# Applying the Concept of Peptide Uniqueness to Anti-Polio Vaccination

Authors: Darja Kanduc; Candida Fasano; Giovanni Capone; Antonella Pesce Delfino; Michele Calabrò; Lorenzo Polimeno
Journal: Journal of Immunology Research
 (2015)

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## Abstract

Background. Although rare, adverse events may associate with anti-poliovirus vaccination thus possibly hampering global polio eradication worldwide. Objective. To design peptide-based anti-polio vaccines exempt from potential cross-reactivity risks and possibly able to reduce rare potential adverse events such as the postvaccine paralytic poliomyelitis due to the tendency of the poliovirus genome to mutate. Methods. Proteins from poliovirus type 1, strain Mahoney, were analyzed for amino acid sequence identity to the human proteome at the pentapeptide level, searching for sequences that (1) have zero percent of identity to human proteins, (2) are potentially endowed with an immunologic potential, and (3) are highly conserved among poliovirus strains. Results. Sequence analyses produced a set of consensus epitopic peptides potentially able to generate specific anti-polio immune responses exempt from cross-reactivity with the human host. Conclusion. Peptide sequences unique to poliovirus proteins and conserved among polio strains might help formulate a specific and universal anti-polio vaccine able to react with multiple viral strains and exempt from the burden of possible cross-reactions with human proteins. As an additional advantage, using a peptide-based vaccine instead of current anti-polio DNA vaccines would eliminate the rare post-polio poliomyelitis cases and other disabling symptoms that may appear following vaccination.


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## Body

## 1. Introduction

Vaccine-associated paralytic poliomyelitis (VAPP) [1] is the consequence of the replication of vaccine-derived polioviruses (VDPVs) that originate by genetic mutations from the strain contained in the oral polio vaccine (OPV). In fact, the poliovirus (PV) genetic mutability [2] appears to be the molecular basis of VAPP, which throws a shadow over the great success represented by vaccination in fighting PV infection [3, 4].Currently, two new monovalent OPVs [5] and the change of the schedule from OPV to the exclusive use of inactivated polio vaccine (IPV) [6] represent options to interrupt PV transmission. However, it has been observed that reduction of exposure to a live attenuated virus such as that contained in OPV will inevitably lead to a decrease in herd immunity to a live microorganism and to natural boosters [7]. Such considerations, along with recent PV infection outbreaks, further complicate the issue of polio eradication [8].In this scenario, new vaccine formulations and renewed research efforts might help to specifically fight PV infection. Recently, we analyzed the peptide overlap between PV1, strain Mahoney, and the human proteome and described a high extent of peptide sharing involving human proteins linked to fundamental cellular functions [9]. These data appeared to be of interest also because PV has been studied mainly at the nucleotide level [10], and, in general, there is a lack of knowledge of the interaction(s) between PV and the human host at the peptide/protein level. Actually, phenetic analyses of PV might help define peptide-based therapeutic approaches against PV infection, given that, for example,(i)
one single amino acid (aa) change, that is, His to Tyr at aa position 142 of virion protein 2 (VP2) or Val to Ile at aa position 160 of virion protein 1 (VP1), on the capsid surface of PV1 Sabin allows the establishment of persistent infections in HEp-2c cell cultures [11];(ii)
replacement of the Ala residue with Asp at aa position 3 is linked to 50% loss of virion protein 4 (VP4) precursor myristoylation and severe reduction in specific infectivity [12];(iii)
the 25th aa, Ile, of PV 2C protein interacts with human reticulon 3, a protein involved in viral replication and/or pathogenesis [13].Moreover, since the 1980s we have known that small aa groupings can play a critical role in neutralizing PV. For example, the discontinuous E4T7T/S8R9 tetrapeptide, which is present in PV VP1, is crucial for neutralizing PV3 by anti-PV3 25-1-14 monoclonal antibody (mAb) [14]. Additional examples of short immune determinants in the immune response against PV are the following ones:(i)
A linear heptapeptide (DNNQTSP) is an Ab-binding site mapped to aa residues 164–170 of VP2 [15].(ii)
Short synthetic peptides (e.g., DNTVRET, RSRSES, RSRSESSIESF, and STTNKDK) from PV VP1 prime the immune system of rabbits for a long-lasting, virus-neutralizing IgG Ab response following a single inoculation of intact virus [16].(iii)
The Immune Epitope Database (IEDB;http://www.immuneepitope.org) [17] includes the continuous linear pentapeptide STTNK (IEDB ID: 61944) [18, 19] and the discontinuous pentapeptide T141E143S312E315P417 (IEDB ID: 91064) [20] as PV-derived epitopes.Following the mathematical quantification of pentapeptide sharing between PV1 and human proteins, we reported that 2,040 out of the 2,204 pentamers composing the PV1 proteome are shared with the human proteome for a total of 18,223 matches, including multiple occurrences [9]. In general, a vast peptide sharing with human proteins is a characteristic of viral proteomes [21]. This peptide commonality suggests the existence of common evolutionary links between entities widely different as viruses andHomo sapiens and, in addition, indicates that potential cross-reactivity may affect antivirus vaccine formulations [22] and serological analyses [23]. Hence, it is reasonable to postulate that vaccines based on peptides unique to a virus and absent in the host proteome would guarantee high specificity and, at the same time, eliminate potential cross-reactivity.Pursuing the objectives of overcoming the difficulties posed by the PV tendency to mutate and eliminating the viral reactivation-related VAPP in order to contribute to the global eradication of poliomyelitis, here we examine PV1 Mahoney polyprotein primary sequence and describe a set of pentapeptides uniquely owned by PV1, endowed with immunologic potential, and conserved among 43 PV strains. We propose that this set of pentapeptides might be used in preclinical andin vivo protocols with the ultimate aim of formulating effective, safe, and universal anti-PV vaccines.


