stability, interactions between MEV and TLR4 and efficient immune recognition of various epitopes.As depicted in Figure 3, the docked complex (MEV-TLR4) structural stability was evaluated during 100 ns MD simulation trajectory.The TLR4 root mean square of deviation (RMSD) of Ca atoms exhibited negligible flexibility during the simulation time highlighting that the TLR4 structure remained stable during 100 ns of simulation time.In addition, the RMSD values of the MEV Ca atoms demonstrated an initial increase and then after 40 ns had smooth fluctuations which indicated structural stability of the MEV.Additionally, local structural flexibility of the TLR4 and the MEV in the complex system was investigated using the computation of the root mean square of fluctuation values (RMSF).
The mean RMSF values of the MEV and TLR4 respectively included 0.80 and 0.49 nm, outlining the protein residues' local stability.The loop regions exhibited more fluctuation levels (Figure 3(d)) and flexibility required for the proper MEV holding at the binding pocket.The MEV and TLR4 complex radius of gyration (Rg) was calculated for the structure compactness estimation, demonstrating an approximate mean Rg value of 4.9 and 3.4 nm, respectively (Figure 3(e) and (f)).The Rg fluctuations reflect the regional and conformational movements and alterations in flexible regions of the

Free energy of the between the MEV and TLR4
To clarify the strength of the contact between MEV and TLR4 structures, the binding free energy between the two molecules was calculated using MMPBSA approach.According to Table 5, the Polar and nonpolar components energies (DE polar ¼ À 9080.93 þ/-539.77kJ/mol, DE non-polar ¼ À 733.36 þ/-34.22kJ/mol) play a key role in the stability of the MEV-TLR4 complex.Our results unraveled that the favorable electrostatic energy component (DE ele ¼ À 11033.72 þ/-778.03kJ/mol) has the main role in the binding process of the MEV to the TLR4.The polar term was then defined as the main driving force in the MEV binding to the TLR4.Accordingly, both hydrophilic and hydrophobic interactions, are crucial for occurring thermodynamically favorable connections between the MEV and TLR4 (DG binding ¼ À 9815.01 þ/-286.99kJ/mol).

Immune response simulation
Both the innate and acquired immune responses are pivotal to encounter C. trachomatis.In this study, the associated MEV construct immunogenic profile was evaluated using the C-IMMSIM immune server.As depicted in Figure 4(a), following primary release of the IgM, followed by IgM þ IgG, IgG1 and IgG1 þ IgG2 antibodies, rapid decrease of antigen (Ag) was observed (Figure 4(a)).The increase in the IgG levels was considerable in response to the disease a crucial factor in responses.Moreover, the appearance of various B cell isotypes (isotype switching) confirmed memory formation plus the release of considerable amounts of interleukins and cytokines (Figure 4

