# EPIPOX: Immunoinformatic Characterization of the Shared T-Cell Epitome between Variola Virus and Related Pathogenic Orthopoxviruses

Authors: Magdalena Molero-Abraham; John-Paul Glutting; Darren R. Flower; Esther M. Lafuente; Pedro A. Reche
Journal: Journal of Immunology Research
 (2015)

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## Abstract

Concerns that variola viruses might be used as bioweapons have renewed the interest in
developing new and safer smallpox vaccines. Variola virus genomes are now widely available, allowing computational characterization of the entire T-cell epitome and the use of such information to develop safe and yet effective vaccines. To this end, we identified 124 proteins shared between various species of pathogenic orthopoxviruses including variola minor and major, monkeypox, cowpox, and vaccinia viruses, and we targeted them for T-cell epitope prediction. We recognized 8,106, and 8,483 unique class I and class II MHC-restricted T-cell epitopes that are shared by all mentioned orthopoxviruses. Subsequently, we developed an immunological resource, EPIPOX, upon the predicted T-cell epitome. EPIPOX is freely available online and it has been designed to facilitate reverse vaccinology. Thus, EPIPOX includes key epitope-focused protein annotations: time point expression, presence of leader and transmembrane signals, and known location on outer membrane structures of the infective viruses. These features can be used to select specific T-cell epitopes suitable for experimental validation restricted by single MHC alleles, as combinations thereof, or by MHC supertypes.


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## Body

## 1. Introduction

Smallpox was a devastating contagious disease that ravaged humankind for millennia, wiping out entire civilizations [1]. The disease was caused by two types of variola virus (VARV), major and minor, which differed greatly in their average mortality rates: 30% versus 1%, respectively. VARV major was the most prevalent form [2, 3]. Systematic vaccination against smallpox began in the early 19th century but the disease lingered until the World Health Organization (WHO) initiated worldwide vaccination campaigns in 1967. The last case was reported in Somalia in 1977 and in May 1980 the WHO declared that smallpox had been eradicated, ceasing vaccination [1, 2]. Eradication was facilitated because there are no animal reservoirs for the virus, as it only infects humans [4].VARV belongs to theOrthopox genus of thePoxviridae family, consisting of large double-stranded DNA viruses that replicate in the cytoplasm of infected cells [5, 6]. Poxviruses are large and complex with ~250 genes and a multistage life cycle, producing different infective forms including intracellular mature virions (IMV) and extracellular enveloped virus (EEV) [5, 6]. Humans can be infected by several poxviruses; the closest to VARV that are also pathogenic to humans are vaccinia (VACV), cowpox (CPXV), and monkeypox (MPXV) viruses [7, 8]. The primary reservoir of MPXV is rodents [9], while CPXV has the broadest animal reservoir range of all poxvirus, including cats, dogs, elephants, and rodents [10]. Historically, VAVC has been considered to emerge after repeated passages from an ancestral CPXV [11]. However, phylogenetic studies question that view and there are some speculations that VACV could be a horsepox virus (HPXV) [12]; yet both, the host and origin of VACV, remain unknown [13]. VACV and CPXV infections in humans are generally mild and self-limiting and can induce cross-protective immunity [14]. The observation that CPXV sufferers did not get smallpox led Edward Jenner in 1798 to introduce a method of vaccination through scarifications withVariolae Vaccinae, Latin, for CPXV [15]. Immunization with CPXV was eventually displaced by VACV vaccine, which was used subsequently for global smallpox vaccination [12].As smallpox was eradicated and vaccination ceased, the global population has become increasingly susceptible to both smallpox and zoonosis by orthopoxviruses [8, 9]. People under 30 have no immunity against these viruses and VACV-induced immunity is waning in those that were vaccinated [16]. Despite recommendations by the WHO, stockpiles of smallpox virus had never been destroyed and there are concerns that unregistered stocks could be used as a weapon of bioterrorism [17]. Several features make smallpox a major terrorist threat. It replicates easily, is aerosolizable, and is highly contagious before, during, and after disease onset. Moreover, smallpox is lethal and disfiguring and has already been used as a biological weapon in North America during the French and Indian Wars [18]. Thus, there is renewed interest in the development of vaccines against smallpox, particularly safer ones, since immunization with VACV can result in serious adverse events and it is considered risky in immunocompromised or immune-suppressed individuals [19].Immune protection against orthopoxviruses requires both B and T cells [20] but the relevance of T cells is paramount. CD8 T cells are required to eliminate infected cells, while help by CD4 T cells is essential to elicit effective humoral responses [21]. Thus, people with dysfunctional humoral responses (e.g., agammaglobulinemia) can be vaccinated with VACV, while those with loss of T cells cannot as they can suffer severe disease [22]. T-cell immune responses are triggered by the recognition of foreign peptides bound to cell surface-expressed major histocompatibility complex (MHC) molecules, also known as human leukocyte antigens, HLA, in humans. CD4 T cells recognize peptides presented by MHC class II (MHC II) molecules while CD8 T cells recognize peptides presented by MHC class I (MHC I) molecules.Advances in both immunology and genomic analysis offer new possibilities for eliciting immune protection without the requirement for live-virus vaccination and attendant complications. The identification of HLA class I and class II restricted T-cell epitopes (CD8 and CD4 T-cell epitopes, resp.) from poxviruses may allow us to develop safe and yet immunogenic peptide-based vaccines. Here, we describe the identification of protein antigens that are shared between several pathogenic orthopoxviruses, including VARV, MPXV, CPXV, and VACV, and T-cell epitopes that are identical in all selected proteins. This information was used to create a freely accessible web resource, EPIPOX: URLhttp://imed.med.ucm.es/epipox/, intended to facilitate the design of epitope-based vaccines against orthopoxviruses.


## 2. Materials and Methods

### 2.1. Orthopoxvirus Sequences and Experimentally Defined T-Cell Epitopes

In this study, we used the entire proteomes of 8 orthopoxviruses: VARV major, strain Bangladesh-1975, GenBank Accession: GB: L22579; VARV major, strain India-1967, GB: NC_00161; Variola major minor, strain Garcia-1966, GB: Y16780; Monkeypox virus, strain Zaire-96-I-16, GB: NC_003310; Cowpox virus strain, strain Brighton Red, GB: AF482758, Vaccinia virus, strain Copenhagen, GB: M35027; Vaccinia virus, strain Tian Tan, GB: AF095689; Vaccinia virus, strain Ankara, GB: U94848. The proteomes were obtained from the various translation features of the relevant GenBank genomic records using BIOPERL [23] (Table 1).Table 1
Orthopoxviruses used in this study.
Virus
Strain
ACC
Genes
VARV major
Bangladesh-1975
L22579
189
VARV major
India-1967
NC_00161
197
VARV minor
Garcia-1966
Y16780
206
MPXV
Zaire-96-I-16
NC_003310
191
CPXV
Brighton Red
AF482758
218
VACV
Copenhagen
M35027
262
VACV
Tian Tan
AF095689
243
VACV
Ankara∗
U94848
157
∗Modified strain that has lost the ability to replicate; VARV: variola virus;  MPXV: monkeypox virus; CPXV: cowpox virus; VACV: vaccinia virus.We also used experimentally defined poxvirus-specific HLA I and HLA II-restricted T-cell epitopes that were retrieved from the IEDB [24] and EPIMHC [25] databases. We only considered unique T-cell epitope sequences with a size of 9 amino acids that were reported to be identified in humans infected with orthopoxviruses or who were vaccinated. We provide a list of experimentally defined T-cell epitopes as supplementary material in Additional File S1 in Supplementary Material available online at http://dx.doi.org/10.1155/2015/738020.


### 2.2. Protein Sequence Analyses and Annotations

We took VARV major, strain Bangladesh-1975, as the reference for subsequent sequence analyses. We identified proteins with leader signals using SIGNALP [26] and transmembrane regions using TMHMM [27]. We identified protein orthologs using BLAST [27]. Briefly, we first BLAST the reference proteins against formatted databases of each of the remaining orthopoxvirus proteomes. We performed BLAST searches with default settings and considered only the description of the first hit and the corresponding alignment. Subsequently, we selected those protein searches that gave hits in each of the proteomes with identities greater than 60% and identified the corresponding orthologs. We used BIOPERL to parse BLAST hits [23].Information on the temporal expression of VACV genes was kindly provided by Dr. Lefkowitz from the Poxvirus Bioinformatics Resource Center [28]. The information consisted on annotations identifying those genes that are expressed early (E), intermediate (I), and late (L) during the life cycle of VACV. This information is provided as supplementary material in Additional File S2. In addition, we identified, from the data provided by Dr. Lefkowitz, gene products associated with the outer membranes of VACV IMV and EEV infective forms, as well as those proteins that are part of the VACV virion or CORE. This information is also included as supplementary material in Additional File S2. Protein annotations obtained for VACV were transferred to protein orthologs.


