Journal Pre-proof

In silico screening of antigenic B-cell derived T-cell epitopes and designing of a multi-
epitope peptide vaccine for Acinetobacter nosocomialis

Rida Sajjad, Sajjad Ahmad, Syed Sikander Azam

PII: S1093-3263(19)30283-9
DOI: https://doi.org/10.1016/j.jmgm.2019.107477
Reference: JMG 107477

To appear in: Journal of Molecular Graphics and Modelling

Received Date: 19 April 2019

Revised Date: 20 August 2019
Accepted Date: 10 October 2019

Please cite this article as: R. Sajjad, S. Ahmad, S.S. Azam, In silico screening of antigenic B-cell derived
T-cell epitopes and designing of a multi-epitope peptide vaccine for Acinetobacter nosocomialis, Journal
of Molecular Graphics and Modelling (2019), doi: https://doi.org/10.1016/j.jmgm.2019.107477.

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1 Title Page
2 Manuscript title:

3 In silico screening of antigenic B-cell derived T-cell epitopes and designing of a multi-epitope
4 peptide vaccine for Acinetobacter nosocomialis

5 Authors: Rida Sajjad 1, Sajjad Ahmad1, Syed Sikander Azam1,

6 Institutional address:
1
7 Computational Biology Lab, National Center for Bioinformatics, Quaid-i-Azam University,
8 Islamabad, Pakistan.
9 Email addresses:

10 Rida Sajjad, Email: ridasajjad32@gmail.com

11 Sajjad Ahmad, Email: sahmad@bs.qau.edu.pk

12 Syed Sikander Azam, Email:syedazam2008@gmail.com, ssazam@qau.edu.pk

13 Corresponding Author:
14 Syed Sikander Azam
15 E-mail: syedazam2008@gmail.com, ssazam@qau.edu.pk
16 Phone: 0092-51-906 44130
17 Computational Biology Lab, National Center for Bioinformatics, Quaid-i-Azam University,
18 Islamabad 45320, Pakistan
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29 ABSTRACT

30 Globally, antibiotic-resistant and tolerated bacterial isolates of Acinetobacter species are

31 imposing high financial cost on health care systems and as such, molecular targets with
32 promising immune protection could provide substantive benefits to human healthcare. Here,
33 we performed an in silico based proteome-wide screening for antigenic B-cell derived T-cell
34 epitopes and their following use to design a multi-epitope peptide vaccine that can effectively
35 engage the host immune system against Acinetobacter nosocomialis SSA3 strain. Epitopes of
36 the fimbrial biogenesis outer membrane usher FimD protein: YQQGINNYL and
37 YRTNYTTVG were revealed appropriate for multi-epitope peptide construct designing. This
38 protein has no homology to the host, essential to the pathogen survival and is localized at the
39 pathogen surface. The predicted epitopes have high affinity for the highly expressed DRB*0101
40 allele in humans based on the lowest IC50 value in MHCPred and have an exo-membrane
41 topology for efficient immune system recognition. The designed multi-epitope peptide vaccine
42 is composed of the mentioned shortlisted antigenic epitopes linked to each other through a
43 GPGPG linker, and an EAAAK linker that joined the multi-epitope peptide to the Cholera B
44 subunit from Vibrio cholera as an adjuvant to increase vaccine construct antigenicity. The
45 vaccine construct was docked and simulated with a transmembrane toll-like receptor (TLR4)
46 that revealed construct stable binding with the TLR4 through the adjuvant, allowing the
47 epitopes exposed to the host immune system essential for generating effective innate and
48 long-lasting adaptive immunity. The designed multi-epitope peptide vaccine may prompt the
49 development of a vaccine to control refractory and deleterious A. nosocomialis infections.

50 Keywords: Acinetobacter nosocomialis; Multi-epitope peptide vaccine; Molecular dynamics

51 simulation.

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57 1. Introduction

58 Acinetobacter is a genus of diverse group of non-fermentative, gram-negative bacilli, isolated

59 frequently in nosocomial infections and has significant prevalence in intensive care units where
60 sporadic, endemic and epidemic cases occur [1], [2], [3].This genus includes 34 unique species,
61 many of them are present far and wide in the environment [2], [4]. The species: Acinetobacter
62 nosocomialis, Acinetobacter baumannii, Acinetobacter calcoaceticus and Acinetobacter pittii
63 are grouped as Acinetobacter calcoaceticus-baumannii (Acb) complex based on high clinical
64 relevance [2], [6]. These species most commonly cause pneumonia, meningitis, septicemia, and
65 urinary tract infections [2], [7]. Often found that the said species are responsible for infections
66 recalcitrant to treatment of multiple drugs thus making these pathogens a continuous healthcare
67 problem in both developing and developed countries over the last 20 years [2], [6], [8]. In
68 particular, infections caused by A. nosocomialis and A. baumannii are the most prevalent in
69 intensive-care units and are of prime concern in modern health-care system [9], [10], [11].
70 Patients infected with A. nosocomialis has 30-days lower mortality compared to patients infected
71 by A. baumannii [12]. Moreover, A. nosocomialis has become the major source of infections
72 transmitted through medical devices, such as foley catheters, cerebrospinal fluid shunts, and
73 vascular catheters, etc. [13], [14] and is responsible for 80% of the hospital-acquired infections
74 [14]. Majority of the first-line antibiotics have reduced efficacy against this pathogen due to its
75 capacity of adapting resistance mechanisms that render the antibiotics less effective [15], [16].
76 Additionally, literature reports the emergence and dissemination of pan-drug resistance (PDR)
77 isolates of the pathogen that are resistant to approximately all antibiotics used in clinics, and as
78 such, a peptide vaccine with both therapeutics and prophylactic applications hold great
79 importance in reducing the current antibiotics crisis [17], [18], [19].

