In-Silico Design and Assessment of OprD Based Mult

bioRxiv preprint doi: https://doi.org/10.1101/2022.05.25.493433; this version posted May 26, 2022.
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In-silico design and assessment of OprD based multi-epitope
vaccine against Acinetobacter baumannii

Kashaf Khalid and Saadia Andleeb*
Department of Industrial Biotechnology, Atta-ur-Rahman School of Applied Biosciences (ASAB),
National University of Sciences & Technology (NUST), Islamabad 44000, Pakistan
* Correspondence: saadiamarwat@yahoo.com
Abstract
Gram-negative, opportunist pathogen Acinetobacter baumannii is notorious for causing a
plethora of nosocomial infections predominantly respiratory diseases and blood-stream
infections. Due to resistance development towards last-resort antibiotics, its treatment is
becoming increasingly difficult. Despite numerous therapeutic developments, no vaccine is
available against this ubiquitous pathogen. It is therefore apropos to formulate a rational vaccine
plan to get rid of the super-bug. Considering the importance of Outer Membrane Porin D (OprD)
as a potential vaccine candidate, we methodically combined the most persistent epitopes present
in the A. baumannii strains with the help of different immunoinformatic approaches to envisage a
systematic multi-epitope vaccine. The proposed vaccine contains highly immunogenic stretches
of linear B-cells, cytotoxic T lymphocyte epitopes, and helper T lymphocyte epitopes of outer
membrane porin OprD. The finalized epitopes proved to be significant as they are conserved in
A. baumannii strains. The final 3D structure of the construct was projected, refined, and verified
by employing several in silico approaches. Apt binding of the protein and adjuvant with the
TLR4 suggested significantly high immunogenic potential of our designed vaccine. MD

bioRxiv preprint doi: https://doi.org/10.1101/2022.05.25.493433; this version posted May 26, 2022. The copyright holder for this preprint (which
simulations showed highly stable composition of the protein. Immune simulations disclosed a
prominent increase in the levels of the immune response. The proposed vaccine model is
proposed to be thermostable, immunogenic, water-soluble, and non-allergenic. However, this
study is purely computational and needs to be validated by follow-up wet laboratory studies to
confirm the safety and immunogenicity of our multi-epitope vaccine.
Keywords:
Acinetobacter baumannii; nosocomial infections; multi-epitope subunit vaccine; bioinformatic
approaches, molecular dynamics simulations, molecular docking
1. Introduction
Gram-negative, opportunist A.baumannii is a ubiquitous coccobacillus, obligate aerobe and one
of the most rampant causes of nosocomial infections typically in immunocompromised patients,
accounting for enhanced mortality rates (Morris et al., 2019). Notorious for causing common
outbreaks and endemics, it has been reported to transfer substantially from patient-to-patient,
colonize on hands of health care workers as well as stay on environmental surfaces for longer
durations, thus leading towards A.baumannii specific endemicity (Morris et al., 2019; Wong et
al., 2016).It is one of the most common sources of myriad diseases that mainly involve
bloodstream infections such as bacteremia, respiratory infections including acute pneumonia and
chronic pneumonia, soft tissue infections, urinary tract infections, meningitis, and less commonly
osteomyelitis (Dexter et al., 2015; Harris et al., 2019; Morris et al., 2019).
Antimicrobial and antibiotic resistance levels of A.baumannii are stated to be four times higher
as compared to other Gram-negative bacteria such as Pseudomonas aeruginosa or Klebsiella
pneumoniae (Du et al., 2019). In this context, Centers for Disease Control and Infection (CDC)
has characterized the multi-drug resistant (MDR) Acinetobacter spp. as an urgent threat hence
emphasizing the need for extensive research on therapeutics development (Giammanco et al.,
2017). Additionally, A.baumannii has been listed by World Health Organization(WHO) among
top-priority dangerous pathogens that are responsible for posing the greatest threat to public
health(Harding et al., 2018).
Burgeoning resistance rates have left us with very few treatment options rendering the existing
treatment approaches as mostly incompetent. Existing approaches usually involve monotherapy
and combinatorial therapy which make use of different antibiotics such as polymyxins,
tigecycline, and sometimes aminoglycosides. However, their undesirable pharmacokinetic
properties, ability to cause toxicity (nephrotoxicity, neurotoxicity), and resistance have led to
clinical failure (Shrivastava et al., 2018). Moreover, some of the antibiotics are only useful if
they are used in combination whereas others are involved in increased fatality rate. Therefore,
new therapeutic options are the pressing need to treat multidrug-resistant A.baumannii infections
(Isler et al., 2018).
Taking this into account, researchers have worked extensively to devise new cost-effective
therapeutic strategies that predominantly involve chemo-immuno therapies and unraveling new
epitopes for active or passive immunization against the MDR pathogen. Newly proposed vaccine
candidates against this pathogen include outer membrane proteins and porins like Omp34
kDa(Jahangiri et al., 2018), OprC and OmpA in combination with pal (Lei et al., 2019), outer
membrane protein nuclease NucAb (Garg et al., 2016) , FilF (Singh et al., 2016), BamA (Singh
et al., 2017), phospholipase D (Zadeh Hosseingholi et al., 2014) , Pili subunit hemagglutinin
(Homenta et al., 2014) and functional exposed amino acid BauA (Sefid et al., 2015).Despite of
these advancements, there have been no FDA approved vaccines in markets due to underlying
limitations such as high toxicity, low immunogenicity ,insolubility when expressed, or complex
compositions.
Many studies have validated the use of OMPs as successful vaccine candidates in GNBs such as
Legionella pneumophila, Mannheimia haemolytica, Aeromonas salmonicida, Anaplasma
marginale, Bartonella henselae, Campylobacter jejuni, and Avian Pathogenic E.coli (APEC)
(Ayalew et al., 2010; Diao et al., 2020; Hove et al., 2020; Moumène et al., 2015). A copious
amount of information has been generated through proteomic analysis on different types of
families of porins such as OmpA and OprD family in Pseudomonas aeruginosa (Chevalier et al.,
2017). OprD is primarily involved in carbapenem uptake. Recent studies have shown
downregulation of OprD and upregulation of efflux systems in the development of mutation
mediated resistance (Chevalier et al., 2017; Zeng et al., 2014). The active contribution of OprD
in causing the resistance makes it an important tool for combating this pathogen.
According to studies, OprD of A.baumannii could act as a putative vaccine candidate
(Kim et al., 2016). However, there is a lack of information on the immunogenic capability of
OprD in A.baumannii . This evidence convinces us to carry out the present study aiming at the
assessment of the capacity of OprD to elicit an immune response through bioinformatics
analysis. In the current study, we selected immunogenic B and T cells of OprD of A.baumannii.
This paper concisely elucidates and explores the in-silico strategies employed in vaccine
designing. Region of OprD having the greatest potential of immunogenicity has been elected as a
novel candidate for the vaccine that could potentially be utilized for designing therapeutic/
prophylactic peptide vaccines against A. baumannii.