### 2.3. Prediction of T-Cell Epitopes

We predicted MHC I and MHC II peptide binding to anticipate potential CD8 and CD4 T-cell epitopes, respectively. Specifically, we predicted peptide-MHC binding from VARV Bangladesh proteins that are shared between all selected orthopoxviruses using 32 HLA I- and 33 HLA II-allele specific position-specific scoring matrices (PSSMs) [29–31]. For a given protein, we considered the top 2% and 4% of scoring peptides to constitute HLA I- and HLA II-binding peptides, respectively. We only predicted binding for peptides of nine residues; most HLA I-restricted peptides are 9 residues in length and while HLA II-restricted peptides vary in length (9–22 amino acids) they have a core of 9 residues that anchor the peptide in the binding groove of HLA II molecules [30, 32]. We also used N-gram language models to identify whether peptides can be generated from the source antigen by proteasomal cleavage [33]. This information is only relevant to HLA I-binding peptides, since most peptides presented by MHC I are derived from antigens degraded by the proteasome [34].


### 2.4. Database Building and Web Server Implementation

Predicted T-cell epitopes and obtained protein annotations were incorporated into a POSTGRES relational database. The database consists of 3 tables (peptides,predictions, andproteins) that are linked through unique keys (Figure 1). Briefly, tablepredictions contains peptide sequences and their MHC restriction elements; tablepeptides includes the peptide molecular weight, its protein accession number, and whether the peptide is cleaved by the proteasome; and tableproteins contains gene product information including temporal expression (E: early, I: intermediate, and L:late), location in the virus (IMV, EEV, and CORE), and the existence of leader and transmembrane regions. We also developed a web front end or GUI to allow ready access to EPIPOX. Behind the interface is a Python script that handles database queries through underlying SQL. The EPIPOX resource is implemented on an Apache Web server under the Mac OSX operating system.Figure 1
EPIPOX database structure. EPIPOX is a relational database consisting of three main tables:peptides,predictions, andproteins. Table names are boxed with double lines. For each table, we show their fields and boxed with single lines the fields that work as table keys. For fields taking discrete nominal values, we show them between square brackets.


## 2.1. Orthopoxvirus Sequences and Experimentally Defined T-Cell Epitopes

In this study, we used the entire proteomes of 8 orthopoxviruses: VARV major, strain Bangladesh-1975, GenBank Accession: GB: L22579; VARV major, strain India-1967, GB: NC_00161; Variola major minor, strain Garcia-1966, GB: Y16780; Monkeypox virus, strain Zaire-96-I-16, GB: NC_003310; Cowpox virus strain, strain Brighton Red, GB: AF482758, Vaccinia virus, strain Copenhagen, GB: M35027; Vaccinia virus, strain Tian Tan, GB: AF095689; Vaccinia virus, strain Ankara, GB: U94848. The proteomes were obtained from the various translation features of the relevant GenBank genomic records using BIOPERL [23] (Table 1).Table 1
Orthopoxviruses used in this study.
Virus
Strain
ACC
Genes
VARV major
Bangladesh-1975
L22579
189
VARV major
India-1967
NC_00161
197
VARV minor
Garcia-1966
Y16780
206
MPXV
Zaire-96-I-16
NC_003310
191
CPXV
Brighton Red
AF482758
218
VACV
Copenhagen
M35027
262
VACV
Tian Tan
AF095689
243
VACV
Ankara∗
U94848
157
∗Modified strain that has lost the ability to replicate; VARV: variola virus;  MPXV: monkeypox virus; CPXV: cowpox virus; VACV: vaccinia virus.We also used experimentally defined poxvirus-specific HLA I and HLA II-restricted T-cell epitopes that were retrieved from the IEDB [24] and EPIMHC [25] databases. We only considered unique T-cell epitope sequences with a size of 9 amino acids that were reported to be identified in humans infected with orthopoxviruses or who were vaccinated. We provide a list of experimentally defined T-cell epitopes as supplementary material in Additional File S1 in Supplementary Material available online at http://dx.doi.org/10.1155/2015/738020.


## 2.2. Protein Sequence Analyses and Annotations

We took VARV major, strain Bangladesh-1975, as the reference for subsequent sequence analyses. We identified proteins with leader signals using SIGNALP [26] and transmembrane regions using TMHMM [27]. We identified protein orthologs using BLAST [27]. Briefly, we first BLAST the reference proteins against formatted databases of each of the remaining orthopoxvirus proteomes. We performed BLAST searches with default settings and considered only the description of the first hit and the corresponding alignment. Subsequently, we selected those protein searches that gave hits in each of the proteomes with identities greater than 60% and identified the corresponding orthologs. We used BIOPERL to parse BLAST hits [23].Information on the temporal expression of VACV genes was kindly provided by Dr. Lefkowitz from the Poxvirus Bioinformatics Resource Center [28]. The information consisted on annotations identifying those genes that are expressed early (E), intermediate (I), and late (L) during the life cycle of VACV. This information is provided as supplementary material in Additional File S2. In addition, we identified, from the data provided by Dr. Lefkowitz, gene products associated with the outer membranes of VACV IMV and EEV infective forms, as well as those proteins that are part of the VACV virion or CORE. This information is also included as supplementary material in Additional File S2. Protein annotations obtained for VACV were transferred to protein orthologs.


## 2.3. Prediction of T-Cell Epitopes

We predicted MHC I and MHC II peptide binding to anticipate potential CD8 and CD4 T-cell epitopes, respectively. Specifically, we predicted peptide-MHC binding from VARV Bangladesh proteins that are shared between all selected orthopoxviruses using 32 HLA I- and 33 HLA II-allele specific position-specific scoring matrices (PSSMs) [29–31]. For a given protein, we considered the top 2% and 4% of scoring peptides to constitute HLA I- and HLA II-binding peptides, respectively. We only predicted binding for peptides of nine residues; most HLA I-restricted peptides are 9 residues in length and while HLA II-restricted peptides vary in length (9–22 amino acids) they have a core of 9 residues that anchor the peptide in the binding groove of HLA II molecules [30, 32]. We also used N-gram language models to identify whether peptides can be generated from the source antigen by proteasomal cleavage [33]. This information is only relevant to HLA I-binding peptides, since most peptides presented by MHC I are derived from antigens degraded by the proteasome [34].


## 2.4. Database Building and Web Server Implementation

Predicted T-cell epitopes and obtained protein annotations were incorporated into a POSTGRES relational database. The database consists of 3 tables (peptides,predictions, andproteins) that are linked through unique keys (Figure 1). Briefly, tablepredictions contains peptide sequences and their MHC restriction elements; tablepeptides includes the peptide molecular weight, its protein accession number, and whether the peptide is cleaved by the proteasome; and tableproteins contains gene product information including temporal expression (E: early, I: intermediate, and L:late), location in the virus (IMV, EEV, and CORE), and the existence of leader and transmembrane regions. We also developed a web front end or GUI to allow ready access to EPIPOX. Behind the interface is a Python script that handles database queries through underlying SQL. The EPIPOX resource is implemented on an Apache Web server under the Mac OSX operating system.Figure 1
EPIPOX database structure. EPIPOX is a relational database consisting of three main tables:peptides,predictions, andproteins. Table names are boxed with double lines. For each table, we show their fields and boxed with single lines the fields that work as table keys. For fields taking discrete nominal values, we show them between square brackets.


## 3. Results and Discussion

### 3.1. Epitope-Vaccine Design against Orthopoxviruses

T-cell adaptive immunity is required for clearance of poxviruses during infection and/or vaccination and can also contribute to protective immunity from subsequent exposures [35, 36]. Moreover, peptides corresponding to VACV-specific CD8 T-cell epitopes can confer protection to mice subjected to lethal VACV challenges [37]. Fueled by the need to develop safer smallpox vaccines, such knowledge has led to the recent identification of many VACV-specific T-cell epitopes [37, 38]. These T-cell epitopes are deposited haphazardly in various specialized databases, including IEDB [24], EPIMHC [25], TEPIDAS [39], and AntiJen [40]. Of relevance for epitope-vaccine design, CD8 T-cells target primarily early and nonstructural gene products [41, 42]. CD4 T cells target late and the most abundant genes products (IMV and EEV membrane proteins and CORE proteins), as do antibodies [42, 43]. While some of the identified VAVC-specific T-cell epitopes are conserved in VARV a rational approach to identifying all potential T-cell epitopes eliciting cross-protective immunity is still required.