80 Strategies for developing conventional vaccines in lowering mortality and morbidity due to
81 infectious diseases have been highly efficacious for several decades [20], [21]. However,
82 conventional vaccines based on whole organisms or large proteins are not encouraged anymore
83 because of the unnecessary antigenic load that produces little protective immunity and
84 complicates the situation by inducing reactogenic and/or allergenic responses [20]. Peptide
85 vaccine, on the other hand, is an attractive alternative for induction of highly targeted immune

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 86 responses and stop onset of allergenic and/or reactogenic reactions [20]. Contrariwise, peptide
87 vaccines are weakly immunogenic and need specific carriers for delivery [20], [22].

88 To circumvent the limitations of peptide vaccines, in the current investigation, we aimed to

89 design a multi-epitope peptide comprise of epitopes from fimbrial biogenesis outer membrane
90 usher FimD protein that is conserved, non-homologous to the host, essential for the pathogen
91 survival, virulent, has exposed surface topology, non-allergenic, antigenic, less number of
92 transmembrane helices, adhesive and has localization in extracellular matrix or outer membrane.
93 Prediction of antigenic B-cell derived T-cell epitopes for designing a multi-epitope peptide is
94 done through different online immune-informatics tools and servers [23], [24], [25], [26].
95 Among them, the most important is VaxiJen that predicts bacterial origin antigens in an
96 alignment-independent fashion [27]. The classification of an antigen is done based on
97 physicochemical properties without the need for alignment and regardless of the protein
98 sequence length [27]. BCPred is commonly employed for predicting linear B-cell epitopes using
99 subsequence kernel method [28]. ProPred-I [29] is used to predict major histocompatibility
100 complex (MHC) class I binding regions in antigens whereas binding regions of MHC class II are
101 predicted by ProPred [30]. The binding affinity of the antigenic peptides for different alleles of
102 MHC-class I and class II are predicted using MHCPred [23]. VirulentPred based on bi-layer
103 cascade Support Vector Machine (SVM) is employed for prediction of bacterial virulent proteins
104 and peptides [23]. The predicted antigenic epitopes in the present study were linked to an
105 adjuvant of Cholera toxin B subunit [31] to design a multi-epitope peptide vaccine construct to
106 enhance immunogenicity of the individual epitope. Further, the vaccine construct was docked
107 with toll-like receptor 4 (TLR4) protein [32] and the complex was subjected to molecular
108 dynamics simulations of 100-ns to decipher complex stability. The screened epitopes of the
109 fimbrial biogenesis outer membrane usher FimD protein and the multi-epitope peptide vaccine
110 construct could be explored in future for development of a vaccine against A. nosocomialis in
111 specific and Acinetobacter genus in general.

112

113

114

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115 2. Materials and Methods

116 The complete flow of the study is illustrated in Fig.1.

117 2.1. Subtractive proteomics-based screening for potential vaccine proteins

118 The amino acid sequence of all proteins (3787) that constitute the complete proteome of A.
119 nosocomialis strain SSA3 was retrieved from the Genome database of the National Center for
120 Biotechnology Information (NCBI) [33]. Initially, CD-HIT (Cluster Database at High Identity
121 with Tolerance) [34] program was used for clustering protein sequences to discard redundant
122 sequences from the dataset that share 60% of sequence identity [35]. Following this, BLASTp
123 [36] against human proteome (Tax id: 9606) and Database of Essential Genes (DEG) [37] was
124 run to discard proteins that showed sequence identity of ≤ 30% and ≥ 30%, respectively [38].
125 The homology check against human proteome was vital to discard host homologous proteins to
126 eliminate the risk of autoimmune reactions [23]. Essentiality analysis was carried out to filter
127 only those proteins that are essential for the growth and survival of the pathogen and are
128 expressed continuously on the pathogen surface so that can be efficiently recognized by the host
129 immune system [39]. In the next step, virulent proteins having sequence identity of ≥ 35% and
130 bit score of ≥ 100 were selected from the shortlisted proteins of the previous step and this was
131 done by running BLASTp against virulence factor database (VFDB) [23], [39], [40]. Virulent
132 proteins are considered as good vaccine targets as they mediate many infectious pathways and
133 activate host immune system [41]. Subcellular localization was performed through PSORTb
134 [42], CELLO [43] and CELLO2GO [44] to filtered proteins of the outer membrane and
135 extracellular matrix. Outer membrane and extracellular proteins constitute secretome and exo-
136 proteome of the pathogen and are localized exposed to the human immune system [23], [39].