Figure 1. General schema of the strategy followed in the present study.
2. Materials and Methods

2.1 Pre-Vaccine Designing Immunoinformatic Analysis
2.1.1 FASTA Sequence Retrieval
The FASTA protein sequence of OprD (ID QFQ03744) of A.baumannii representative strain
ATCC 19606 was downloaded from National Center for Biotechnology Information (NCBI).
Additional bioinformatic analysis was carried out, as described in the following section.
2.1.2. Signal peptide and localization prediction
To identify possible signal peptide in the sequence, online tools SignalP 5.0 (Almagro
Armenteros et al., 2019) available at http://www.cbs.dtu.dk/services/SignalP-5.0/ and LipoP

(Juncker et al., 2003) at http://www.cbs.dtu.dk/services/LipoP/ were used. SignalP 5.0
encompasses artificial neural networks to improve the prediction performance of signal peptide
(Almagro Armenteros et al., 2019). LipoP 1.0 locates signal peptidase I and II cleavage sites
within the protein sequence (Juncker et al., 2003). Predictions about protein localization were
performed by employing a template free algorithm, DeepLoc which also achieves high accuracy
by employing deep neural networks (Almagro Armenteros et al., 2017). Two different servers
were employed for prediction of putative transmembrane domains (TMBs): PRED-TMBB and
BOCTOPUS2. PRED-TMBB enables its users to pinpoint trans-membrane strands of GNB
through the method of Hidden Markov Model (HMM) (Tsirigos et al., 2016).Meanwhile, other
online tool, BOCTOPUS2 predicts topology by detecting the backbone hydrogen bonding
restraints that could be used to form large size TMBs (Hayat and Elofsson, 2012).
2.1.3 Analysis of conserved region
A preliminary analysis of OprD protein sequence of A.baumannii as representative for all the
possible strains of bacterium was conducted using BLASTp. BLAST search was performed with
non-redundant protein sequences (nr) database of bacteria using blosum62 matrix. The retrieved
sequences were aligned by multiple sequence alignment (MSA) using BLAST software, to
obtain the conserved regions. To evaluate the reliability of this protein sequence, it was used as a
query for performing BlastP analysis against non-redundant database restricted to Homo sapiens
(taxid:9606). Non-human homologous proteins in other GNBs were also identified and
interpreted to assess the inter-species and intra-species conservation of the selected protein.
2.1.4. Mapping of continuous B-cell epitopes

B-lymphocytes in immune system detect and attach themselves to B-cell epitopes (BCEs)
harbored within foreign molecules. Prediction of BCEs is important in designing the vaccine
construct as well as diagnostic tests (Larsen et al., 2006). Several servers were used to discover
potential B-Cell Epitopes. BepiPred-2.0 webserver was exploited to forecast the linear B-cells
epitopes by employing random forest algorithm. As compared to other tools, this method is
outstanding for sequence-based predictions (Jespersen et al., 2017). For precise estimation of the
linear BCEs, Hidden Markov model, Thornton’s method, and Support Vector Machine (SVM)
methods were utilized by ABCpred, BCPREDS, and SVMTriP, respectively (Saha and Raghava,
2006; Yao et al., 2020). Using various tools to find BCEs generates good quality results (Faria et
al., 2011).
2.1.5 Evaluation of Cytotoxic T lymphocytes (CTL)
Identifying specific peptide patterns that elicit strong MHC restricted cytotoxic T cell response is
a vital step to formulate vaccine (Zhao et al., 2003) . To effectively forecast the CTLs, a
webserver NetCTL 1.2 was harnessed in which information is generated by artificial neural
networks (ANN) and matrix methods pertaining to TAP transport efficiency, MHC class I
affinity and proteasomal cleavage (Larsen et al., 2007). A default parameter 0.75 was used as a
threshold value.
2.1.6 Helper T-Lymphocyte (HTL) Epitope Mapping
Identification of HTLs was achieved by the webserver NetMHCII 2.3. This server calculates
binding affinities of the peptides to MHC II molecules including 7 mouse H2 alleles, 20 HLA-
DQ,9 HLA-DP, and 25 HLA-DR class II alleles. It has been reported to be a highly effective tool
for accurately measuring binding affinities of peptides towards MHC class II binding molecules.
The results are shown in IC50 nM units along with the percentage rank to a set of 1,000,000
random natural peptides. IC50 binding values of < 500 nM represent strong binding affinities so
this criteria was chosen (Jensen et al., 2018).
2.2 Multi-Epitope Subunit Vaccine Designing
Correct positioning of the nominated epitopes plays a significant role in yielding maximum
immunization. Improper joining of peptides without linkers and adjuvant may lead to the
synthesis of a completely new protein with unknown properties (Farhadi et al., 2015). To curb
such errors, selected B-cell, HTL, and CTL epitopes were progressively connected by means of
suitable linkers viz. GPGPG to connect linear B cell epitopes with HTLs and AAY to connect
CTLs. Apart from enhancing the immunogenicity of the construct, these linkers are also involved
in better epitope presentation and averting the production of junctional epitopes (Tahir ul Qamar
et al., 2020). Adding an appropriate adjuvant is fundamental in boosting immunogenic behavior
of the vaccine (Sun et al., 2018).Capability of 548AA long GroEl HSP60 of Salmonella typhi as
an immunogenic protein has been promising (Chitradevi et al., 2013). Therefore, its protein
sequence (AN: NP_458769.1) was retrieved from NCBI and saved in the FASTA format. It was
attached to amino terminus of the designed construct via EAAAK linker which is a rigid helical
linker that does not permit interaction of construct with other areas of proteins, thus resulting in
stability (Choi et al., 2019).
2.3. Post-Design Analysis and Validation
2.3.1. Assessment of Physicochemical Aspects and Peptide Solubility
The physicochemical aspects of designed vaccine construct was predicted by the ExPasy
Protparam webserver. The parameters calculated were the total number of residues, molecular
weight in kDa, in-vivo and in-vitro half-life, aliphatic index, theoretical pI, instability index, and
grand average of hydropathicity (GRAVY) (Gasteiger et al., 2005). To gauge the solubility of
the peptide, Protein-Sol webserver was employed. This webserver uses a fast protein sequence-
based calculation of solubility. The prediction output is given in the format of 0-1 range with
values greater than the average value of 0.45 being considered as soluble (Hebditch et al., 2017).
2.3.2. Immunogenicity Profiling
Allertop v 2.2 was employed to investigate the allergenicity of the vaccine. It uses a machine
learning method that classifies protein using the k-nearest neighbors (kNN) algorithm and thus
shows accuracy level of 85.3% at 5-fold cross-validation (Dimitrov et al., 2014). VaxiJen v 2.0
and ANTIGENpro tools to measure antigenic behavior of the peptide were utilized. VaxiJen
depicts the immunogenic potential of construct with an accuracy ranging from 70% to 89%
(Flower et al., 2017). According to several reports, ANTIGENpro can predict the protein
antigenicity with an accuracy greater than 75.5% (Magnan et al., 2010).
2.3.3. Determination of the Secondary Structure
To determine the secondary structure of the polypeptide, a webserver PSIPRED was employed
which performs processing of position specific scoring matrix using two feed forward neural
network with an accuracy of 84.2% (McGuffin et al., 2000).
2.3.4 Derivation of 3-Dimensional Structure of Vaccine
To forecast the 3D shape of the peptide, webserver I-TASSER (Iterative Threading ASSEmbly
Refinement) was utilized that performs the structure folding and remodeling through Monte
Carlo simulations which are based on the improved knowledge-based force field. It consists of
three key methodologies: (i) hydrogen-bonding networks (ii) basic statistical potentials, and (iii)
threading-based restraints from LOMETS (Yang et al., 2014). It is reported to be a top-ranked
server for carrying out structural predictions for the last five CASP experiments (Zhang, 2008).
2.3.5. Improvement of Derived Protein Structure
The initial models are further sent for improvement that includes accurate and quality
transformation of initial models into structures that are comparable with experimental structures
(Feig, 2017). ModRefiner server http://zhanglab.ccmb.med.umich.edu/ModRefiner was utilized
for preliminary refinements to achieve an overall improvement in the physical structure (Xu and
Zhang, 2011). Next, a webserver GalaxyRefine http://galaxy.seoklab.org/cgi-
bin/submit.cgi?type=REFINE was employed to further enhance the structural quality. This
method involves remaking and repacking of side chains as well as overall structure relaxation by
molecular dynamics simulations. It has refinement (Heo et al., 2013).
2.3.6 Structure Validation
Evaluation and confirmation of the structural quality of the protein construct are ensured via
several quality assessment tools such as Ramachandran plot analysis for viewing torsion angles
distribution in the structure, clash scores for all-atom contact analysis, aberrations in structural
conformity, and rotamers analysis. These indicators have proved to be highly valuable in
determining the accuracy of local and global three-dimensional structures of protein (Pražnikar et
al., 2019). In light of this, many online web servers were utilized to confirm the refined
structures. These include (i) ProSA-web https://prosa.services.came.sbg.ac.at/ (Wiederstein and
Sippl, 2007) (ii) ERRAT https://servicesn.mbi.ucla.edu/ERRAT/ (Dym et al., 2012) (iii)
MolProbity http://molprobity.biochem.duke.edu/ (Chen et al., 2010). PROCHECK
https://servicesn.mbi.ucla.edu/PROCHECK/ (Roman Laskowski et al., 1983) was also employed