### 3.2. Shared Orthopoxvirus Proteins for Cross-Protective Immunity

Nearly all orthopoxviruses can protect against challenge with another orthopoxvirus [14]. This exquisite cross-protective immunity is likely a result of direct antigenic similarity between poxviruses. Therefore, prior to defining potential T-cell epitopes we identified shared antigens between pathogenic orthopoxviruses. Identification of shared antigens is also relevant to reducing the experimental burden associated with T-cell identification. Human pathogen orthopoxviruses have large genomes encompassing over 180 open reading frames (ORF) with the exception of VACV Ankara strain, which has only 157 genes and lacks the ability to replicate [44] (Table 1). Using VARV major, strain Bangladesh-1975, as a reference, we identified 124 ORFs that are shared between 8 different complete genomes from several orthopoxviruses, including VARV minor, CPXV, MPXV, and several VACV strains (Additional File S3). Despite the criterion for selection being 60% identity, all 124 selected proteins have an average identity ≥ 85% as shown in Additional File S3. These proteins are prime candidates to induce cross-protective immunity although they need to be targeted by the immune system. Interestingly, within the selected proteins there are 8 known immunogens that conferred >60% protection to VACV in animal models (Table 2) [45]. Six of these immunogens are IMV or EEV proteins carrying transmembrane regions and/or are being late gene products. Interestingly, among the selected 124 proteins we found 26 additional proteins with transmembrane regions that could also be prime vaccine subunits candidates (Table 3). Some of these proteins also have leader signal sequences (Table 3). Viral proteins with leader sequences follow the cell secretory pathway and are thus also important targets to consider for vaccine design [46, 47].Table 2
Orthopoxvirus proteins contributing to cross-protective immunity.
VACC: GI|ORF
VARV: GI|ORF
MPXV: GI|ORF
CPXV: GI|ORF
LOC1/EXP2/TM3/LD4
335424|L1R
438991|M1R
17974993|M1R
20153082|V099
IMV/late/yes/no
335455|D8L
439016|F8L
17975018|E8L
20153106|V119
IMV/late/yes/no
335500|A27L
439052|A31L
17975052|A29L
20153143|V156
IMV/late/no/no
335508|A33R
439057|A36R
17975058|A35R
20153149|V162
EEV/early/yes/no
335549|B5R
439084|B6R
17975080|B6R
20153177|V190
EEV/#/yes/yes
335438|H3L
439004|I3L
17975006|H3L
20153094|V107
IMV/late/yes/no
335477|A10L
439032|A11L
17975034|A11L
20153122|V135
CORE/#/no/no
335341|C7L
438926|D11L
17974926|D10L
20153015|V028
U/early/no/no
Table shows GenBank identification numbers (GI) and open reading frame names (ORF) for VACC (strain Copenhagen), VARV (strain Bangladesh-1975), MPXV (strain Zaire-96-I-16), and CPXV (strain Brighton Red).1LOC: location, 2EXP: temporal expression, 3TM: transmembrane, and 4LD: leader signal. NS: nonstructural gene. #: information not available. U: unknown. List of proteins was obtained from [45]. Annotations 1, 2, 3, and 4 obtained as indicated elsewhere in Section 2.Table 3
Shared orthopoxvirus proteins with transmembrane and/or leader sequences.
VARV GI|ORF
MPXV GI|ORF
CPXV GI|ORF
VACV GI|ORF
IDEN1 (%)
TM2
LEAD3
EXP4
LOCATION5
GI:439084|B6R
GI:17975080|B6R
GI:20153177|V190
GI:335549|B5R
93.1
Yes
Yes
L
EEV membrane∗
GI:439016|F8L
GI:17975018|E8L
GI:20153106|V119
GI:335455|D8L
94.7
Yes
No
L
IMV membrane∗
GI:438990|H9R
GI:17974992|G10R
GI:20153081|V094
GI:335423|G9R
98.1
Yes
No
L
U
GI:438919|D4R
GI:17974919|D3R
GI:20153007|V020
GI:335333|C11R
88.8
Yes
Yes
U
U
GI:439085|B7R
GI:17975081|B7R
GI:20153178|V191
GI:335550|B6R
93.1
Yes
No
U
U
GI:439035|A14L
GI:17975037|A14L
GI:20153125|V138
GI:335483|A13L
88.6
Yes
No
L
IMV membrane
GI:438946|C8L
GI:17974949|C10L
GI:20153036|V049
GI:335366|F4L
97.6
Yes
No
E
U
GI:438977|K5L
GI:17974979|I5L
GI:20153068|V081
GI:335409|I5L
94.9
Yes
No
L
IMV membrane
GI:438967|E8R
GI:17974969|F7R
GI:20153058|V071
GI:335395|E8R
97.4
Yes
No
L
U
GI:439004|I3L
GI:17975006|H3L
GI:20153094|V107
GI:335438|H3L
95.8
Yes
No
L
IMV membrane∗
GI:439014|F6R
GI:17975016|E6R
GI:20153104|V117
GI:335453|D6R
99.0
Yes
No
L
U
GI:439003|I2R
GI:17975005|H2R
GI:20153093|V106
GI:335437|H2R
99.2
Yes
No
L
U
GI:439056|A35L
GI:17975057|A34L
GI:20153148|V161
GI:335506|A32L
98.1
Yes
No
L
U
GI:439000|L5L
GI:17975002|L5L
GI:20153090|V103
GI:335433|J5L
98.1
Yes
No
L
U
GI:439058|A37R
GI:17975059|A36R
GI:20153150|V163
GI:335509|A34R
98.1
Yes
No
L
EEV membrane
GI:438991|M1R
GI:17974993|M1R
GI:20153082|V095
GI:335424|L1R
99.2
Yes
No
L
IMV membrane∗
GI:439057|A36R
GI:17975058|A35R
GI:20153149|V162
GI:335508|A33R
93.0
Yes
No
E
EEV membrane∗
GI:438951|C13L
GI:17974954|C15L
GI:20153041|V054
GI:335373|F9L
97.5
Yes
No
L
U
GI:439038|A17L
GI:17975040|A17L
GI:20153129|V142
GI:335486|A16L
97.0
Yes
No
L
U
GI:438974|K2L
GI:17974976|I2L
GI:20153065|V078
GI:335405|I2L
99.3
Yes
No
L
U
GI:439008|I7R
GI:17975010|H7R
GI:20153098|V111
GI:335442|H7R
95.2
Yes
No
L
U
GI:438982|H3L
GI:17974984|G2L
GI:20153073|V086
GI:335414|G3L
95.8
Yes
No
L
U
GI:439042|A22L
GI:17975044|A21L
GI:20153134|V147
GI:335490|A21L
96.9
Yes
No
U
U
GI:439059|A38R
GI:17975061|A38R
GI:20153152|V165
GI:335512|A36R
92.3
Yes
No
E, L
EEV membrane
GI:439031|A10L
GI:17975033|A10L
GI:20153121|V134
GI:335476|A9L
89.0
Yes
Yes
E, L
U
GI:439033|A12R
GI:17975035|A12R
GI:20153123|V136
GI:335481|A11R
98.5
Yes
No
L
U
GI:439036|A15L
GI:17975038|A15L
GI:20153126|V139
GI:335484|A14L
97.8
Yes
No
L
IMV membrane
GI:439067|A46R
GI:17975066|A43R
GI:20153159|V172
GI:335522|A43R
92.3
Yes
Yes
U
U
GI:439039|A18L
GI:17975041|A18L
GI:20153130|V143
GI:335487|A17L
98.0
Yes
No
L
IMV membrane
GI:439077|J7R
GI:17975076|B2R
GI:20153172|V185
GI:335539|A56R
82.1
Yes
Yes
E, L
EEV membrane
GI:439062|A41L
GI:17975063|A40L
GI:20153155|V168
GI:335516|A38L
94.7
Yes
Yes
U
U
Table shows GenBank identification numbers (GI) and open reading frame names (ORF) for VARV: strain Bangladesh-1975, MPXV: strain Zaire-96-I-16, CPXV: strain Brighton Red, and VACV: strain Copenhagen.1IDEN: average identity between the selected proteins. 2TM: transmembrane. 3LEAD: leader signal. 4EXP: temporal expression (E: early, I: intermediate, and L: late). 5LOCATION: location.  ∗Proteins known to induce protective immunity (see Table 2). Annotations were obtained as indicated elsewhere in Section 2. U: information not found.