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137

138 Fig. 1. Complete workflow of the study for designing a multi-epitope vaccine construct against A. nosocomialis. Complete proteome of the pathogen
139 was retrieved from the genome database of NCBI and analyzed in subtractive proteomics to prioritized proteins suitable for vaccine designing.
140 Following, physicochemical characterization (molecular weight, number of transmembrane helices) of the screened proteins was done to ensure
141 selection of proteins that can be utilized easily in bench work analysis. Immunoinformatics investigation was carried out further for identification of
142 appropriate B-cell derived T-cell epitopes for designing a multi-epitope peptide vaccine construct. Finally, molecular docking and simulations studies
143 were carried out to decipher vaccine construct binding conformation at the binding site of tested TLR4 immune receptor and understand complex
144 dynamics versus time.
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145 2.2. Physiochemical characterization of potential vaccine proteins

146 Physicochemical characterization of potential vaccine proteins was performed to select the most
147 suitable candidates for experimental analysis [23], [39]. For this purpose, ExPASY server [45]
148 was utilized first to screen proteins weighing <110 kDa as small size proteins are easy to purify
149 [23], [39]. The filtered proteins were then subjected to HMMTOP [46] and TMHMM [47] to
150 estimate the number of transmembrane helices each protein harbored [23], [39]. Proteins with
151 less number of transmembrane helices are preferred because such proteins can be easily cloned
152 and express [23], [39]. Further characterization was done using SPAAN [48] to select adhesion-
153 like proteins. Adhesive proteins are surface localized to facilitate adhesion to the host tissues to
154 initiate bacterial colonization and infectious cycle [49]. The allergenicity of the proteins was
155 evaluated through Allertop server [50] to eliminate proteins from analysis that are predicted as
156 allergic and may cause allergic responses [51].

157 2.3. Epitope mapping

158 The non-allergenic proteins were then subjected to antigenicity evaluation phase where the
159 antigenic potential of the proteins was evaluated first by setting the cut-off value > 0.4 in
160 VaxiJen v2.0 [27]. The antigenic proteins were analyzed for B-cell epitopes using BCPred [28]
161 with threshold score ≥ 0.8 while epitope length was set to 20-mer [38]. The topology of the B-
162 cell epitopes was analyzed using HMMTOP [46], and those having exposed topology were
163 subjected to T-cell epitope prediction. Prophred [30] and Prophred I [29] were used to map
164 binding alleles of the major histocompatibility complex I (MHC-I) and MHC-II, respectively
165 [23]. Only T-cell epitopes having the highest number of common interacting MHC molecules
166 [23], [39] were selected for MHCpred [52]. MHCpred was used to predicts epitopes with IC50 <
167 100 nm for the DRB*0101 allele [23], [39]. Human leukocyte antigen (HLA) DRB*0101 is the
168 most commonly prevalent major histocompatibility complex (MHC)-II allele in worldwide
169 population and antigens binding to this allele could lead efficient immune recognition and
170 immune response generation [23], [39].

171 2.4. Structure prediction and evaluation

172 For the purpose to view the topology of predicted epitopes on their respective protein structure,
173 the three dimensional (3D) structure of epitope proteins was predicted using a comparative

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174 approach. The following tools: Phyre2 [53], Swiss-Model [54], Raptor X [55], Modeller [56] and
175 I-Tasser [57] were used. The predicted structures were evaluated using PROSA [58],
176 PROCHECK [59], Verify-3D [60] and ERRAT [61]. The most suitable structure after selection
177 then underwent energy minimization using UCSF Chimera for 1500 rounds [35].

178 2.5. Pepitope analysis

179 The shortlisted predicted antigenic epitopes and their respective protein 3D structures were used
180 in Pepitope server [62] to make sure the exo-membranous topology of the epitopes [23].

181 2.6. Interactome evaluation

182 The epitopes protein was then analyzed in Search Tool for the Retrieval of Interacting
183 Genes/Proteins (STRING) [63] to decipher high interacting nodes (confidence score of 0.9) of
184 the protein at the cellular level [35].

185 2.7. Construction of a multi-epitope peptide vaccine construct

186 To enhance antigenicity of individual epitope, a multi-epitope peptide was constructed [20]. A
187 linker, EAAAK, was used to link the Cholera toxin B subunit (UniProtKB -
188 E9RIX3 (E9RIX3_VIBCL) [31] as an adjuvant to the end of the N-terminal of the construct,
189 while a GPGPG linker was employed for linking epitopes [64]. The Allertope [50] and VaxiJen
190 [27] servers were used to re-confirm the allergenicity and antigenicity of the construct,
191 respectively. Expasy server [45] was used further to evaluate different physiochemical properties
192 of the construct. The 2D and 3D structure of the construct were predicted through Phyre2 [53].
193 The construct structure was refined through Galaxy Refine (http://galaxy.seoklab.org/cgi-
194 bin/submit.cgi?type=REFINE)[65].