to analyze the Ramachandran Plot. The Ramachandran plots help in analyzing the dispersal of
(φ, Ψ) torsion angles of the protein backbone to evaluate the protein structures (Sobolev et al.,
2020).
2.3.7 Mapping of discontinuous B-cell epitopes (Conformational)
Distantly located residues on the primary structure of a protein that fall into proximity due to
folding of a protein structure constitute >90% of discontinuous B-cell epitopes (Mukonyora,
2015). An online tool, ElliPro http://tools.iedb.org/ellipro/ was employed to infer conformational
BCEs. Ellipro is considered the state of the art webserver as it gave an AUC value of 0.732
(Ponomarenko et al., 2008).
2.3.8 Molecular Dynamics (MD) Analysis
GROMACS 5.0 software was used to have a deep understanding of structural integrity of protein
in a life-like simulated environment (Abraham et al., 2015). OPLS-AA force field was applied to
the system and overall charge was noted when pdb file was used as an input. SPC/E water model
was used to solvate the cubic system and genion tool was employed to add ions into the system
to nullify the overall charge. It is pertinent to stabilize the temperature using NVT ensemble and
pressure under NPT ensemble. Subsequently, MD simulations were performed for 10
nanoseconds (ns) and resulting trajectories of root-mean square deviation (RMSD) and root-
mean square fluctuations (RMSF) were examined.
2.3.9. Molecular Docking Analysis
Only appropriate attachment of immunogenic molecules to the specific immune receptors can
evoke a proper immune response. TLR4 is involved not only in generating suitable immune
response and long-lasting immunity but also plays a specific role in A. baumannii infections in
vitro and in vivo (Monem et al., 2020; Pulido et al., 2020; Shi et al., 2020). Therefore, TLR4 was
selected as an immune receptor for binding to the vaccine construct. TLR4 binding pockets were
forecasted by the CASTp v 3.0 server (Computed Atlas of Surface Topography of proteins)
accessible at http://sts.bioe.uic.edu/castp/. Docking analysis of the designed construct with TLR4
(PDB ID:4G8A), was conducted by a python-based web server HADDOCK v 2.4 (High
Ambiguity Driven protein-protein DOCKing) available at
https://wenmr.science.uu.nl/haddock2.4/. Interactions are measured in the terms of inter and
intramolecular energies that involve Van der Waal forces and electrostatic energies (Ambrosetti
et al., 2020). Three steps are involved in the docking protocol viz, stringent body energy
minimization, an intermediately-flexible fine-tuning in torsion angle space, and an ultimate
refinement in the explicit solvent (De Vries et al., 2010). To accurately drive the docking
protocol, active and passive residues were procured using the online web-tool CPORT
(Consensus Prediction Of interface Residues in Transient complexes)
https://milou.science.uu.nl/services/CPORT/ (Xue et al., 2016). Thus, the data collected after
successfully predicting the active and passive residues was used to guide the prediction-driven
docking in HADDOCK v 2.4 to better explore the interactions among the vaccine construct and
TLR4 as well as the adjuvant and TLR4. The structure of adjuvant GroEl (AN: NP_458769.1)
was modeled by employing SWISS-Model (https://swissmodel.expasy.org/). To calculate the
protein binding affinities, PRODIGY web-server (PROtein binDIng enerGY prediction) (Xue et
al., 2016) accessible at https://bianca.science.uu.nl/prodigy/ was exploited.
2.3.10. Codon analysis and mRNA expression of the target protein
Codon optimization is essential to promote copious yields of gene product in the host. Codon
adaptation as well as the reverse translation was performed to improve the gene expression levels
in the host. Therefore, codon utilization of the intended construct in Escherichia coli K12 strain
was performed by the JCat (Java Codon adaptation tool) server at http://www.jcat.de/ (Grote et
al., 2005). Moreover, three additional opportunities were availed to avoid cleavage sites of
restriction enzymes, rho-independent transcription terminators, and prokaryotic ribosome
binding. The results show enhanced GC content besides Codon Adaptation Index (CAI), which
indicates expression levels of protein. CAI score of 1 is considered ideal value however values
greater than 0.8 are deemed desirable. To enhance efficacy and promote expression of the
designed peptide in host, it was cloned in E.coli vector by integrating two restriction sites viz.,
Nde I and Xho I at the amino and carboxy-terminus of the designed construct, respectively.
Finally, the adjusted sequence was incorporated into the pET30a (+) expression vector via the
SnapGene software.
2.3.11. Estimation of the immune response
A webserver C-ImmSim available at http://150.146.2.1/C-IMMSIM/index.php generates
simulations of the responses generated by the immune system upon stimulation by the final
vaccine construct. It produces simulations for both humoral and cellular immune responses that
are markedly tantamount to the actual immune responses generated by the human body (Rapin et
al., 2010) . Three simulated injections having no LPS were given at three time intervals 1,42 and
84 using default settings of simulation volume and simulation steps set at 10 and 100
respectively and the random seed at 12345 (Majid and Andleeb, 2019).
3. Results
3.1. Pre-Vaccine Design Analysis
3.1.1. Sequence retrieval and primary in-silico analysis