### 3.3. T-Cell Epitome from Pathogenic Orthopoxvirus Proteins

We targeted the shared orthopoxvirus proteins for T-cell epitopes prediction using 32 and 33 HLA I- and HLA II-specific profile matrices (details in Material and Methods). The alleles targeted for peptide binding prediction are shown in Additional File S4. We selected these alleles because there are experimental peptide-binding data for them, which is required to make accurate peptide-MHC binding predictors [48]. Incidentally, these HLA alleles are frequently expressed in the general population and targeting them for epitope prediction permits the development of epitope-based vaccines covering the entire population. These HLA allelic variants can have overlapping peptide-binding repertoires and can be clustered accordingly in supertypes [49, 50]. Selecting promiscuous peptide-binders to multiple HLA molecules facilitates the development of vaccines with a minimum number of peptides [49–51].We predicted a total of 18726 HLA I-restricted and 32722 HLA II-restricted orthopoxvirus specific T-cell epitopes, all being identical between all orthopoxviruses considered in this study. In Additional File S4 we provide numbers of T-cell epitopes predicted by each HLA-specific profile used in this study. We predicted more CD4 than CD8 T-cell epitopes because we used a more permissive peptide-binding threshold for MHC II molecules (4% of top scoring peptides) than for MHC I molecules (2% of top scoring peptides) since peptide-binding prediction to MHC II molecules is considerably less accurate than to MHC I molecules [46]. Interestingly, we identified only 8106 unique HLA I-restricted T-cell epitope sequences and a few more (8483) unique HLA II-restricted T-cell epitope sequences. Therefore, there is a considerable overlap between the peptide binding repertoires of HLA molecules, which is larger for HLA II molecules than for HLA I molecules. HLA I-restricted peptides bound on average to 2.3 distinct HLA I molecules, while HLA II-restricted peptides bound on average to 3.8 distinct HLA II molecules. This is due to the fact that peptide-binding to MHC II molecules is more degenerate than to MHC I molecules [29, 30]. In Additional File S5, we provide all distinct predicted peptides with the HLA molecules that they were predicted to bind. Interestingly, there is also some overlap between HLA I- and HLA II-restricted peptides. In particular, we find that there are 2452 peptides that are predicted to be restricted by both HLA I and HLA II molecules. Thus, in total the predicted T-cell epitome consisted of just 14137 unique sequences among all predicted T-cell epitopes.We compared the predicted T-cell epitome with experimentally defined poxvirus-specific HLA-restricted T-cell epitopes deposited in the IEDB [24] and EPIMHC [25]. We retrieved 170 HLA I and 9 HLA II-restricted T-cell epitopes meeting our criteria (see Additional File S1) but we only considered for comparison 85 HLA I- and 8 HLA II-restricted T-cell epitopes that we identified here to be conserved in all orthopoxviruses considered in this study. Of those, 72 HLA I- and 6 HLA II-restricted T-cell epitopes were found within our predicted T-cell epitome. Moreover, we predicted the experimentally verified restriction element in > 80%. The experimentally determined and shared epitopes that were not predicted (a minority) either are restricted by noncovered alleles or were simply not predicted. In Table 4, we summarize the data showing the verified and predicted HLA restriction elements. In sum, we readily predicted most of the experimentally verified T-cell epitopes. Considering that on average 10% of predicted T-cell epitopes can be experimentally verified [52], we shall expect that there are many more valid T-cell epitopes remaining to be validated within the T-cell epitome predicted in this study.Table 4
Experimentally identified T-cell epitopes within the shared T-cell epitome predicted from pathogenic orthopoxvirus proteins.
(a)
CD8 T-cell epitopes
VARV GI
VARV ORF
Experimental HLA I restriction
Predicted HLA I restriction
A
LMRRIAVV
439013
F5R
HLA-A2
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
HLA-A6802
YLLSLFSTL
439056
A35L
HLA-A2
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
HLA-A6802
YLAKLTALV
438985
H5R
HLA-A2
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
HLA-A6802
NLLCHIYSL
438979
K7L
HLA-A2
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
HLA-Cw0702
IVIEAIHTV
439072
J2R
HLA-A0201
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
HLA-Cw0304
SLSAYIIRV
439004
I3L
HLA-A0201
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
HLA-A0207
YLDGQLARL
438965
E6R
HLA-A0201
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
HLA-A0207
YLPEVISTI
438988
H7L
HLA-A0201
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
HLA-Cw0102
TYNDHIVNL
439072
J2R
HLA-A2301
HLA-A2301
HLA-A2402
HLA-A2403
HLA-A2405
HLA-A2407
HLA-Cw0702
RPPSFYKPL
439046
A25R
HLA-B7
HLA-B0702
HLA-B3501
HLA-B5101
HLA-B5301
HLA-B5401
HLA-Cw0102
ALDEKLFLI
439045
A24R
HLA-A0201
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
HLA-A0207
FPYEGGKVF
438968
E9L
HLA-B0702
HLA-B0702
HLA-B1502
HLA-B3501
HLA-B5101
HLA-B5301
HLA-B5401
RLYDYFTRV
438973
K1L
HLA-A0201, HLA-A2
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
ILDDNLYKV
438985
H5R
HLA-A0201, HLA-A2
HLA-A0201
HLA-A0202
HLA-A0205
HLA-A0207
HLA-Cw0702
LLSYYVVYV
439009
F1R
HLA-A2
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
FLIDLAFLI
438960
E1L
HLA-A2
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
HLA-Cw0304
FPRSMLSIF
438994
M4R
HLA-B07:02
HLA-B0702
HLA-B3501
HLA-B4402
HLA-B5301
HLA-B5401
YLFDFVISL
438996
L1R
HLA-A2
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
YLIKLIEPV
439009
F1R
HLA-A0201, HLA-A2
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
SPSNHHILL
439025
A4L
HLA-B07:02
HLA-B0702
HLA-B3501
HLA-B5101
HLA-B5301
HLA-B5401
YPSNKNYEI
439033
A12R
HLA-B07:02
HLA-B0702
HLA-B3501
HLA-B5101
HLA-B5301
HLA-B5401
MLMETMFFI
439007
I6R
HLA-A2
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
ILNPVASSL
438998
L3R
HLA-A2
HLA-A0201
HLA-A0202
HLA-A0205
HLA-A0206
HLA-B1501
FPSVFINPI
438968
E9L
HLA-B0702
HLA-B0702
HLA-B3501
HLA-B5101
HLA-B5301
HLA-B5401
KYQSPVNIF
439043
A21R
HLA-A24, HLA-class I
HLA-A2301
HLA-A2402
HLA-A2403
HLA-A2405
HLA-A2407
YLFGGFSTL
438980
K8R
HLA-A2
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
YLYETYHLI
438981
H1L
HLA-A2
HLA-A0201
HLA-A0202
HLA-A0205
HLA-A0206
VLYNGVNYL
439009
F1R
HLA-A2
HLA-A0201
HLA-A0202
HLA-A0205
HLA-A0206
LIQEIVHEV
439029
A8L
HLA-A0201
HLA-A0201
HLA-A0202
HLA-A0205
HLA-A0206
VELGSGNSF
439043
A21R
HLA-B3701
HLA-B1501
HLA-B1502
HLA-B4402
HLA-Cw0304
RMIAISAKV
438934
P1L
HLA-A2
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0206
FILGIIITV
439036
A15L
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
LLSKNTFYL
438981
H1L
HLA-A2
HLA-A0201
HLA-A0202
HLA-A0205
HLA-A0207
RPRDAIRFL
438961
E2L
HLA-B0702
HLA-B0702
HLA-B3501
HLA-B5301
HLA-B5401
KLFNKVPIV
438996
L1R
HLA-A2
HLA-A0201
HLA-A0202
HLA-A0206
HLA-A6802
SLFMILCTR
438979
K7L
HLA-A0301, HLA-A1101
HLA-A1101
HLA-A3101
HLA-A3301
HLA-A6801
ILNDEQLNL
439029
A8L
HLA-A0201
HLA-A0201
HLA-A0205
HLA-A0207
YLLGDSDSV
439038
A17L
HLA-A2
HLA-A0201
HLA-A0205
HLA-A0206
QLMYALEPR
438985
H5R
HLA-A0301, HLA-A1101
HLA-A3101
HLA-A3301
HLA-A6801
LMDENTYAM
439066
A45R
HLA-A2
HLA-A0205
HLA-A0207
HLA-Cw0702
GLLLGCFWV
438955
C17L
HLA-A2
HLA-A0202
HLA-A0203
HLA-A0206
LLSHFYPAV
438952
C14L
HLA-A2
HLA-A0201
HLA-A0205
HLA-A6802
NLFTFLHEI
439045
A24R
HLA-class I
HLA-A0201
HLA-A0203
HLA-A0206
GLFDFVNFV
439070
A49R
HLA-A2, HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0206
YLGPRVCWL
439038
A17L
HLA-A2
HLA-A0202
HLA-A0205
HLA-A0206
ILKSLGFKV
438980
K8R
HLA-A2
HLA-A0202
HLA-A0203
HLA-A0205
DEVASTHDW
439025
A4L
HLA-B4403
HLA-B4402
HLA-B5801
FLVIAINAM
439029
A8L
HLA-A2, HLA-A0201
HLA-A0206
HLA-Cw0304
LSDLKKTIY
439040
A19R
HLA-A1, HLA-class I
HLA-A0101
HLA-B1501
LLYFKVFGI
439019
N1L
HLA-A2
HLA-A0202
HLA-A0205
SSNIMSESY
439014
F6R
HLA-A0101, HLA-A3002
HLA-A0101
HLA-B1501
DTRGIFSAY
439032
A11L
HLA-A26, HLA-class I
HLA-A0101
HLA-B5801
QIDVEKKIV
439029
A8L
HLA-A0201
HLA-A0207
HLA-A6802
YLFRCVDAV
439030
A9R
HLA-A2
HLA-A0205
HLA-A0206
KIEDLINQL
438973
K1L
HLA-A0201
HLA-A0203
HLA-A0207
FTIDFKLKY
439009
F1R
HLA-A1, HLA-A2601, HLA-A2902
HLA-A0101
HLA-Cw0702
WLKIKRDYL
439074
J4R
HLA-B0801
HLA-B0801
AINVEKIEL
473688
L6R
HLA-A0201
HLA-A0207
RIFVRVYNV
439007
I6R
HLA-A2
HLA-A0202
SIIDLIDEY
438942
C4R
HLA-B1501
HLA-A0203
EERHIFLDY
439009
F1R
HLA-B4403
HLA-B4402
ILSDENYLL
439028
A7L
HLA-A2, HLA-A0201
HLA-A0205
HISALKRRY
439027
A6R
HLA-A0101, HLA-A2902
HLA-A0101
FLNISWFYI
438968
E9L
HLA-A2, HLA-A02:01
HLA-A0205
SEVKFKYVL
439002
I1L
HLA-B44
HLA-B4402
KLLLWFNYL
438980
K8R
HLA-A2
HLA-A0203
YIDISDVKV
438973
K1L
HLA-A0201
HLA-A0207
VWINNSWKF
439013
F5R
HLA-A24, HLA-A2301, HLA-A2402
HLA-Cw0702
VLPFDIKKL
439014
F6R
HLA-A0201
HLA-Cw0102
VETSISDYY
439043
A21R
HLA-B3701
HLA-B1501
SQIIDISLR
439022
A1L
HLA-A0301, HLA-A1101
HLA-B2705
HDVYGVSNF
439045
A24R
HLA-B4403
HLA-B4402
(b)
CD4 T-cell epitope
VARV GI
VARV ORF
Experimental HLA II restriction
Predicted HLA II restriction
FLIDLAFLI
438960
E1L
HLA-class II
DRB1∗0101
DRB1∗0102
DRB1∗0301
DRB1∗0311
DRB1∗0401
DRB1∗1201
DRB1∗1302
DRB1∗1502
DRB3∗0101
DRB5∗0101
IHWQIISSE
439072
J2R
DRB1∗04:05
DRB1∗0405
DRB1∗0406
DRB1∗0410
DRB1∗1304
DRB3∗0101
YIDAYVSRL
439021
N3L
DRB1∗15:01
DRB1∗0901
DRB1∗1103
DRB3∗0202
LMDENTYAM
439066
A45R
HLA-class II
DRB1∗1103
IDAYVSRLL
439021
N3L
DRB1∗15:01
DRB1∗1201
RMIAISAKV
438934
P1L
HLA-class II
DRB1∗0407
T-cell epitopes in this table are a subset of those provided in Additional File S1.