195 2.8. Codon adaptation and in silco Cloning

196 An increased rate of foreign genes expression within the host can be achieved through codon
197 adaptation of the construct sequence, especially when the host’s codon usage is different from
198 that of source organism of the foreign gene [66]. A codon that fails to adapt can result in less rate
199 of expression in the host. For this reason, the Java Codon Adaptation Tool (JCAT) [67] was used
200 to adapt the codon usage of the vaccine peptide construct to Escherichia coli K12 strain. The

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201 adapted sequence was then cloned into vector pET28a (+) via SnapGene
202 (https://www.snapgene.com/).

203 2.9. Molecular docking and dynamics simulations

204 Molecular docking of the vaccine construct was performed with TLR4 molecule (PDB ID:
205 4G8A) using ClusPro 2.0 [68]. TLR4 was favored as a receptor molecule in docking over MHC
206 molecules due to its ability of generating rapid innate immune response followed by long lasting
207 adaptive immunity. The MHC based response take several days to become operational.
208 Simulation run of 100-ns was performed for the docked complex using AMBER 16. System
209 topologies were recorded using ff14SB force field [69]. The system was neutralized by adding 3
210 Na+ ions and then placed in TIP3P water box of padding distance 12 Å. Later, preprocessing was
211 performed that can be divided into seven steps: first, hydrogen atoms of the system were
212 minimized for 500 cycles, followed by water box minimization for 1000 cycles. Carbon alpha
213 atoms were minimized for 1000 cycles and lastly non-heavy atoms were minimized for 300
214 cycles with restraint of 5 kcal/mol-Å2 and 100 kcal/mol-Å2, respectively. Moving ahead, the
215 system was heated for 20 picoseconds (ps) at 300 K. To maintain temperature, Langevin
216 dynamics [70] was used with gamma value of 1. For constraint on hydrogen bonds, SHAKE
217 algorithm [71] was used, while for heating, constant-temperature, constant volume (NVT)
218 ensemble was employed. The system was then subjected to 100-ps with time step of 2
219 femtoseconds (fs) in the pre-equilibration phase, while for pressure equilibration; the isothermal-
220 isobaric (NPT) ensemble was used with 5 kcal/mol-Å2 restraint on alpha-carbon atoms. After
221 this, with restraint of 1 kcal/mol-Å2 after every 10-ps, the pressure phase was extended for
222 additional 50-ps. Lastly, the system was equilibrated for 1-ns. The total production run was of
223 100-ns using Berendsen algorithm [72] with an NVT ensemble. A cut-off of 8.0Å was employed
224 to nonbonded interactions, while SHAKE algorithm was used for hydrogen atoms. Simulation
225 trajectories were analyzed using CCPTRAJ [73].

226

227

228

229

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230 3. Results and Discussion

231 3.1. Proteome subtraction for potential subunit vaccine targets

232 Subtractive proteomics is a step-by-step approach to analyze the complete proteome of bacterial
233 pathogens for proteins relevant for therapeutic applications [74]. This approach has now been
234 extensively explored for identification of pathogen-specific drug targets in many studies [75],
235 [76]. However, its application for screening subunit surface proteins harboring antigenic peptides
236 is limited to several pathogens and can be used in combination with immunoinformatics tools for
237 predicting antigenic epitopes [77]. Herein, subtractive proteomics was amalgamate with reverse
238 vaccinology (RV) for antigenic epitope mapping in potential subunit vaccine candidates against
239 A. nosocomialis reference strain SAA3. The pathogen proteome contains 3787 proteins of which
240 3675 proteins are orthologous and constitute the core proteome of the pathogen. A set of 112
241 proteins was found paralogous, and thus discarded from further analysis. Orthologous proteins
242 represent the core genome of an organism and are evolutionarily conserved for cellular functions
243 vital for viability [23], [39], [74]. On other side, paralogous proteins are duplicate sequences and
244 are evolutionary not conserved [23], [39], [74]. Orthologous proteins dataset are therefore
245 considered attractive candidates for both drug and vaccine and used in this research work for
246 screening potential vaccine targets [23], [39], [74]. The core proteome was then subjected to
247 BLASTp against reference human proteome to filter only those proteins that show no homology
248 to the host. Homologous proteins share similar functions and screening of any protein that is
249 homologous to the host could lead to auto-immune reactions [23], [39], [74]. The analysis
250 revealed 3,091 proteins as non-homologous, while 584 proteins were homologs to the human
251 proteins therefore discarded. Next in the process, proteins that were significant for survival of the
252 pathogen were extracted as function of essential proteins are vital to sustaining life of these
253 pathogens [23], [39], [74]. Further, as these proteins are essential for survival they will
254 continuously express in the cell that will further ensure their antigenic determinants recognition
255 by the host immune system to activate both humoral and cell medial immunity [41].
256 Essentiality analysis found 803 as essential, while the rest of 2,288 were considered non-
257 essential hence discarded. Virulence is the potential of bacterial pathogens to cause infection in
258 host or damage host tissues [23], [39]. Bacteria achieved virulence for survival effectiveness in
259 adverse environmental conditions for the following reasons: (i) niche colonization in the host, (ii)