The complete protein sequence of OprD (AN: QFQ03744) 438 amino acid in length, belonging
to representative strain ATCC 19606 of A.baumannii was downloaded in FASTA format from
NCBI. The protein was predicted to be extracellular by Deeploc. Signal peptide was identified at
the position 1 to 22 of the sequence with position 22-23 as a cleavage site which could be
cleaved by signal peptidase. BOCTOPUS2 and PRED-TMBB predicted eight transmembrane
strands. BLASTp analysis was carried out using the accession number as a query sequence
against which no hit specific to the taxid:9606 Homo sapiens was found. However, the protein
sequence covered 726 strains of Acinetobacter spp and thus suggested wide-spread presence of
this protein sequence amongst numerous strains of A.baumannii. Results revealed 93.76% to
100% similarity among A.baumannii strains by covering 726 strains of A.baumannii and found
40 hits of OprD which shared ≥97% identity, 100% query coverage, and E-value of 0, thus
proving as a universally conserved A. baumannii antigen. OprD is also found in Pseudomonas
aeruginosa and Klebsiella pneumoniae strains but with lesser identity (36-40% and 37%
respectively).
3.1.2. Prediction of linear B-cells
The selection of the most recurrent and immunogenic epitopes was done carefully to design the
vaccine construct. BCPred, ABCpred, SVMTrip and BepiPred 2.0 predicted a total of 74
epitopes. and final recruitment of selected epitopes in the development of a new vaccine
construct (Table 1).
3.1.3. Prediction of Cytotoxic T Lymphocytes (CTLs)

Overall, 20 CTL (9-mer) epitopes were identified with the assistance of NetCTL 1.2 webserver.
Default settings were selected for the calculation of epitopes. Finally, 02 epitopes were carefully
chosen based on their high-affinity scores (Table 1).
3.1.4. Prediction of Helper T Lymphocytes (HTLs)
The webserver NetMHCII 2.2 envisaged MHC-II epitopes that illustrate high binding scores
towards the HLA alleles in the form of IC50 values (in nM). For final incorporation into the
vaccine construct, a sum of two epitopes were opted (Table 1).
Table 1 The final selected epitopes, their position, and Placement ID in the vaccine design
Epitope Placement ID Sequence Position Conservation

(Start-End)
B-Cell B1 LTRDKKQGAKDTSSV 47-61 100%
B2 ANAHTYSATGTVAPN 256-270 100%
Cytotoxic-T C1 NADGFQSIY 317-325 100%
Lymphocyte
C2 ASDAYQGAY 412-421 100%
Helper-T H1 KLTLAVLISAAIISS 6-21 100%
Lymphocyte
H2 VQSGFAKDASLRIRN 393-408 100%
3.2 Construction of final vaccine with different adjuvants and linkers
To design an efficient vaccine, it is necessary to make use of a potent adjuvant that would result
in a boosted immunogenicity potential of the construct. A total of 06 peptides were arranged and
the GroEl adjuvant from Salmonella typhimurium was incorporated into the amino terminus of
the B-cell epitopes using the EAAAK linker. MHC-1, MHC-II epitopes, and B cell epitopes
were connected via specific linkers, AAY, and GPGPG as they do not modify the conformation
of vaccine construct. EAAAK linker helps in the attachment of the adjuvant at the N-terminus of
B-cell epitope. The remaining BCEs and HTLs were joined together with the help of GPGPG
linkers. For proper fitting of the CTL epitopes, AAY linkers were utilized. At the carboxy
terminus of peptide, six-His tag was attached to promote the process of identification and
purification (Fig. 2)
Figure 2. Schematic diagram and amino acid sequence of the final vaccine construct. (a) Arrangement of selected
epitopes along with adjuvant and appropriate linkers. (b) Blue ink represents the amino acid sequences of epitopes
and adjuvant whereas black ink represents the linkers and 6-His tag.
3.3 Post-Vaccine Design Analysis and Validation
3.3.1. Analysis of physicochemical features
The physicochemical aspects were estimated using the FASTA format in the ExPASY
ProtParam webserver to know about the basic characteristics of the vaccine. The molecular
weight was calculated to be 69.9 kDa. The isoelectric point value (pI) was found to be 5.51,
which illustrates that the protein is slightly acidic. ProtParam declared the protein as stable by
identifying the instability index (II) value as 28.26.II values greater than 40 signify instability.
The GRAVY value was noted to be −0.026 exhibiting the hydrophilic nature of the protein and
thus the possibility of interactions with the neighboring water molecules. Half-life of the
designed construct was evaluated to be 30 hours in mammalian reticulocytes in vitro, >20 hours
in yeast, and >10 hours in E. coli in vivo. The protein displays the property of being soluble
when expressed by exhibiting the solubility score of 0.611. Furthermore, the protein construct
was indicated to be thermostable by showing the aliphatic index value of 74.85.
3.3.2. Antigenicity, Toxicity, Allergenicity,
The overall antigenicity of the construct was estimated to be 0.64 in bacteria and 0.55 in virus
model by VaxiJen. AntigenPro estimated the antigenicity score to be 0.88. The VaxiJen score at
a threshold value of 0.4 was also determined excluding the adjuvant for which the antigenicity
score was estimated 0.78 in the bacteria model and 0.477 in the virus model. It is clear from the
results that the vaccine construct itself can elicit the immune response whether an adjuvant is
attached. The protein was proposed to be a non-allergen by AllerToP v 2.0.
3.3.3. Secondary Structure
The final construct consisted of 670 residues with 34.48% of the random coil, 54.03 % of the
alpha-helical structure, and 11.49 % of the extended strand (Fig. 3)