Discussion
Despite some advances in the C. trachomatis infection eradication, the ability of this bacterium to cause substantial health problems has remained considerable.Until today, there has not been any protective MEV to prevent the C. trachomatis infection or associated disorders such as autoimmune responses (Murray & McKay, 2021;Phillips, 2019;Witkin et al., 2017).In this study, an MEV candidate was designed using potentially immunogenic epitopes of C. trachomatis and the CTB adjuvant (TLR4 agonist) by the employment of appropriate linkers.The CTB was attached to the N-terminal region of the MEV construct using the EAAAK linker.The GPGPG glycine rich linker was used to bind proteins epitopes for increase the accessibility and motility of adjoining domains and the construct solubility.Various proteins were adopted including MOMP, PmpD, Virulence plasmid protein pGP2-D, CPAF and OmcB.These virulence proteins participate in the infection severity and are also critical for the bacterium (Steiert et al., 2023;Zhong, 2017) (Russi et al., 2018).An MEV candidate was designed against breast cancer using cancer-testis antigen BORIS which used L7/L12 ribosomal protein from mycobacterium or TLR-4/MD-2 agonist and elicited CTL, INF-c and B cell epitopes (Mahdevar et al., 2022).Another study revealed that an MEV using the SARS-CoV-2 non-structural proteins and b-defensin adjuvant could elicit CD8 þ and CD4 þ T cells (Safavi et al., 2020).Our study predicted CTL and HTL epitopes were screened in terms of antigenicity, immunogenicity and promiscuous traits (CTL and HTL epitopes overlapping) which were similar to those of previous studies on other diseases such as melanoma, mammary carcinoma and breast cancer (Mahdevar et al., 2021;Safavi et al., 2019aSafavi et al., , 2019b)).Our candidate was non-allergen using the AllerTOP v.2.0 server and had good physicochemical characters using ExPASy server.Moreover, the MEV was stable according to the molecular weight of 64.769 kDa and instability index of 32.40, where according to instructions, the instability index< 40 means a stable condition of the designed MEV.
The IEDB-Immune Epitope Database was employed for prediction of the T cell epitopes.The MEV theoretical pI and the GRAVY indices respectively included 9.83 and À 0.287 where the lower GRAVY score reflects polar nature and proper water solubility.Furthermore, the MEV was thermostable considering aliphatic index of 79.90.The half-life of the MEV was >30 h in mammalian reticulocytes, >20 h in yeast and >10 h in E. coli.This demonstrates the time needed for the protein reaching 50% of its concentration in the cells.These conditions were similar to those by MEV candidates against severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) and Toxoplasma gondii.The potential of MEV candidate binding to the host targets or its molecular modeling is achieved by several immuno-informatics tools.
Using the Ramachandran plot analysis, 92.7% of residues were placed in the favored region, 5.4% in allowed region and only 1.9% in outlier region which confirm the quality of the MEV 3D structure.The ERRAT score confirmed the MEV total quality and also the Z-score provided by the ProSA included À 5.28 which validated the 3D structure.
Protein epitopes can be recognized by TLR2 or TLR4 molecules (Aboudounya & Heads, 2021;Halajian et al., 2022).TLR4 is expressed in various immune cell types such as immature DCs, granulocytes, monocytes and macrophages (Demirci et al., 2020).Hence, TLR4 was selected to boost the immune responses and docked with the MEV structure which is activated through direct interaction.This activation leads to further induction of inflammatory responses in immune cells such as macrophages via NF-jB pathway (Demirci et al., 2020).Moreover, the negative charge of the MEV has the advantage in order not to aggregate or bind to the cell membrane.Noticeably, TLR4-deficient mice have been more susceptible to viral and bacterial infections.An in silico study by Aslam et al, revealed that MEV composed of Type III secretion system translocon proteins could elicit CTL, HTL, B cells responses and the docking with the CTB (TLR4 agonist) resulted in CAI score of 0.97 at highest level of expression in E. coli bacterium (Aslam et al., 2021).In an in vivo study by Tifrea et al, a recombinant subunit MOMP MEV candidate of C. trachomatis was able to protect the mice against infection, pathogenesis and infertility (Tifrea et al., 2020).
In our study, the immune simulation analysis using C-IMMSIM server demonstrated the rapid antibody responses initiated by IgM, mitigated the antigen sharply.Additionally, B cells population increase and isotype switching exhibited memory immunity.The activation of Th and Tc cells and production of cytokines was also indicative of proper immune provocation.Moreover, development of memory T cells inferred prolonged cellular immunity.
A recent study has used an MEV against Chlamydia pneumonia using subtractive and core proteins and reverse vaccinology in which binding to the TLR4 was confirmed and similar to study MHC-I and MHC-II binding and expression in E. coli host was demonstrated (Noor et al., 2022).Their study exhibited considerable innate and acquired responses using C-IMMSIM server.Similarly, our MEV construct elicited considerable B and T cells responses with memory immunity simulated in silico using the same server.
Our findings verified the docked complex (MEV-TLR4) structural stability during 100 ns MD simulation trajectory.The MEV-TLR4 complex RMSD of Ca atoms increases over time verifying continues enhancing trend up to 20 ns and then enters a plateau exhibiting a stable complex formation, while the RMSD score converged within �0.1 nm.Additionally, low rate of conformational or structural alterations in the flexible loop regions of the MEV-TLR4 complex, as observed by the RMSF pattern at various time intervals, indicated the stability, without effect on the efficient binding.The analysis of MD simulation revealed that the MEV-TLR4 complex was stable and inseparable during time span of MD simulation (Figure 3).
Various MEV candidates have been designed for bacterial and viral agents and also cancerous cells.The advantages of a MEV over the whole bacterial proteins or attenuated bacterial cells mostly include higher immunogenic capacity, lower costs and shorter time of production, easier construct manipulation, lack of allergenicity and excess antigenic load, lack of non-specific host receptor binding and thereby providing safety in terms of cross-reactivity (Aslam et al., 2021;Bahrami et al., 2019;Honkila et al., 2018;Nash et al., 2020).In a study by Hong Yu et al, outer membrane complex (COMC) provoked higher levels of neutralizing antibody and protection compared to recombinant OMPs in mouse model (Yu et al., 2020).In this study, the MD simulation unraveled that the Polar and nonpolar components energies (DE polar ¼ À 9080.931þ/-539.77kJ/mol, DE non-polar ¼ À 733.363 þ/-34.22kJ/mol) play a key role in the stability of the MEV-TLR4 complex.Our results demonstrated that the favorable electrostatic energy component (DE ele ¼ À 11033.721þ/-778.03kJ/mol) has the main role in the binding process of the MEV to the TLR4.The polar term was then defined as the main driving force in the MEV binding to the TLR4.Accordingly, both hydrophilic and hydrophobic interactions, are crucial for occurring thermodynamically favorable connections between MEV candidate and TLR4 (DG binding ¼ À 9815.016þ/-286.99kJ/mol).