## 2. Methods

The primary aa sequence of human PV1 polyproteins, strain Mahoney (NCBI taxonomic identifier: 12081; Swiss-Prot/UniProtKB entry: P03300), consisting of 11 viral proteins, 2,209 aa long [25] (further details at http://www.uniprot.org/uniprot/P03300), was analyzed for aa sequence similarity to the human proteome at the pentapeptide level. In brief, the viral polyprotein was dissected into 5-mers sequentially overlapped by four residues: MGAQV, GAQVS, AQVSS, QVSSQ, VSSQK, and so forth; then, each viral pentapeptide was used as a probe to scan the human proteome for instances of the same pentapeptide, as already described [9]. The similarity analysis used the Protein Information Resource (PIR) peptide match program (http://pir.georgetown.edu/) [26].PV-derived epitopes were retrieved from the Immune Epitope Database (IEDB;http://www.immuneepitope.org) [17]. Only PV epitopes that had been experimentally validated in the human host were considered in this study.Consensus peptide sequences were defined by ClustalW multialignment analysis (http://www.uniprot.org/program/?query=clustalw&sort=score) [24] of sequences from 43 PV strains retrieved from UniProt database (http://www.uniprot.org/) on the basis of the following characteristics: (1) described in scientific literature; (2) corresponding to the entire PV polyprotein; (3) derived from PV1 and PV3; (4) derived from PV variants isolated from VAPP or acute flaccid paralysis (AFP) patients or from immunocompromised patients with residual paralysis. Description and references of the 43 PV sequences used for multialignment analysis are reported in detail at http://www.uniprot.org/; the relative Swiss-Prot/UniProtKB entries are P03300, P03301, P03302, P06209, Q9Q281, Q9Q280, Q71AZ9, Q5TLH5, D1YSI9, D1YSJ1, D1YSJ2, D1YSJ3, D1YSJ4, D1YSJ5, D1YSJ6, D1YSJ7, D1YSJ9, D1YSK1, D1YSK2, D1YSK3, D1YSK4, D1YSK5, D1YSK6, D1YSK7, D1YSK8, D1YSK9, D1YSL0, D1YSL1, D1YSL2, D1YSL3, D1YSL4, D1YSL5, D2X673, D8L541, B4YUL3, B4YUL4, Q84792, C5HJY2, C5HJY3, D1GE40, D2E679, D1GE41, and D2XUS9.