Figure 3. PSIPRED generated secondary structure of the designed peptide. (a) E denotes extended strand,
h: alpha helix, c: random coil, and b: Beta turn. (b) Elements of secondary structure (blue: alpha helix,
red: extended strand, green: beta turn, yellow: random coil
3.3.4. Derivation of the Tertiary Structure
I-TASSER server predicted five models based on top ten threading templates which exhibited
good alignment according to their z-scores ranging from 1.71-15.61. The predicted models
showed c-scores in the range of -2.50 to -0.45. The normal value range of the C-score is -5 to 2,
where values closer to 2 represent higher confidence in the structure. The predicted structure
showing the maximum c-score of -0.45 was chosen for additional refinements (Fig 4a). The TM
score and RMSD value were documented to be 0.66 and 3.0 ± 4.6, respectively. A model
demonstrates precise topology if the TM value is greater than 0.5. Values lesser than 0.17
indicate nonspecific similarity.

Figure 4. 3-D structure prediction, improvement, and validation. (a) 3-D structure modeled by I-TASSER
(b) structure refined by ModRefiner (c) Improved structure by GalaxyRefine (d) Ramachandran Plot
depicting allowed and disallowed regions (e) Z-score scatter plot
3.3.5. Refinement of Protein 3-D Structure
Rectification of the primary vaccine model was attained by the ModRefiner server (Fig 4b)
followed by subsequent refinements by GalaxyRefine (Fig. 4c). After several refinements, a final
model was selected based on distinct parameters which include RMSD (0.286), GDT-HA
(0.9873) and MolProbity (2.046). Poor rotamers value and clash score was observed to be 0.0
and 10.7, respectively. Ramachandran plot score was noted to be 94.5%. Therefore, further
immunoinformatic analysis were performed on this carefully chosen model.
3.3.6. Structure Validation
According to the Ramachandran plot analysis, 94.46 % of protein residues were found to be
present in the favored region (Fig. 4d). This value is consistent with the GalaxyRefine score of
94.5%. Moreover, 8.7% residues were observed in the allowed region and only 1% of the
residues were detected in the disallowed region. These percentages symbolize the good quality of
our predicted model. To determine the presence of any possible errors in the refined model or
validate the global quality of the structures and, it is necessary to determine ERRAT and ProSA-
web scores. Thus, the Z-score of -9.54 (Fig. 4e) and the quality factor of 87.6% further endorse
the good quality of our model.
3.3.7. Predicted Discontinuous Epitopes
A sum of 7 discontinuous B-cell epitopes having 127 residues in total and the immunogenicity
scores ranging from 0.641 to 0.891 was identified. Varying sizes of the conformation epitopes
were documented extending from five to thirty-nine residues (Table 2, Figure 5).
Table 2. Discontinuous epitopes present in the protein as predicted by the ElliPro. A total of 127 residues
lie in the 7 conformational B-cell epitopes
No. Residues No. of Score

residues
1 A:L633, A:T634, A:L635, A:A636, A:V637, A:L638, A:I639, 35 0.966
A:S640, A:A641, A:A642, A:I643, A:I644, A:S645, A:S646,
A:A647, A:A648, A:Y649, A:V650, A:Q651, A:S652, A:G653,
A:F654, A:A655, A:K656, A:D657, A:A658, A:S659, A:L660,
A:R661, A:I662, A:R663, A:N664, A:H667, A:H669, A:H670
2 A:H665, A:H666, A:H668 3 0.965
3 A:A619, A:K620, A:D621, A:A622, A:S623, A:L624, A:R625, 14 0.92
A:I626, A:R627, A:N628, A:A629, A:A630, A:Y631, A:K632
4 A:A550, A:A551, A:A552, A:K553, A:L554, A:T555, A:R556, 69 0.775

A:D557, A:K558, A:K559, A:Q560, A:G561, A:A562, A:K563,
A:D564, A:T565, A:S566, A:S567, A:V568, A:G569, A:P570,
A:G571, A:P572, A:G573, A:A574, A:N575, A:A576, A:H577,
A:T578, A:Y579, A:S580, A:A581, A:T582, A:G583, A:T584,
A:V585, A:A586, A:P587, A:N588, A:G589, A:P590, A:G591,
A:P592, A:G593, A:K594, A:L595, A:T596, A:L597, A:A598,
A:V599, A:L600, A:I601, A:S602, A:A603, A:A604, A:I605,
A:I606, A:S607, A:S608, A:G609, A:P610, A:G611, A:P612,
A:G613, A:V614, A:Q615, A:S616, A:G617, A:F618
5 A:G198, A:Y199, A:L200, A:S201, A:P202, A:Y203, A:F204, 112 0.684
A:I205, A:N206, A:K207, A:P208, A:E209, A:T210, A:G211,
A:A212, A:V213, A:E214, A:L215, A:E216, A:S217, A:P218,
A:F219, A:L221, A:A223, A:D224, A:K225, A:K226, A:I227,
A:S228, A:N229, A:I230, A:R231, A:E232, A:M233, A:L234,
A:P235, A:V236, A:L237, A:E238, A:A239, A:V240, A:A241,
A:K242, A:A243, A:G244, A:K245, A:P246, A:L247, A:L248,
A:I250, A:A251, A:E252, A:D253, A:V254, A:E255, A:G256,
A:E257, A:A258, A:L259, A:A260, A:T261, A:L262, A:V263,
A:V264, A:N265, A:T266, A:M267, A:R268, A:G269, A:I270,
A:V271, A:K272, A:V273, A:A274, A:A275, A:V276, A:K277,
A:A278, A:P279, A:G280, A:G282, A:D283, A:R284, A:G298,
A:T299, A:V300, A:I301, A:S302, A:E303, A:E304, A:I305,
A:G306, A:M307, A:E308, A:L309, A:E310, A:K311, A:A312,
A:T313, A:L314, A:E315, A:D316, A:L317, A:G318, A:Q319,
A:A320, A:K321, A:I325, A:N326, A:K327, A:D328, A:T329
Figure 5. Conformational B-cell epitopes projected as by ElliPro. (a-g) Depiction of conformational B-
cell epitopes of the OprD protein of A. baumannii from different angles. The yellow portion shows the
epitopes, rest of the protein is symbolized by grey sticks.
3.3.8. Molecular dynamics analysis of multi-epitope vaccine
To assess the movement of atoms and stability of protein, molecular dynamics simulation was
performed using the GROMACS server. The designed system underwent equilibrations
(potential energy, pressure, and temperature) followed by energy minimization. As a result,
graphs revealed that system reached a desirable temperature of 299.7 K (Fig. 6a) and an average
pressure of 0.92 bar with a total drift value of 0.62(Fig. 6b). To further analyze the overall
stability of the structure and how much the conformation was changed between two time points,
RMSD graph was analyzed. It showed that the protein remained highly stable. Initially the
fluctuations started from 0.12 nm and go up to 1.10 nm in time of 8.8 ns. These slight
oscillations show that model maintains stability over the period (Fig. 6c). To further analyze the
conformational stability, RMSF plot was generated and analyzed in which the highest peak of
fluctuation was observed to be at 1.37 nm, while the lowest peak of fluctuation was at 0.15 nm.
The overall graph showed very slight oscillations where higher peaks in graph depict regions of
higher flexibility (Fig. 6d).