Conclusion
In this study, an in silico designed MEV candidate efficacy from C. trachomatis MOMP, PmpD, OmcB, CPAF and Pgp-2D proteins was verified using immunoinformatics tools.The construct antigenicity, allergenicity, MHC-I and MHC-II binding epitopes, CTL and IFN-c-induction were predicted.The MEV construct had enough water solubility at high expression levels in E. coli.In the next step, the MEV' structural mapping and characterization, 3D structure homology modeling and refinement and its interaction with the TLR4 were also confirmed using molecular docking approach.Moreover, MD simulation verified the structural stability of the TLR4-MEV complex and the MEV candidate high affinity to bind to the TLR4.The MEV construct had enough antigenicity and non-allergenicity with ability to binding to MHC-I and MHC-II molecules, stimulation of T cells and B cells and INF-c release.The immune simulation confirmed acceptable responses of both the humoral and cellular arms.Future experimental studies are required to verify these findings.

Figure 1 .
Figure 1.Total flowchart of the procedure for designing the MEV candidate.

Figure 2 .
Figure 2. (a) Schematic profile of the MEV candidate with 588 residues length.A CTB as an adjuvant was attached to the N-terminal region of the MEV using EAAAK linker, followed by six CTL, five HTL and five INF-c epitopes integrated by GPGPG and KK linkers.(b) 3D structure model of the MEV protein.(c) ProSA value of 3D MEV model showing Z-score (-5.28).(d) Ramachandran plot assessment of refined structure.(e) The ERRAT overall quality factor of 3D MEV model.

Figure 3 .
Figure 3. (a) MEV (green cartoon)-immune receptor (light blue cartoon) complex after 100 ns simulations time.(b) Interface residues related to MEV (magentas line) and (brown line) are labeled.Illustration of the molecular dynamic equilibration for simulation outputs.(c) Root mean squared deviations (RMSDs) of Ca for TLR4 and subunit MEV.(d) Root mean squared fluctuations (RMSFs) of Ca atoms for TLR4 and subunit MEV.(e) and (f) Radius of gyration of the TLR4 and MEV candidate, respectively.
(c) and (d)).The B cells population enhancement was also in correlation to the IgG increase (Figure 4(b)-(d)).The immunologic provocation related to the MEV was dramatic and long-lasting considering memory B cells and T cells development.The capacity of eliciting HTLs and CTLs responses and associated memory Tc and Th were also observed (Figure 4(e)-(i)).High levels of INF-c and IL-2 alongside T H 1 and memory T cells was indicative of proper immune responses against C. trachomatis through activating various cells such as macrophages, natural killer (NK) cells, Tc and B cells.Noticeably, development of active Th, Tc and B cells was also indicative of acceptable provocation of responses.Overall, the MEV construct was capable of stimulating acceptable durable antibacterial host responses.

Figure 4 .
Figure 4. Immune responses simulation following the MEV construct exposure in silico.(a) Antigen (Ag) count along with antibody titers with specific subclasses.(b) Cytokines responses.(c and d) B cells population.(e-g) Th (helper T) cells population.(h and i) Tc (cytotoxic T) cells population.

Table 1 .
Final CTL epitopes for the MEV construction.

Table 2 .
Final HTL epitopes for the MEV construction.

Table 3 .
Antigenicity, allergenicity and physiochemical traits assessments of the final MEV protein primary sequence.

Table 4 .
Linear (A) and discontinuous (B) B cell epitopes of the final 3D structure of the C. trachomatis MEV candidate.
A. Peptides (linear B cell epitopes)

Table 5 .
Binding free energy calculation for the MEV candidate-TLR-4 complex.
a Electrostatic contribution.b van der Waals contribution.c Polar contribution of the solvation effect.d Non-polar contribution of solvation effect.e DE polar