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260 evasion from the host immune system, (iii) suppression of the host immune system, (iv) entry
261 into and exit out the host cells, (v) obtain nutrition from the host [78]. BLASTp against VFDB
262 illustrated 94 virulent proteins that meet the selection criteria. For extracting proteins that lie in
263 the extracellular matrix and outer membrane of the pathogen, subcellular localization was
264 performed that identify 5 proteins having exposed surface topology (Table.1). These five
265 proteins have either outer membrane or extracellular localization in any of the two out of three
266 subcellular localization tools. These proteins are as follows: multidrug efflux RND transporter
267 subunit AdeA, EmrA/EmrK family multidrug efflux transporter periplasmic adaptor subunit,
268 outer membrane channel subunit AdeK, fimbrial biogenesis outer membrane usher FimD
269 protein, and 3-deoxy-7-phosphoheptulonate synthase. Multidrug efflux RND transporter subunit
270 AdeA protein is involved in β-lactam and cationic antimicrobial peptide (CAMP) resistance
271 pathways [79]. EmrA/EmrK family multidrug efflux transporter periplasmic adaptor subunit
272 performs its function as drug transporter [80]. The outer membrane channel subunit AdeK is part
273 of AdeIJK complex and belongs to resistance nodulation cell division (RND) family efflux pump
274 and contribute to resistance against β -lactams, tetracycline, chloramphenicol, flouroquinolones,
275 pyronine, fusidic acid, trimethoprim, erythromycin, rifampin, and sodium dodecyl sulphate [81].
276 Fimbrial biogenesis outer membrane usher FimD protein is involved in pilus synthesis by
277 recruiting FimA subunits across the outer membrane [82]. The enzyme 3-deoxy-7-
278 phosphoheptulonate synthase take part in phenylalanine, tyrosine and tryptophan amino acid
279 biosynthesis [83].

280 3.2. Physiochemical characterization of potential subunit vaccine targets

281 Characterizing potential subunit vaccine proteins based on physiochemical properties is

282 significant as they aid in selection of proteins that can be easily analyzed experimentally [23],
283 [39]. Importantly, proteins with molecular weight < 110 kDa are usually purified readily
284 compared to those having higher molecular weight [23], [39]. Likewise, cloning and expression
285 of proteins are easy when the proteins have less than two transmembrane helices [23], [39]. All
286 the shortlisted five proteins were found to have low molecular weight and less number of
287 transmembrane helices. Adhesive proteins increase pathogen potential of attachment to host
288 tissues and in turn enhances pathogen colonization to start infectious cycle [35]. Only two
289 proteins: multidrug efflux RND transporter subunit AdeA and fimbrial biogenesis outer

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290 membrane usher FimD protein were found adhesive while the remaining were discarded because
291 of their non-adhesive nature.

292 Table 1. Subcellular localization of the pathogen virulome for prioritizing outer membrane
293 (secretome) and extracellular (exoproteome) proteins that can be readily recognized by the MHC
294 molecules and generate robust immune responses.

Length
Protein of CELLO CELLO2GO PSORTB
Accession no.
protein
Multidrug efflux
RND transporter Outer
Outer Cytoplasmic
>Wp_016805174 periplasmic adaptor 396 membrane/
subunit-AdeA membrane membrane
extracellular

EmrA/EmrK family
multidrug efflux
Outer Outer
>Wp_004710210 transporter 383 Unknown
membrane membrane
periplasmic adaptor
subunit
Multidrug efflux
RND transporter
Outer Outer Outer
>Wp_016803831 AdeIJK outer 484
membrane membrane membrane
membrane channel
subunit-AdeK
Fimbrial biogenesis
Outer Outer Outer
>Wp_016805529 outer membrane usher 852
membrane membrane membrane
FimD protein
3-deoxy-7-
Outer
>Wp_016805332 phosphoheptulonate 351 Extracellular Cytoplasmic
membrane
synthase
295
296

297 Lastly, the allergenicity of the selected two proteins was investigated to make sure that these
298 proteins are free from peptides responsible for inducing adverse allergic reactions. Both the
299 proteins were predicted non-allergic and thus further subjected to epitope mapping phase (Table.
300 2).

301

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302 Table 2. The predicted physicochemical properties of mentioned proteins.

Molecular Trans-membrane
Adhesion
Protein Weight Helices Antigenicity Allertop
Probability
(kDa) HMMTOP/THMM
Multidrug efflux
RND transporter Non-
43 0/0 0.5 0.6
periplasmic adaptor allergen
subunit-AdeA
EmrA/EmrK family
multidrug efflux
transporter 40 1/1 - 0.7 Allergen
periplasmic adaptor
subunit
Multidrug efflux
RND transporter
Non
AdeIJK outer 52 0/0 - 0.6
allergen
membrane channel
subunit-AdeK
Fimbrial biogenesis
Non-
outer membrane 94 0/0 0.6
0.5 allergen
usher FimD protein
3-deoxy-7-
phosphoheptulonate Non-
40 0/0 - 0.4
synthase allergen