Figure 6. Molecular dynamics simulation of vaccine (a) Temperature equilibration (at 100 picoseconds)
of the simulated environment obtained via classical NVT ensemble shows average value of 300 (K) (b)
Pressure equilibration (at 100 picoseconds) of the simulated environment obtained via classical NPT
ensemble shows average pressure of 0.92 bar (c) RMSD plot of the vaccine shows slight oscillations,
confirming the overall protein stability (d) RMSF plot revealing flexibility of the protein through high
fluctuation peaks.
3.3.9 Molecular Docking

HADDOCK v 2.2 was used harnessed to perform the molecular docking using the data-driven
approach. To understand the contacts between the immune receptor and the TLR4, the binding
pockets of the protein were estimated by the CASTp 3.0 server. As a result, a binding pocket
with a molecular surface area of 2933.3 Å2 and a molecular surface volume of 7317.6 Å3 was
identified. This pocket having the mouth molecular surface of 418.2 Å2 and the molecular
surface circumference sum of 348.1 Å could act as a potential interacting surface. In order to
gain insights into the residues actively participating in the interaction, CPORT gave a list of
active residues
A2,K3,L4,T6,E8,V39,A43,A44,G45,A46,A108,S200,A202,A203,I204,S205,A256,S257,I260,R
261,H263,H264 from the B chain of the GroEL adjuvant, T6, E8, E29, V33, A36, A37, V39,
A43, A44, G45, A46, A47, A49, K108, E109, D112, A114, E199, K277, F279, G280, D281,
M285, Q288, T329, I330, D332, Y476, E481, N485,M486, M489, G490, D493, I513, E516,
C517, M518, V519 from the A chain of the chimeric protein and E26, E41, L42, N43, Y45, K46,
I47, P48, N50, L51, F53, S54, T55, H67, L68,S70, Y71, S72, F73, S75, F76, E78, L79, S99,
S100, S103, S124, Y189, T191, L305,S384, G408, V409, V440, K475, A477, D500, S502,
N524, S526, L533, D534, D548, S550, M555,S557, N573, T575, A580 from the B chain of
TLR4. These active residues were fed to server to drive docking protocol smoothly and
effectively. The HADDOCK results generated several docked complexes out of which the
clusters having the lowest RMSD values (protein-TLR4 complex (0.5 Å), adjuvant-TLR4
complex (11.6 Å) with the best poses and least intermolecular energies among the construct-
TLR4 complex (−317.5 Kcal/mol), adjuvant-TLR4 complex (−389.2 Kcal/mol) were carefully
chosen. According to the results generated by the PRODIGY, the relative binding free energies
(ΔG) of both complexes; construct-TLR4 (-16.7Kcal/mol) and adjuvant-TLR4(-11.8Kcal/mol)

exhibit the capacity of construct and adjuvant to bind to each other properly and thus playing role
in instigating the TLR4 receptor response by making suitable conformational changes.
Additionally, a total of 11 hydrogen bonds between the vaccine-TLR4 complex as well as 14
hydrogen bonds between the adjuvant-TLR4 complex indicate strong bonding. The examination
of the number of interfacial contacts (IC) per property between the two complexes was (ICs
charged-charged: 13, ICs polar-polar: 14, ICs apolar-apolar:23) in case of vaccine and TLR4
complex whereas for the adjuvant and TLR4 complex values are (ICs charged-charged: 14, ICs
apolar-apolar: 20, ICs polar-polar:1) (Figure 7, Figure 8).
Figure 7. Molecular docking of the designed construct with TLR-4. (a) Binding of TLR-4 (Hot-pink) with
vaccine model (Yellow). (b)The interface of binding residues. (c) Interacting bonds among the vaccine
and TLR-4
Figure 8. Molecular docking of GroEl adjuvant with TLR-4. (a) Binding of TLR-4 (Hot-pink) with GroEl
adjuvant (Cyan). (b)The interface of binding residues. (c) Interacting bonds among the vaccine and TLR-
3.3.10. cDNA and mRNA Analysis for Cloning and Expression Prediction
The frequency of codon usage of the recombinant protein as well as the expression host needs to
be similar. For this purpose, codon optimization of the designed construct is performed utilizing
the JCat (Java Codon Adaptation Tool). The improved sequence was 2033 nucleotides long
having a GC content of 50.54% and Codon Adaptation index of 0.99 (Fig. 9). GC values
fluctuating from 30 to 70 % are optimal and indicative of high gene expression in the host.
Moreover, E. coli pET30a (+) vector was used for integration and optimal expression of the
designed construct. For successful integration and cloning of the sequence into the vector, two
restriction sites Xho I and Nde I were added with the help of SnapGene software (Fig. 10).
Figure 9. Adapted codons of the final vaccine protein. Codon adaptation index of optimized codon is 0.99
and GC value is found out to be 50.54%.