### 3.4. EPIPOX Database and Web Server

We developed a relational database based upon the predicted T-cell epitome and a web-based resource to facilitate online access and to query the database. We named this resource EPIPOX and made it available for free public use (URL:http://imed.med.ucm.es/epipox/). EPIPOX is ade facto analysis pipeline of viral T-cell epitomes. The content of the EPIPOX database is organized in three tables (peptides,predictions, andproteins) (Figure 1). The tablepredictions contains all predicted T-cell epitopes, consisting of 18726 HLA I- and 32722 HLA II-restricted peptides, each identified by its sequence and restriction element. Peptide sequences in this table are not unique as each peptide can bind to numerous HLA I molecules. Peptide sequences are, however, unique in the tablepeptides.This table contains 14137 sequences comprising the whole predicted epitome regardless of the restriction elements. Antigen annotations in EPIPOX are found within the tableproteins (Figure 1). We only included annotations that are relevant to epitope vaccine design, such as temporal expression of gene products and location in relevant structures of the virus such as the EEV and IVM membranes and CORE. Early expressed proteins and highly expressed proteins are generally thought to be more immunogenic, particularly with regard to CD8 T cells [53, 54]. On the other hand, highly abundant late proteins that are located in membrane structures of the poxvirus appear to be the main focus of the antibody and CD4 T-cell response [42, 43]. In the tableproteins, we also provide annotations on whether the proteins have transmembrane region or leader signal sequence, as proteins with these features often interact with host cells and are important targets for subunit vaccine design [46, 47].The EPIPOX web interface (Figure2) allows querying of the database combining any annotation field in the database, as described above. For intuitive use, the interface is divided in two main sections. In the first section (SEARCH), users select proteins and restriction elements for epitope retrieval. In this section, EPIPOX also provides the option to query the database for promiscuous T-cell epitopes binding to three HLA I supertypes (A2, A3, and B7). The alleles belonging to these supertypes are present in 88% of the population regardless of their ethnic groups. Selecting promiscuous peptides restricted by these 3 supertypes facilitates maximizing the population coverage of vaccines with minimum numbers of peptides [49, 50, 55]. In the second section (LIMIT), users can select annotation criteria to restrict the results. As an example, in Figure 3, we show the page resulting from a sample query consisting of promiscuous peptides from CORE binding to the A2 supertype. From the EPIPOX output, users can also access additional information available from the Virus Pathogen Resource database (Figure 3) [56].Figure 2
EPIPOX input page. The input page of EPIPOX is divided in two main sections for intuitive use. In the first part (SEARCH), users select HLA molecules and proteins to retrieve T-cell epitopes (multiple selection is allowed) while in the second part the user can limit the search output according to various criteria. These criteria include temporal expression of gene products (E: early; I: intermediate; L: late), location of proteins in relevant structures of the virus (CORE, IMV, and EEV), and the presence of leader and transmembrane regions. In addition, users can select only those peptides with a relative score above some selectable value. HLA-specific profiles used to score T-cell epitopes can reach a maximum score, which is used to set the relative score in percentage of each peptide. For HLA I-restricted epitopes, users can also restrict the search to those epitopes potentially generated by the proteasome.Figure 3
EPIPOX result page. The figure shows a slice of the output resulting from promiscuous CORE protein peptides binding to the A2 supertype. The output consists of a tabulated list, with information on each of the fields of the search query (columns). From fieldSOURCE NAME (1), users can access proteins from the Virus Pathogen Database (http://www.viprbrc.org/) (1) and by clicking on the epitope sequence, fieldSEQ (2), users will get the amino acid sequence of the protein showing the peptide in bold (2).EPIPOX is related somewhat to certain existing databases. On the one hand, it shares features with generic epitope databases such as EPIMHC [25], AntiJen [40], and IEDB [24] and on the other hand it shares features with poxvirus genome annotation-orientated databases such as the Poxviridae database [28] (no longer operating) and the Virus Pathogen Resource (http://www.viprbrc.org/) [56]. This later resource contains information on virus sequences, functional annotations and epitopes derived from IEDB [24]. However, the Virus Pathogen Resource does not allow selection of epitopes or antigens by criteria that are relevant to epitope vaccine design. In fact, EPIPOX is the only dedicated immunologic resource that has been designed to facilitate the rational selection of epitopes and antigens for subunit vaccine design.


## 3.1. Epitope-Vaccine Design against Orthopoxviruses

T-cell adaptive immunity is required for clearance of poxviruses during infection and/or vaccination and can also contribute to protective immunity from subsequent exposures [35, 36]. Moreover, peptides corresponding to VACV-specific CD8 T-cell epitopes can confer protection to mice subjected to lethal VACV challenges [37]. Fueled by the need to develop safer smallpox vaccines, such knowledge has led to the recent identification of many VACV-specific T-cell epitopes [37, 38]. These T-cell epitopes are deposited haphazardly in various specialized databases, including IEDB [24], EPIMHC [25], TEPIDAS [39], and AntiJen [40]. Of relevance for epitope-vaccine design, CD8 T-cells target primarily early and nonstructural gene products [41, 42]. CD4 T cells target late and the most abundant genes products (IMV and EEV membrane proteins and CORE proteins), as do antibodies [42, 43]. While some of the identified VAVC-specific T-cell epitopes are conserved in VARV a rational approach to identifying all potential T-cell epitopes eliciting cross-protective immunity is still required.