303

304 3.3. B and T-cell epitope mapping

305 Epitope identification is central for understanding many immunological applications including
306 vaccine development [23], [39]. As a part of vaccine development, it is important to map
307 epitopes in the filtered two proteins sequence that have the potential to invoke robust immune
308 responses. Antigenicity of the proteins was evaluated first that revealed both the proteins as
309 antigenic: multidrug efflux RND transporter subunit AdeA (0.65), and fimbrial biogenesis outer
310 membrane usher FimD protein (0.56). The potential of these antigenic proteins to stimulate
311 humoral immunity was followed, which unveiled 20-mer B-cell epitopes for multidrug efflux
312 RND transporter subunit AdeA and for fimbrial biogenesis outer membrane usher FimD protein.
313 The potential of an epitope to bind to MHC-I and MHC-II molecules of the cellular immunity
314 forms the basis for the epitope-based vaccine development because they are safe, easy to produce
315 and induce precise and broader immune responses [20], [23], [39]. Only fimbrial biogenesis

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316 outer membrane usher FimD protein revealed five T-cell epitopes and two of which:
317 YQQGINNYL and YRTNYTTVG belonged to both classes of MHC. MHCPred was used to
318 determine the binding strength of the epitopes to DRB1*0101. The epitopes; RGNAKTNA (IC50
319 53.83), YQQGINNYL (IC50 13.58), NRSETTKQ (IC50 44.46), YRTNYTTVG (IC5032.66) and
320 VLVHAPDA (IC50 17.42) having IC50 less than 100nM were considered i.e. both the 9-mer
321 epitopes of the protein bind to both MHC classes are virulent and antigenic. These five 9-mer
322 epitopes were subjected to Allertop server for checking the allergenicity to avoid occurrence of
323 severe kind of complication due to minor allergies[49]. Out of five epitopes, two epitopes were
324 thus selected and were subjected to further studies as shown in Table 3.

325 Table 3. B-cell derived T-cell epitopes for the core fimbrial biogenesis outer membrane usher
326 FimD protein.

IC50
Epitopes elicit B and Total Alleles value
Protein Allergenicity
T-Cell immunity binding
(nM)

YQQGINNYL 18 13.58 Non-allergen

Fimbrial biogenesis outer
membrane usher FimD
protein Non-allergen
YRTNYTTVG 16 32.66

327 3.4. Structure prediction and evaluation

328 Structure of fimbrial biogenesis outer membrane usher FimD protein harboring our selected
329 epitopes was predicted. The evaluation of the predicted structures is tabulated in Table 4. Model
330 generated by Phyre2 was selected as the best model since 89% of the residues for this model
331 were mapped in the most favorable region, while 10.4%, 0.5%, and 0.5% were in the additional
332 allowed region, generously allowed region, and disallowed region of the Ramachandran plot,
333 respectively. Moreover, the protein has a high ERRAT quality score of 91.002, Verify-3D of
334 89% and PROSA Z-score of -9.35. In contrast, the models generated by Swiss-Model, Raptor
335 X, I-TASSER, and Modeller were not selected mainly because of the high distribution of
336 residues in the disallowed region of the Ramachandran graph. The Swiss-Model predicted
337 structure has low percent of residues in the favored region i.e. 60%. The Raptor X structure,
338 on other hand, was not considered because of low ERRAT value in addition to high percent of

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339 residues in the disallowed region of Ramachandran. Comparatively, to the Phyre2 model, I-
340 TASSER model has low number of residues in the favored region of Ramachandran as well as
341 low value for Verify-3D, and ERRAT. The structure predicted by Modeller was found close
342 in term of quality; however, was not preferred as it has 2% fewer residues in the favored
343 region of the Ramachandran plot. Minimization of the selected model was performed in UCSF
344 Chimera with RMSD of 0.2 Å.

345 Table 4. Structure quality evaluation of fimbrial biogenesis outer membrane usher FimD protein
346 predicted by different software.

Evaluation Parameter Phyre2 Swiss-Model RaptorX I-TASSER Modeller

(3RFZ) (3RFZ) (3RFZ) (3RFZ) (6E14)
PROSA -9.35 -9.49 -9.01 -9.02 -9.2
Ramachandran 89% 60% 82.9% 81% 88%
Verify3D 89% 80% 82% 74.77% 87.89%
ERRAT 91.002 86.684 57.457 74.9403 89.003
347

348 3.5. Pepitope analysis

349 Using Pepitope server, the exo-membrane topology of the selected epitopes was visualized. Both
350 the epitopes were found not folded in the protein 3D structure and have exo-membrane
351 topologies as shown in the Fig.2.

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352

353 Fig.2. Exo-membrane topology view of epitopes: YRTNYTTVG (blue) and YQQGINNYL (red)
354 on the surface of the fimbrial biogenesis outer membrane usher FimD protein (gray cartoon).
355 3.6. Interactome evaluation

356 The fimbrial biogenesis outer membrane usher FimD protein was analyzed in STRING database
357 for evaluating the protein interacting network at cellular basis with strong interaction score of ≥
358 0.9. STRING is a biological database of known and predicted protein-protein interactions. It
359 provides system-level understanding of the cellular process and is an intuitive platform for
360 annotating functional, structural, and evolutionary properties of proteins. This was vital to
361 understand protein interactions direct or indirect indispensable for its functionality. As shown in
362 the Fig.3, fimbrial biogenesis outer membrane usher FimD protein has direct interactions with
363 three proteins: F911-02226, HMPREF0010-00598, and F911-01973. All these interacting
364 proteins are revealed to play functions in pili biosynthetic pathway.