Figure 10. In silico restriction cloning of final protein into pET30a (+) vector. Red fragment signifies the
inserted gene sequence of the proposed vaccine whereas black segment specifies backbone of E. coli
vector. Cloned sequence is bounded by restriction enzymes Xho I and Nde I. 6x-His tag is positioned at
C-terminus.
3.3.11 Estimation of the Immune Responses
The collective immune responses after three times exposure to the antigen, produced by the final
vaccine construct indicate a considerable spike in the levels of IgM as well as B-cell populations.
Furthermore, an extortionate gush in the levels of IgM+ and IgG, IgG1, and IgG2 was also
observed (Figure 11a). Recurrent exposures to the vaccine helped in the development of memory
cells. Along with strong memory development, helper T cells and cytotoxic T cell populations
showed decent levels of the increment (Figure 11 b, c, d).

Figure 11. Simulated immune responses instigated by chimeric protein (a) Antibodies generated because
of successive antigen injections; other immune cells are denoted by colored peaks (b) considerable shifts
were observed in population levels of B-cells after three injections (c) Increments of helper-T
lymphocytes (d) Cytotoxic T-cell populations per state after the injections. Cells that are not presented
with the antigen are shown by the resting state while T-cells that demonstrate tolerance to the antigen
because of repeated exposures are indicated by the anergic state.
4. Discussion
A. baumannii has emerged as an extremely cumbersome pathogen across the world becoming the
leading cause of nosocomial and neonatal deaths (Peleg et al., 2008). It has successfully
developed pan-drug resistance and thus appeared as one of the most difficult pathogens to treat
or control (Styles et al., 2020). Almost 30 and 76% percent of the deaths are due to A.baumannii
infections which can allegedly increase depending upon the severity of the patients (Ballouz et
al., 2017). Different types of potential vaccine candidate have been discovered and many subunit
vaccines consisting of protein or set of proteins have been studied against A. baumannii that
include Outer Membrane Proteins (OMPs), Outer membrane Vesicles (OMVs), Inactivated
Whole Cells Vaccines (IWCs), Ata, bap and several other subunit vaccines (Singh et al., 2016).
However, due to the complex configurations, enhanced toxicity, and evocation of non-specific
immune response, none of these vaccines could make their way to market or even the clinical
trials (Ni et al., 2017a). More apt and rational vaccine design to combat this pathogen is the need
of the hour. Therefore, we designed a multi-epitope vaccine for the first time using innocuous
and more immunogenic regions of the outer membrane porin OprD.
The goal of this study was to introduce a novel multi-epitope vaccine design by exploiting
numerous in-silico approaches against A. baumannii. OprD is a vaccine candidate protein
predicted by the in-silico analysis of complete genomes and several strains of MDR A.
baumannii. Complex proteome analysis of complete genomes and antibiotic-resistant strains of
A. baumannii was performed by (Ni et al., 2017b). Outer membrane porins being profound in
nature could instigate a considerable amount of immune response and induce immunization
when encountered with A. baumannii. Eight porin proteins having potential roles as protective
antigens and virulence factors were recognized, out of which one was OprD. Several studies
support the prospective role of OprD as a successful vaccine candidate (Li et al., 2014). This
porin participates in the transport of basic amino acids and carbapenem uptake. It is pertinent to
use bioinformatics tools for carefully choosing the regions of OprD that confer immunogenicity.
OprD is present in a wide array of clinical strains of A.baumannii (Ni et al., 2017a; Zhu et al.,
2019). Using this protein would help in effectively targeting the complete range of pathogenic
strains of A. baumannii.
This study focuses on generating a multi-epitope vaccine from a single representative protein of
OprD. Latest computational tools for the development of vaccines have made it convenient to
carefully select rationally apt epitopes from the proteomes of pathogenic microbes. Employment
of such tools could help us pave ways towards designing an effective vaccine strategy. To have a
reliable vaccine model, it is germane to bring into account all the facets of vaccine development
viz. secondary and tertiary structure, antigenicity, allergenicity, flexibility, accessibility, and
hydrophilicity of the vaccine. Sticking to only one of these characteristics would not generate the
needed results.
The antibody-mediated or the humoral immune response is prompted by the recognition of
distinct features of the pathogen considered as non-self by the host (Chaplin, 2010). These
distinct features are antigenic determinants, also known as epitopes that are specifically detected
by the antigen-binding site of the antibodies present in the host's immune system (Cruse and
Lewis, 1999). There have been studies supporting the role of B cells and T cells immunity
against A.baumannii infections (Chen, 2020; Morris et al., 2019). The role of the TLR4 pathway
has also been reported in detail along with the potential roles of other TLRs in mediating the
interaction between antigens and the host immune system (Kim et al., 2013). We first projected
T cell and B cell epitopes of the protein and combined them with the aid of specific linkers to
obtain a multi-epitope vaccine as a result. These spacers play a key role in improving vaccine
design (Shey et al., 2019). Formerly reported cleavable linkers, AAY, GPGPG, and EAAK were
fused into the final vaccine design with AAY and GPGPG connecting the forecasted peptides
and EAAK linking the adjuvant to the N terminal of the B cell epitopes (Dong et al., 2020). A
six-His tag was joined at the C-terminus to ensure purification at the later stages. Several
computations based on immunoinformatic analysis revealed that the designed vaccine model
contains high-affinity MHC class I and class II epitopes and linear B cell epitopes in bulk
amounts. The absence of allergenic features in this model further endorses the safety of it as a
vaccine candidate.
The molecular weight of our construct was observed to be 69.9 kDa with the capability of being
soluble when expressed. Apart from defining the bioactivity of the protein, the solubility of
protein also plays a crucial role in the biochemical and functional analysis (Ahmad et al., 2018).
The protein was detected to be slightly acidic with a theoretical pI of 5.51. Similarly, it was
noted that the protein will be stable upon expression, hence reinforcing the competence of the
designed construct. The protein was also observed to be hydrophobic due to the presence of
aliphatic side chains as indicated by the aliphatic index value. All these features designate the
protein as has ability to withstand high temperatures and thus well suited for utilization in the
endemic parts.
Information regarding the secondary, as well as the tertiary structure, is vital in vaccine
designing (Shey et al., 2019). Secondary structure analysis revealed that the protein is largely
comprised of coils (43%), with only 7.16% of the residues disordered. The 3D structure of the
vaccine showed notable refinement and thus resulted in the attainment of the desirable properties
such as Ramachandran plot values. Rama-favored regions had a score of 94.5 % with rare
residues in the outlier region thus suggesting the high quality of the model.
Energy minimization was performed to stabilize the overall conformation as well as minimize
the potential energy of vaccine. During energy minimization, anomalous parts of the structure
are repaired hence giving rise to a more stable protein structure that would behave more
efficiently in the life-like cellular environments. The predicted RMSD of the vaccine-TLR-4
complex was predicted to be 0.5Å, which confirms the firmness of the complex.
Several studies have demonstrated the role of TLR4 in protecting against A.baumannii
infections. Therefore, a data-driven docking analysis was carried out to assess the possible
interactions between the designed construct and TLR4. Results suggest that the designed
construct could effectively stimulate the immune response. Binding energies elucidate the strong
binding of TLR4 with the construct. It is strongly recommended to further investigate the
interactions between TLR4 and the construct in vitro as well as in vivo.
The results obtained by immune simulations complied with the actual immune responses. A
continuous spike in immune responses was observed after giving repeated exposures to the
antigen.IgG1, IgG3, IgE immune responses have been reported against A.baumannii in different
reports (Ansari et al., 2019; Cha et al., 2018). We observed a spike in B and T cell populations,
as well as a considerable amount of peak in Th cell populations, was also noted.
The first and foremost step for the validation of a potential vaccine candidate is to inspect it for
immunoreactivity (Gori et al., 2009). For this purpose, it is necessary to express the target
protein in an appropriate host. E. coli is a renowned expression system to attain maximum
protein expression levels (Chen, 2012) .To achieve high-level expression in E.coli (strain K12),
codon optimization was executed. The values of the CAI (0.99) and GC content (50.54%) were
well-suited to obtain an optimum expression.
5. Conclusion
Novel and effective strategies are required to cope with difficult-to-treat A. baumannii infections.
This study makes use of several in-silico tools to design a rational vaccine that consists of
multiple B and T cell (CTL and HTL) epitopes. Our designed model is more antigenic and
immunogenic with decreased cytotoxic effects which could trigger antibodies associated
protection.
6. Disclosure statement
No conflict of interest was reported by the authors.
Acknowledgment
The authors would like to acknowledge the computational help to run molecular dynamics
simulations by Lab Engineer Muhammad Hassan Khan from Research Center for Modeling and
Simulation (RCMS), National University of Sciences and Technology (NUST), Islamabad,
Pakistan.
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bioRxiv preprint doi: https://doi.org/10.1101/2022.05.25.493433; this version posted May 26, 2022.