## 3.2. Shared Orthopoxvirus Proteins for Cross-Protective Immunity

Nearly all orthopoxviruses can protect against challenge with another orthopoxvirus [14]. This exquisite cross-protective immunity is likely a result of direct antigenic similarity between poxviruses. Therefore, prior to defining potential T-cell epitopes we identified shared antigens between pathogenic orthopoxviruses. Identification of shared antigens is also relevant to reducing the experimental burden associated with T-cell identification. Human pathogen orthopoxviruses have large genomes encompassing over 180 open reading frames (ORF) with the exception of VACV Ankara strain, which has only 157 genes and lacks the ability to replicate [44] (Table 1). Using VARV major, strain Bangladesh-1975, as a reference, we identified 124 ORFs that are shared between 8 different complete genomes from several orthopoxviruses, including VARV minor, CPXV, MPXV, and several VACV strains (Additional File S3). Despite the criterion for selection being 60% identity, all 124 selected proteins have an average identity ≥ 85% as shown in Additional File S3. These proteins are prime candidates to induce cross-protective immunity although they need to be targeted by the immune system. Interestingly, within the selected proteins there are 8 known immunogens that conferred >60% protection to VACV in animal models (Table 2) [45]. Six of these immunogens are IMV or EEV proteins carrying transmembrane regions and/or are being late gene products. Interestingly, among the selected 124 proteins we found 26 additional proteins with transmembrane regions that could also be prime vaccine subunits candidates (Table 3). Some of these proteins also have leader signal sequences (Table 3). Viral proteins with leader sequences follow the cell secretory pathway and are thus also important targets to consider for vaccine design [46, 47].Table 2
Orthopoxvirus proteins contributing to cross-protective immunity.
VACC: GI|ORF
VARV: GI|ORF
MPXV: GI|ORF
CPXV: GI|ORF
LOC1/EXP2/TM3/LD4
335424|L1R
438991|M1R
17974993|M1R
20153082|V099
IMV/late/yes/no
335455|D8L
439016|F8L
17975018|E8L
20153106|V119
IMV/late/yes/no
335500|A27L
439052|A31L
17975052|A29L
20153143|V156
IMV/late/no/no
335508|A33R
439057|A36R
17975058|A35R
20153149|V162
EEV/early/yes/no
335549|B5R
439084|B6R
17975080|B6R
20153177|V190
EEV/#/yes/yes
335438|H3L
439004|I3L
17975006|H3L
20153094|V107
IMV/late/yes/no
335477|A10L
439032|A11L
17975034|A11L
20153122|V135
CORE/#/no/no
335341|C7L
438926|D11L
17974926|D10L
20153015|V028
U/early/no/no
Table shows GenBank identification numbers (GI) and open reading frame names (ORF) for VACC (strain Copenhagen), VARV (strain Bangladesh-1975), MPXV (strain Zaire-96-I-16), and CPXV (strain Brighton Red).1LOC: location, 2EXP: temporal expression, 3TM: transmembrane, and 4LD: leader signal. NS: nonstructural gene. #: information not available. U: unknown. List of proteins was obtained from [45]. Annotations 1, 2, 3, and 4 obtained as indicated elsewhere in Section 2.Table 3
Shared orthopoxvirus proteins with transmembrane and/or leader sequences.
VARV GI|ORF
MPXV GI|ORF
CPXV GI|ORF
VACV GI|ORF
IDEN1 (%)
TM2
LEAD3
EXP4
LOCATION5
GI:439084|B6R
GI:17975080|B6R
GI:20153177|V190
GI:335549|B5R
93.1
Yes
Yes
L
EEV membrane∗
GI:439016|F8L
GI:17975018|E8L
GI:20153106|V119
GI:335455|D8L
94.7
Yes
No
L
IMV membrane∗
GI:438990|H9R
GI:17974992|G10R
GI:20153081|V094
GI:335423|G9R
98.1
Yes
No
L
U
GI:438919|D4R
GI:17974919|D3R
GI:20153007|V020
GI:335333|C11R
88.8
Yes
Yes
U
U
GI:439085|B7R
GI:17975081|B7R
GI:20153178|V191
GI:335550|B6R
93.1
Yes
No
U
U
GI:439035|A14L
GI:17975037|A14L
GI:20153125|V138
GI:335483|A13L
88.6
Yes
No
L
IMV membrane
GI:438946|C8L
GI:17974949|C10L
GI:20153036|V049
GI:335366|F4L
97.6
Yes
No
E
U
GI:438977|K5L
GI:17974979|I5L
GI:20153068|V081
GI:335409|I5L
94.9
Yes
No
L
IMV membrane
GI:438967|E8R
GI:17974969|F7R
GI:20153058|V071
GI:335395|E8R
97.4
Yes
No
L
U
GI:439004|I3L
GI:17975006|H3L
GI:20153094|V107
GI:335438|H3L
95.8
Yes
No
L
IMV membrane∗
GI:439014|F6R
GI:17975016|E6R
GI:20153104|V117
GI:335453|D6R
99.0
Yes
No
L
U
GI:439003|I2R
GI:17975005|H2R
GI:20153093|V106
GI:335437|H2R
99.2
Yes
No
L
U
GI:439056|A35L
GI:17975057|A34L
GI:20153148|V161
GI:335506|A32L
98.1
Yes
No
L
U
GI:439000|L5L
GI:17975002|L5L
GI:20153090|V103
GI:335433|J5L
98.1
Yes
No
L
U
GI:439058|A37R
GI:17975059|A36R
GI:20153150|V163
GI:335509|A34R
98.1
Yes
No
L
EEV membrane
GI:438991|M1R
GI:17974993|M1R
GI:20153082|V095
GI:335424|L1R
99.2
Yes
No
L
IMV membrane∗
GI:439057|A36R
GI:17975058|A35R
GI:20153149|V162
GI:335508|A33R
93.0
Yes
No
E
EEV membrane∗
GI:438951|C13L
GI:17974954|C15L
GI:20153041|V054
GI:335373|F9L
97.5
Yes
No
L
U
GI:439038|A17L
GI:17975040|A17L
GI:20153129|V142
GI:335486|A16L
97.0
Yes
No
L
U
GI:438974|K2L
GI:17974976|I2L
GI:20153065|V078
GI:335405|I2L
99.3
Yes
No
L
U
GI:439008|I7R
GI:17975010|H7R
GI:20153098|V111
GI:335442|H7R
95.2
Yes
No
L
U
GI:438982|H3L
GI:17974984|G2L
GI:20153073|V086
GI:335414|G3L
95.8
Yes
No
L
U
GI:439042|A22L
GI:17975044|A21L
GI:20153134|V147
GI:335490|A21L
96.9
Yes
No
U
U
GI:439059|A38R
GI:17975061|A38R
GI:20153152|V165
GI:335512|A36R
92.3
Yes
No
E, L
EEV membrane
GI:439031|A10L
GI:17975033|A10L
GI:20153121|V134
GI:335476|A9L
89.0
Yes
Yes
E, L
U
GI:439033|A12R
GI:17975035|A12R
GI:20153123|V136
GI:335481|A11R
98.5
Yes
No
L
U
GI:439036|A15L
GI:17975038|A15L
GI:20153126|V139
GI:335484|A14L
97.8
Yes
No
L
IMV membrane
GI:439067|A46R
GI:17975066|A43R
GI:20153159|V172
GI:335522|A43R
92.3
Yes
Yes
U
U
GI:439039|A18L
GI:17975041|A18L
GI:20153130|V143
GI:335487|A17L
98.0
Yes
No
L
IMV membrane
GI:439077|J7R
GI:17975076|B2R
GI:20153172|V185
GI:335539|A56R
82.1
Yes
Yes
E, L
EEV membrane
GI:439062|A41L
GI:17975063|A40L
GI:20153155|V168
GI:335516|A38L
94.7
Yes
Yes
U
U
Table shows GenBank identification numbers (GI) and open reading frame names (ORF) for VARV: strain Bangladesh-1975, MPXV: strain Zaire-96-I-16, CPXV: strain Brighton Red, and VACV: strain Copenhagen.1IDEN: average identity between the selected proteins. 2TM: transmembrane. 3LEAD: leader signal. 4EXP: temporal expression (E: early, I: intermediate, and L: late). 5LOCATION: location.  ∗Proteins known to induce protective immunity (see Table 2). Annotations were obtained as indicated elsewhere in Section 2. U: information not found.