16
365

366 Fig. 3. Cellular interacting network of Fimbrial biogenesis outer membrane usher FimD protein.
367 3.7. Multi-epitope peptide construction, 3D structure prediction and refinement

368 As reported that individual peptide has the limitation of poor antigenicity and coverage of T-cell
369 alleles, therefore, to circumvent these, the shortlisted antigenic epitopes were formulated in
370 multi-epitope peptide vaccine construct. The construct consists of epitopes linked through a
371 GPGPG linker, while a EAAAK linker was used to link epitopes peptide with the Cholera toxin
372 B subunit as an adjuvant shown in Fig. 4. Cholera toxin has been reported as an adjuvant and has
373 strong mucosal immunogenic properties. The B subunit of Cholera toxin is non-toxic portion and
374 has strong affinity for monosialotetrahexosylganglioside that is expressed in variety of cell types
375 including the epithelial cells of the gut, dendritic cells, macrophages as well as B-cells and can
376 lead to strong innate and adaptive immune responses against the antigen to which it is attached
377 [31]. After construct designing, Phyre2 server was used to predict the secondary and tertiary
378 model of the construct as illustrated in Fig.5. The construct was then subjected to GalaxyRefine
379 for the refining the 3D structure of construct. The structure quality was seen improved after
380 treatment in GalaxyRefine as depicted by Ramachandran plot. Before treatment, it was observed
381 that 80.1% residues of the construct are in the most favored region of the Ramachandran, while
382 after refinement, this is increased to 89.0%. Furthermore, according to Ramachandran plot, 6.6%
383 residues were mapped in the allowed region and disallowed region contains 2.2 %. Fig.6
384 represents the Ramachandran plot for unrefined and Galaxy refined model.

17
385

386 Fig. 4. Schematic diagram of the multi-epitope peptide vaccine construct. It consists of B subunit
387 adjuvant of cholera toxin linked through EAAK linker to the multi-epitopes peptide. The
388 epitopes are joined together by GPGPG linker for efficient immune presentation.

389
390 Fig. 5. Secondary (A) and tertiary structure (B) of the vaccine construct. The adjuvant is shown
391 in cyan, epitope YQQGINNYL in green, epitope YRTNYTTVG in blue, GPGPG linker in
392 violet, and EAAAK linker in magenta.

18
393
394 Fig. 6. (A) Ramachandran plot of the unrefined model. (B) Ramachandran plot of the refined
395 model generated by GalaxyRefine. The black squares in the figure represent the Psi and Phi
396 torsion angles of the 3D structure of the vaccine construct, the red, yellow, pale and white region
397 represent the core structure of the secondary structure elements, favored region, generously
398 allowed region and disallowed region, respectively.

399 3.8. Codon Adaptation and in silco Cloning

400 The adapted codons as per codon usage of E. coli strain K12 was achieved using JCAT
401 improving GC-contents to 48.6% with codon adaptive index of 1.0. This ensures that the vaccine
402 construct could be efficiently expressed in the host E. coli. The construct was further cloned into
403 two restriction sites: Xhol and Ndel of pET28a (+) vector as shown in Fig.7. In the cloned
404 vector, the vaccine construct is indicated by blue color with 6-histidine residues present on both
405 end. This 6-histidine tag is useful in purification process [66].

406

19
407

408 Fig. 7. In silico cloning of the vaccine construct sequence in pET28a (+) vector as highlighted in
409 blue color between the Xhol and Ndel restriction sites tagged with 6xHis.

410 3.9. Molecular docking of Vaccine Construct with TLR4

411 In order to check the preferred binding mode of the designed vaccine construct with respect to
412 the receptor TLR4 molecule, molecular docking is performed. Molecular docking of the vaccine
413 construct with the TLR-4 receptor was performed with ClusPro 2.0 that totally generated 30
414 complexes. The best complex with lowest energy score of -1187 kcal/mol was selected.
415 Visualization of the complex revealed the binding of the vaccine construct at the pocket formed

20
416 by chain A, B, and C. The binding conformation and interactions of the best-docked complex are
417 illustrated in Fig.8.

418

419 Fig. 8. (A) Docked complex of the multi-epitope peptide vaccine construct (shown in yellow
420 cartoon) at the binding side of TLR-4. The spheres in green and blue represent chain C and chain
421 A, respectively. Cyan sphere is chain B and magenta depicts chain D (B) Interacting residues of
422 TLR4 chain A (blue), chain B (cyan) and chain C (green) with the vaccine construct (yellow
423 cartoon).