The copyright holder for this preprint (which

In-silico design and assessment of OprD based multi-epitope

vaccine against Acinetobacter baumannii

Department of Industrial Biotechnology, Atta-ur-Rahman School of Applied Biosciences (ASAB),

National University of Sciences & Technology (NUST), Islamabad 44000, Pakistan

Gram-negative, opportunist pathogen Acinetobacter baumannii is notorious for causing a

plethora of nosocomial infections predominantly respiratory diseases and blood-stream

infections. Due to resistance development towards last-resort antibiotics, its treatment is

becoming increasingly difficult. Despite numerous therapeutic developments, no vaccine is

TLR4 suggested significantly high immunogenic potential of our designed vaccine. MD

proposed to be thermostable, immunogenic, water-soluble, and non-allergenic. However, this

confirm the safety and immunogenicity of our multi-epitope vaccine.

Acinetobacter baumannii; nosocomial infections; multi-epitope subunit vaccine; bioinformatic

approaches, molecular dynamics simulations, molecular docking

of the most rampant causes of nosocomial infections typically in immunocompromised patients,

as compared to other Gram-negative bacteria such as Pseudomonas aeruginosa or Klebsiella

health(Harding et al., 2018).

treatment approaches as mostly incompetent. Existing approaches usually involve monotherapy

tigecycline, and sometimes aminoglycosides. However, their undesirable pharmacokinetic

(Isler et al., 2018).

Legionella pneumophila, Mannheimia haemolytica, Aeromonas salmonicida, Anaplasma

downregulation of OprD and upregulation of efflux systems in the development of mutation

According to studies, OprD of A.baumannii could act as a putative vaccine candidate

assessment of the capacity of OprD to elicit an immune response through bioinformatics

prophylactic peptide vaccines against A. baumannii.

Figure 1. General schema of the strategy followed in the present study.

2. Materials and Methods

2.1.1 FASTA Sequence Retrieval

2.1.2. Signal peptide and localization prediction

Armenteros et al., 2019) available at http://www.cbs.dtu.dk/services/SignalP-5.0/ and LipoP

(Juncker et al., 2003) at http://www.cbs.dtu.dk/services/LipoP/ were used. SignalP 5.0

BOCTOPUS2. PRED-TMBB enables its users to pinpoint trans-membrane strands of GNB

2.1.3 Analysis of conserved region

2.1.4. Mapping of continuous B-cell epitopes

2.1.5 Evaluation of Cytotoxic T lymphocytes (CTL)

2.1.6 Helper T-Lymphocyte (HTL) Epitope Mapping

this criteria was chosen (Jensen et al., 2018).

2.2 Multi-Epitope Subunit Vaccine Designing

et al., 2020). Adding an appropriate adjuvant is fundamental in boosting immunogenic behavior

stability (Choi et al., 2019).

2.3. Post-Design Analysis and Validation

2.3.1. Assessment of Physicochemical Aspects and Peptide Solubility

2.3.2. Immunogenicity Profiling

antigenicity with an accuracy greater than 75.5% (Magnan et al., 2010).

2.3.3. Determination of the Secondary Structure

network with an accuracy of 84.2% (McGuffin et al., 2000).

2.3.4 Derivation of 3-Dimensional Structure of Vaccine

threading-based restraints from LOMETS (Yang et al., 2014). It is reported to be a top-ranked

2.3.5. Improvement of Derived Protein Structure

(Feig, 2017). ModRefiner server http://zhanglab.ccmb.med.umich.edu/ModRefiner was utilized

Zhang, 2011). Next, a webserver GalaxyRefine http://galaxy.seoklab.org/cgi-

bin/submit.cgi?type=REFINE was employed to further enhance the structural quality. This

molecular dynamics simulations. It has refinement (Heo et al., 2013).

2.3.6 Structure Validation

structures. These include (i) ProSA-web https://prosa.services.came.sbg.ac.at/ (Wiederstein and

Sippl, 2007) (ii) ERRAT https://servicesn.mbi.ucla.edu/ERRAT/ (Dym et al., 2012) (iii)

MolProbity http://molprobity.biochem.duke.edu/ (Chen et al., 2010). PROCHECK

https://servicesn.mbi.ucla.edu/PROCHECK/ (Roman Laskowski et al., 1983) was also employed