## 3.3. T-Cell Epitome from Pathogenic Orthopoxvirus Proteins

We targeted the shared orthopoxvirus proteins for T-cell epitopes prediction using 32 and 33 HLA I- and HLA II-specific profile matrices (details in Material and Methods). The alleles targeted for peptide binding prediction are shown in Additional File S4. We selected these alleles because there are experimental peptide-binding data for them, which is required to make accurate peptide-MHC binding predictors [48]. Incidentally, these HLA alleles are frequently expressed in the general population and targeting them for epitope prediction permits the development of epitope-based vaccines covering the entire population. These HLA allelic variants can have overlapping peptide-binding repertoires and can be clustered accordingly in supertypes [49, 50]. Selecting promiscuous peptide-binders to multiple HLA molecules facilitates the development of vaccines with a minimum number of peptides [49–51].We predicted a total of 18726 HLA I-restricted and 32722 HLA II-restricted orthopoxvirus specific T-cell epitopes, all being identical between all orthopoxviruses considered in this study. In Additional File S4 we provide numbers of T-cell epitopes predicted by each HLA-specific profile used in this study. We predicted more CD4 than CD8 T-cell epitopes because we used a more permissive peptide-binding threshold for MHC II molecules (4% of top scoring peptides) than for MHC I molecules (2% of top scoring peptides) since peptide-binding prediction to MHC II molecules is considerably less accurate than to MHC I molecules [46]. Interestingly, we identified only 8106 unique HLA I-restricted T-cell epitope sequences and a few more (8483) unique HLA II-restricted T-cell epitope sequences. Therefore, there is a considerable overlap between the peptide binding repertoires of HLA molecules, which is larger for HLA II molecules than for HLA I molecules. HLA I-restricted peptides bound on average to 2.3 distinct HLA I molecules, while HLA II-restricted peptides bound on average to 3.8 distinct HLA II molecules. This is due to the fact that peptide-binding to MHC II molecules is more degenerate than to MHC I molecules [29, 30]. In Additional File S5, we provide all distinct predicted peptides with the HLA molecules that they were predicted to bind. Interestingly, there is also some overlap between HLA I- and HLA II-restricted peptides. In particular, we find that there are 2452 peptides that are predicted to be restricted by both HLA I and HLA II molecules. Thus, in total the predicted T-cell epitome consisted of just 14137 unique sequences among all predicted T-cell epitopes.We compared the predicted T-cell epitome with experimentally defined poxvirus-specific HLA-restricted T-cell epitopes deposited in the IEDB [24] and EPIMHC [25]. We retrieved 170 HLA I and 9 HLA II-restricted T-cell epitopes meeting our criteria (see Additional File S1) but we only considered for comparison 85 HLA I- and 8 HLA II-restricted T-cell epitopes that we identified here to be conserved in all orthopoxviruses considered in this study. Of those, 72 HLA I- and 6 HLA II-restricted T-cell epitopes were found within our predicted T-cell epitome. Moreover, we predicted the experimentally verified restriction element in > 80%. The experimentally determined and shared epitopes that were not predicted (a minority) either are restricted by noncovered alleles or were simply not predicted. In Table 4, we summarize the data showing the verified and predicted HLA restriction elements. In sum, we readily predicted most of the experimentally verified T-cell epitopes. Considering that on average 10% of predicted T-cell epitopes can be experimentally verified [52], we shall expect that there are many more valid T-cell epitopes remaining to be validated within the T-cell epitome predicted in this study.Table 4
Experimentally identified T-cell epitopes within the shared T-cell epitome predicted from pathogenic orthopoxvirus proteins.
(a)
CD8 T-cell epitopes
VARV GI
VARV ORF
Experimental HLA I restriction
Predicted HLA I restriction
A
LMRRIAVV
439013
F5R
HLA-A2
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
HLA-A6802
YLLSLFSTL
439056
A35L
HLA-A2
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
HLA-A6802
YLAKLTALV
438985
H5R
HLA-A2
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
HLA-A6802
NLLCHIYSL
438979
K7L
HLA-A2
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
HLA-Cw0702
IVIEAIHTV
439072
J2R
HLA-A0201
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
HLA-Cw0304
SLSAYIIRV
439004
I3L
HLA-A0201
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
HLA-A0207
YLDGQLARL
438965
E6R
HLA-A0201
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
HLA-A0207
YLPEVISTI
438988
H7L
HLA-A0201
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
HLA-Cw0102
TYNDHIVNL
439072
J2R
HLA-A2301
HLA-A2301
HLA-A2402
HLA-A2403
HLA-A2405
HLA-A2407
HLA-Cw0702
RPPSFYKPL
439046
A25R
HLA-B7
HLA-B0702
HLA-B3501
HLA-B5101
HLA-B5301
HLA-B5401
HLA-Cw0102
ALDEKLFLI
439045
A24R
HLA-A0201
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
HLA-A0207
FPYEGGKVF
438968
E9L
HLA-B0702
HLA-B0702
HLA-B1502
HLA-B3501
HLA-B5101
HLA-B5301
HLA-B5401
RLYDYFTRV
438973
K1L
HLA-A0201, HLA-A2
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
ILDDNLYKV
438985
H5R
HLA-A0201, HLA-A2
HLA-A0201
HLA-A0202
HLA-A0205
HLA-A0207
HLA-Cw0702
LLSYYVVYV
439009
F1R
HLA-A2
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
FLIDLAFLI
438960
E1L
HLA-A2
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
HLA-Cw0304
FPRSMLSIF
438994
M4R
HLA-B07:02
HLA-B0702
HLA-B3501
HLA-B4402
HLA-B5301
HLA-B5401
YLFDFVISL
438996
L1R
HLA-A2
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
YLIKLIEPV
439009
F1R
HLA-A0201, HLA-A2
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
SPSNHHILL
439025
A4L
HLA-B07:02
HLA-B0702
HLA-B3501
HLA-B5101
HLA-B5301
HLA-B5401
YPSNKNYEI
439033
A12R
HLA-B07:02
HLA-B0702
HLA-B3501
HLA-B5101
HLA-B5301
HLA-B5401
MLMETMFFI
439007
I6R
HLA-A2
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
ILNPVASSL
438998
L3R
HLA-A2
HLA-A0201
HLA-A0202
HLA-A0205
HLA-A0206
HLA-B1501
FPSVFINPI
438968
E9L
HLA-B0702
HLA-B0702
HLA-B3501
HLA-B5101
HLA-B5301
HLA-B5401
KYQSPVNIF
439043
A21R
HLA-A24, HLA-class I
HLA-A2301
HLA-A2402
HLA-A2403
HLA-A2405
HLA-A2407
YLFGGFSTL
438980
K8R
HLA-A2
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
YLYETYHLI
438981
H1L
HLA-A2
HLA-A0201
HLA-A0202
HLA-A0205
HLA-A0206
VLYNGVNYL
439009
F1R
HLA-A2
HLA-A0201
HLA-A0202
HLA-A0205
HLA-A0206
LIQEIVHEV
439029
A8L
HLA-A0201
HLA-A0201
HLA-A0202
HLA-A0205
HLA-A0206
VELGSGNSF
439043
A21R
HLA-B3701
HLA-B1501
HLA-B1502
HLA-B4402
HLA-Cw0304
RMIAISAKV
438934
P1L
HLA-A2
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0206
FILGIIITV
439036
A15L
HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0205
HLA-A0206
LLSKNTFYL
438981
H1L
HLA-A2
HLA-A0201
HLA-A0202
HLA-A0205
HLA-A0207
RPRDAIRFL
438961
E2L
HLA-B0702
HLA-B0702
HLA-B3501
HLA-B5301
HLA-B5401
KLFNKVPIV
438996
L1R
HLA-A2
HLA-A0201
HLA-A0202
HLA-A0206
HLA-A6802
SLFMILCTR
438979
K7L
HLA-A0301, HLA-A1101
HLA-A1101
HLA-A3101
HLA-A3301
HLA-A6801
ILNDEQLNL
439029
A8L
HLA-A0201
HLA-A0201
HLA-A0205
HLA-A0207
YLLGDSDSV
439038
A17L
HLA-A2
HLA-A0201
HLA-A0205
HLA-A0206
QLMYALEPR
438985
H5R
HLA-A0301, HLA-A1101
HLA-A3101
HLA-A3301
HLA-A6801
LMDENTYAM
439066
A45R
HLA-A2
HLA-A0205
HLA-A0207
HLA-Cw0702
GLLLGCFWV
438955
C17L
HLA-A2
HLA-A0202
HLA-A0203
HLA-A0206
LLSHFYPAV
438952
C14L
HLA-A2
HLA-A0201
HLA-A0205
HLA-A6802
NLFTFLHEI
439045
A24R
HLA-class I
HLA-A0201
HLA-A0203
HLA-A0206
GLFDFVNFV
439070
A49R
HLA-A2, HLA-A0201
HLA-A0202
HLA-A0203
HLA-A0206
YLGPRVCWL
439038
A17L
HLA-A2
HLA-A0202
HLA-A0205
HLA-A0206
ILKSLGFKV
438980
K8R
HLA-A2
HLA-A0202
HLA-A0203
HLA-A0205
DEVASTHDW
439025
A4L
HLA-B4403
HLA-B4402
HLA-B5801
FLVIAINAM
439029
A8L
HLA-A2, HLA-A0201
HLA-A0206
HLA-Cw0304
LSDLKKTIY
439040
A19R
HLA-A1, HLA-class I
HLA-A0101
HLA-B1501
LLYFKVFGI
439019
N1L
HLA-A2
HLA-A0202
HLA-A0205
SSNIMSESY
439014
F6R
HLA-A0101, HLA-A3002
HLA-A0101
HLA-B1501
DTRGIFSAY
439032
A11L
HLA-A26, HLA-class I
HLA-A0101
HLA-B5801
QIDVEKKIV
439029
A8L
HLA-A0201
HLA-A0207
HLA-A6802
YLFRCVDAV
439030
A9R
HLA-A2
HLA-A0205
HLA-A0206
KIEDLINQL
438973
K1L
HLA-A0201
HLA-A0203
HLA-A0207
FTIDFKLKY
439009
F1R
HLA-A1, HLA-A2601, HLA-A2902
HLA-A0101
HLA-Cw0702
WLKIKRDYL
439074
J4R
HLA-B0801
HLA-B0801
AINVEKIEL
473688
L6R
HLA-A0201
HLA-A0207
RIFVRVYNV
439007
I6R
HLA-A2
HLA-A0202
SIIDLIDEY
438942
C4R
HLA-B1501
HLA-A0203
EERHIFLDY
439009
F1R
HLA-B4403
HLA-B4402
ILSDENYLL
439028
A7L
HLA-A2, HLA-A0201
HLA-A0205
HISALKRRY
439027
A6R
HLA-A0101, HLA-A2902
HLA-A0101
FLNISWFYI
438968
E9L
HLA-A2, HLA-A02:01
HLA-A0205
SEVKFKYVL
439002
I1L
HLA-B44
HLA-B4402
KLLLWFNYL
438980
K8R
HLA-A2
HLA-A0203
YIDISDVKV
438973
K1L
HLA-A0201
HLA-A0207
VWINNSWKF
439013
F5R
HLA-A24, HLA-A2301, HLA-A2402
HLA-Cw0702
VLPFDIKKL
439014
F6R
HLA-A0201
HLA-Cw0102
VETSISDYY
439043
A21R
HLA-B3701
HLA-B1501
SQIIDISLR
439022
A1L
HLA-A0301, HLA-A1101
HLA-B2705
HDVYGVSNF
439045
A24R
HLA-B4403
HLA-B4402
(b)
CD4 T-cell epitope
VARV GI
VARV ORF
Experimental HLA II restriction
Predicted HLA II restriction
FLIDLAFLI
438960
E1L
HLA-class II
DRB1∗0101
DRB1∗0102
DRB1∗0301
DRB1∗0311
DRB1∗0401
DRB1∗1201
DRB1∗1302
DRB1∗1502
DRB3∗0101
DRB5∗0101
IHWQIISSE
439072
J2R
DRB1∗04:05
DRB1∗0405
DRB1∗0406
DRB1∗0410
DRB1∗1304
DRB3∗0101
YIDAYVSRL
439021
N3L
DRB1∗15:01
DRB1∗0901
DRB1∗1103
DRB3∗0202
LMDENTYAM
439066
A45R
HLA-class II
DRB1∗1103
IDAYVSRLL
439021
N3L
DRB1∗15:01
DRB1∗1201
RMIAISAKV
438934
P1L
HLA-class II
DRB1∗0407
T-cell epitopes in this table are a subset of those provided in Additional File S1.