21
424 3.10. Molecular Dynamics Simulations of the Multi-epitope peptide-TLR4 Complex
425 The time-dependent dynamics of the docked multi-epitope peptide with the TLR4 receptor was
426 performed to extract information about the conformational adjustments of TLR4 and the vaccine
427 construct. This is analysis was vital for several important aspects: (i) first to understand whether
428 the peptide construct is stable at the docked site, (ii) to make sure that the induced
429 conformational movements of both TLR4 receptor and the designed vaccine construct is not
430 affecting the binding stability of the docked molecules, and (iii) the epitopes of the vaccine
431 construct are exposed to the host immune system for efficient recognition that will lead to robust
432 immune response. Four different statistical parameters were evaluated based on 100-ns of
433 simulation trajectories as shown in Fig.9. The conformational stability of the complex was
434 evaluated first through the root mean square deviation (RMSD) [66], [84]. RMSD as a function
435 of time was estimated to decipher atomic displacement of Cα atoms of the complex. An average
436 RMSD of 3.2 Å with maximum of 4.4 Å seen at 36 ns was recorded for the studied system.
437 Different variations have been visualized and as such snapshots at 10ns, 20 ns, 30 ns, 40 ns, 50
438 ns, 60 ns, 70 ns, 80 ns, 90 ns, and 100 ns were extracted to investigate the system. The vaccine
439 construct remained firmly bound throughout the simulations at the docked site of TLR4. The
440 RMSD variations of the TLR4 receptor were seen attributed to the moving vaccine construct at
441 TLR4 active site in an attempt of achieving a proper and stable binding conformation. Both
442 epitopes of the vaccine construct were seen exposed and not folded within the TLR4. The
443 structural mobility of the complex was investigated by splitting the total Root mean square
444 fluctuation (RMSF) [66], [84] value into TLR4 residues. The average RMSF calculated for the
445 system is 1.6 Å that is in general favor of protein residues stability. Residues that considerably
446 fluctuates are due their presence in the loop region and loop regions are highly flexible for proper
447 holding the ligand. Residues from Glu1 to Glu579 of the protein were observed to have RMSF in
448 range between 2 Å and 3 Å, Ser604 has an RMSF value of 7 Å, Ser614 to Lys729 have an
449 average RMSF higher than 3 Å, Leu746 have RMSF value of 6 Å and Phe750 to Leu848
450 RMSF value is less than 4 Å. The third statistical parameter calculated for the complex was
451 Radius of Gyration (Rg) [66], [84]. Higher the Rg value for a system, loos tightening of the
452 protein structure will be in contrast to the lower Rg value that implies tight packing of the
453 protein. The mean Rg estimated for the system is 66 Å with maximum value of 66 Å observed at
454 18 ns. It was seen that Rg variations are simply due to vaccine construct movements in the

22
455 receptor protein pocket that induces structure adjustments in the loop regions which seems
456 necessary to properly recognize the construct and bind it in the pocket. Lastly, β-factor [66], [84]
457 for the complex was calculated. β-factor is closely linked to the RMSF and estimates the spatial
458 displacement of atoms around their mean position. The atomic displacement is because of local
459 thermal and vibrational movements. The mean β-factor for the system reported the mean value of
460 90.08 Ų for system. Hence, the same trend and consistency of results were observed in RMSF
461 and β-factor. The overall behavior of the system was stable as shown in Fig. 10.

462

463 Fig.9. Trajectories analysis of the vaccine construct with the TLR-4 receptor (A) RMSD, (B)
464 RMSF, (C) Radius of Gyration, and (D) Beta-Factor.

23
465

466 Fig. 2. Trajectories snapshots at different ns of simulations: (a) 10 ns , (b) 20 ns, (c) 30 ns, (d) 40
467 ns, (e) 50 ns, (f) 60 ns, (g) 70 ns, (h) 80 ns, (i) 90 ns, and (j) 100 ns. The TLR4 receptor is shown
468 in orchid color whereas the vaccine construct is represented in golden new cartoon. The
469 construct was seen in continuous movement at the docked site which can be explained as an
470 approach to achieve proper binding conformation and increased stability.

24
471 4. Conclusions

472 This work demonstrates fimbrial biogenesis outer membrane usher FimD protein as an
473 attractive subunit vaccine target as well as for harboring antigenic B-cell derived T-cell epitopes.
474 The shortlisted epitopes of the protein were further revealed as virulent, non-allergenic, have an
475 exo-membrane topology and high affinity for the DRB*0101 allele. The multi-epitope peptide
476 vaccine construct is showing high affinity and stable binding conformation at the docked side of
477 TLR4. Further, MD simulations revealed increasing affinity of the interacting TLR4 and vaccine
478 construct for each other as the simulation progress with exposed epitopes for activation of both
479 cellular and humoral immunity. The results of the present study are promising and we expect the
480 proposed vaccine construct to show practical applications in controlling antibiotics resistant
481 infections caused by A. nosocomialis subject to experimental validations.

482 Conflict of Interest

483 The authors declare that they have no conflict of interest.
484 Acknowledgments

485 Authors are highly grateful to the Pakistan-United States Science and Technology Cooperation
486 Program (Grant No. Pak-US/2017/360) and the Higher Education Commission (HEC) for
487 granting the financial assistance.

488

489

490

491

492

493

494

495

496

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 Highlights

A multi-epitope peptide vaccine for Acinetobacter nosocomialis.

Two epitopes of fimbrial biogenesis outer membrane usher protein were used in
vaccine design.
We expect the designed recombinant vaccine to be effective for A. nosocomialis
infections.