## 3.4. EPIPOX Database and Web Server

We developed a relational database based upon the predicted T-cell epitome and a web-based resource to facilitate online access and to query the database. We named this resource EPIPOX and made it available for free public use (URL:http://imed.med.ucm.es/epipox/). EPIPOX is ade facto analysis pipeline of viral T-cell epitomes. The content of the EPIPOX database is organized in three tables (peptides,predictions, andproteins) (Figure 1). The tablepredictions contains all predicted T-cell epitopes, consisting of 18726 HLA I- and 32722 HLA II-restricted peptides, each identified by its sequence and restriction element. Peptide sequences in this table are not unique as each peptide can bind to numerous HLA I molecules. Peptide sequences are, however, unique in the tablepeptides.This table contains 14137 sequences comprising the whole predicted epitome regardless of the restriction elements. Antigen annotations in EPIPOX are found within the tableproteins (Figure 1). We only included annotations that are relevant to epitope vaccine design, such as temporal expression of gene products and location in relevant structures of the virus such as the EEV and IVM membranes and CORE. Early expressed proteins and highly expressed proteins are generally thought to be more immunogenic, particularly with regard to CD8 T cells [53, 54]. On the other hand, highly abundant late proteins that are located in membrane structures of the poxvirus appear to be the main focus of the antibody and CD4 T-cell response [42, 43]. In the tableproteins, we also provide annotations on whether the proteins have transmembrane region or leader signal sequence, as proteins with these features often interact with host cells and are important targets for subunit vaccine design [46, 47].The EPIPOX web interface (Figure2) allows querying of the database combining any annotation field in the database, as described above. For intuitive use, the interface is divided in two main sections. In the first section (SEARCH), users select proteins and restriction elements for epitope retrieval. In this section, EPIPOX also provides the option to query the database for promiscuous T-cell epitopes binding to three HLA I supertypes (A2, A3, and B7). The alleles belonging to these supertypes are present in 88% of the population regardless of their ethnic groups. Selecting promiscuous peptides restricted by these 3 supertypes facilitates maximizing the population coverage of vaccines with minimum numbers of peptides [49, 50, 55]. In the second section (LIMIT), users can select annotation criteria to restrict the results. As an example, in Figure 3, we show the page resulting from a sample query consisting of promiscuous peptides from CORE binding to the A2 supertype. From the EPIPOX output, users can also access additional information available from the Virus Pathogen Resource database (Figure 3) [56].Figure 2
EPIPOX input page. The input page of EPIPOX is divided in two main sections for intuitive use. In the first part (SEARCH), users select HLA molecules and proteins to retrieve T-cell epitopes (multiple selection is allowed) while in the second part the user can limit the search output according to various criteria. These criteria include temporal expression of gene products (E: early; I: intermediate; L: late), location of proteins in relevant structures of the virus (CORE, IMV, and EEV), and the presence of leader and transmembrane regions. In addition, users can select only those peptides with a relative score above some selectable value. HLA-specific profiles used to score T-cell epitopes can reach a maximum score, which is used to set the relative score in percentage of each peptide. For HLA I-restricted epitopes, users can also restrict the search to those epitopes potentially generated by the proteasome.Figure 3
EPIPOX result page. The figure shows a slice of the output resulting from promiscuous CORE protein peptides binding to the A2 supertype. The output consists of a tabulated list, with information on each of the fields of the search query (columns). From fieldSOURCE NAME (1), users can access proteins from the Virus Pathogen Database (http://www.viprbrc.org/) (1) and by clicking on the epitope sequence, fieldSEQ (2), users will get the amino acid sequence of the protein showing the peptide in bold (2).EPIPOX is related somewhat to certain existing databases. On the one hand, it shares features with generic epitope databases such as EPIMHC [25], AntiJen [40], and IEDB [24] and on the other hand it shares features with poxvirus genome annotation-orientated databases such as the Poxviridae database [28] (no longer operating) and the Virus Pathogen Resource (http://www.viprbrc.org/) [56]. This later resource contains information on virus sequences, functional annotations and epitopes derived from IEDB [24]. However, the Virus Pathogen Resource does not allow selection of epitopes or antigens by criteria that are relevant to epitope vaccine design. In fact, EPIPOX is the only dedicated immunologic resource that has been designed to facilitate the rational selection of epitopes and antigens for subunit vaccine design.


## 4. Conclusions and Future Development

The availability of the VARV genomes enables the use of predictive tools that reveal entire T-cell epitomes and facilitate the development of epitope-based vaccines. However, in large and complex viruses, such as VARV, the potential T-cell epitome can be so sizeable that it will challenge experimental validation. Therefore, in this work we applied a rational strategy to limit the list of potential T-cell epitopes. First, we reduced the number of antigens by half by simply selecting those that are conserved among pathogenic orthopoxviruses related to VARV. Second, we enriched the antigens with annotations such as temporal expression and location. Lastly, we created a resource andde facto analysis pipeline (EPIPOX) with which to interrogate the resulting T-cell epitome and enable users to select immunologically relevant subsets of T-cell epitopes suitable for experimental validation.We expect that this work and EPIPOX will be instrumental in developing safer smallpox vaccines and thereby in preventing zoonosis caused by other orthopoxviruses, including MPXV, which is also a potential terrorist bioweapon. In the future, we plan to enhance EPIPOX with validated and/or experimentally determined epitopes, upgrade protein annotations with functional information, and include additional features such as TAP transport [57], ERAAP cleavage [58], and T-cell epitope immunodominance. In sum, we would expect EPIPOX to establish itself as a facilitating resource of true utility ininter alia immunoinformatic characterization of viral genomics and computational reverse vaccinology.



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*Source: 738020-2015-10-28.xml*