## 3. Results and Discussion

### 3.1. Identification of Pentapeptides Unique to PV Type 1, Strain Mahoney

Using the procedure described under Section2, we searched the human PV1, strain Mahoney, primary sequence for pentapeptides not shared with human proteins. We used pentapeptides as probes since a pentapeptide is a minimal functional unit in immunology [27], thus representing an appropriate length unit in measuring the qualitative/quantitative parameters of immunological phenomena [28].Table1 reports the pentapeptide platform that characterizes PV1 Mahoney polyprotein when compared to theHomo sapiens proteome. We find that 164 pentapeptides are unique to the viral polyprotein and absent in human proteins. In a few instances, viral 5-mers consecutively overlap (Table 1, pentapeptides in bold), thus forming 6-, 7-, and 8-mer stretches (e.g., PV163–169QNMYYHY, PV446–453NYYTHWAG, PV783–789AYSHFYD, PV1496–1498KGWIVNI, PV1594–1600GIHDNVA, PV1699–1706RTLMYNFP, and PV1750–1756EIQWMRP).Table 1
Pentapeptides unique to PV type 1, strain Mahoney, and absent in the human proteome.
Posa
Sequenceb,c
Posa
Sequenceb,c
Posa
Sequenceb,c
Posa
Sequenceb,c
Posa
Sequenceb,c
Posa
Sequenceb,c
11
GAHEN
393
MIPLN
746
PAKWD
969
YYPAR
1399
CHQPA
1750
EIQWM
29
TINYY
403
KNTMD
748
KWDDY
974
YQSHI
1403
ANFKR
1751
IQWMR
72
NIEAC
404
NTMDM
749
WDDYT
996
CHHGV
1413
CGKAI
1752
QWMRP
73
IEACG
408
MYRVQ
752
YTWQT
1014
FADIR
1418
QLMDK
1762
YPIIN
104
YGRWP
414
NDNPH
763
FYTYG
1021
YAYEE
1437
IVNER
1810
YVGNK
106
RWPEY
436
LSHTM
783
AYSHF
1084
ITRNY
1458
PIQYK
1840
TEQMC
107
WPEYL
446
NYYTH
784
YSHFY
1105
VSPWQ
1475
ECIND
1849
MYGTD
130
CRFYT
447
YYTHW
785
SHFYD
1136
TEACN
1489
VRNYC
1933
VAMRM
145
RGWWW
448
YTHWA
835
KIRVY
1139
CNAAK
1490
RNYCE
1935
MRMAF
146
GWWWK
449
THWAG
841
KPKHI
1188
STIHQ
1496
KGWIV
1946
FHKNP
148
WWKLP
464
SMMAT
847
VWCPR
1190
IHQSC
1497
GWIVN
1947
HKNPG
149
WKLPD
465
MMATG
857
AYYGP
1204
FNNVR
1498
WIVNI
1966
WSKIP
163
QNMYY
494
HVIWD
863
VDYKD
1205
NNVRW
1513
NRAMT
1979
AFDYT
164
NMYYH
495
VIWDI
880
TYGFG
1209
WLSIQ
1532
VYVMY
1983
TGYDA
165
MYYHY
497
WDIGL
881
YGFGH
1231
LEHTI
1534
VMYKL
1993
WFEAL
179
VQCNA
507
MVVPW
883
FGHQN
1291
FDGYK
1536
YKLFA
2028
YCVKG
199
MCLAG
510
PWISN
884
GHQNK
1311
GADMK
1588
GEFTM
2070
KMIAY
242
RFCPV
518
RQTTN
894
GYKIC
1314
MKLFC
1592
MLGIH
2073
AYGDD
244
CPVDY
534
FYQTR
895
YKICN
1326
FIPPM
1594
GIHDN
2115
TWENV
271
TNNCA
556
SACND
897
ICNYH
1340
FTSNY
1595
IHDNV
2131
KYPFL
292
KHNNW
557
ACNDF
912
VSTMW
1341
TSNYV
1596
HDNVA
2148
SIRWT
293
HNNWG
558
CNDFS
914
TMWDR
1368
RFAFD
1660
TETND
2158
TQDHV
295
NWGIA
568
DTTHI
934
ARCNC
1369
FAFDM
1677
MFVPV
2168
LAWHN
323
PMCC
569
TTHIG
942
VYYCE
1371
FDMDI
1699
RTLMY
2201
LYRRW
324
PMCCE
588
MIDNT
948
RRKYY
1388
DMTMA
1700
TLMYN
326
CCEFN
665
CVSII
959
PTFQY
1389
MTMAT
1701
LMYNF
327
CEFNG
708
RFDME
961
FQYME
1394
EMCKN
1702
MYNFP
391
DTMIP
735
QIMYV
962
QYMEA
1398
NCHQP
1742
SYFTQ
aaa position along the human PV type 1, strain Mahoney, primary sequence.
baa sequences given in one-letter code.
cConsecutively overlapping pentapeptides forming 7- and 8-mer stretches unique to PV are given in bold.


### 3.2. Analysis of the Immunologic Potential of Peptides Unique to PV

Next, the pentapeptides described in Table1 were analyzed for their immunologic potential as follows. PV-derived epitopes were retrieved from IEDB, and the epitopes that had been experimentally validated in the human host were analyzed for the presence of pentapeptides unique to PV1 (see Table 1).As reported in Table2, the search through IEDB produced a final list of 78 viral epitopes derived from PV1 Mahoney, PV3 Sabin, and PV3 (P3/LEON/37 and P3/LEON 12A (1)B). Epitopes derived from PV2 strains were not considered since only data from immunoassays in mice, rats, and/or rabbits were available in IEDB for PV2 strains at the time of this study. Following sequence analysis, it was found that 20 of the 78 epitopes (i.e., IEDB IDs: 31814, 48785, 58511, 59797, 71769, 79272, 79480, 99910, 100138, 100244, 100349, 100382, 100536, 100576, 100631, 100667, 100672, 146181, 146248, and 146390; see IEDB IDs in bold in Table 2) have a total of 12 viral pentapeptides that are absent in the human proteome (pentapeptides in capital letters in Table 2). That is, a first conclusion from Table 2 is that 12 out of the 164 pentapeptides unique to PV1, strain Mahoney, are part of 20 PV epitopic sequences endowed with an immunologic potential in the human host.Table 2
Twelve pentapeptides unique to PV1, Mahoney strain, and absent in the human proteome are distributed among twenty PV-derived epitopes recognized by human sera and/or T cells.
IEDB IDa
Epitope sequenceb,c
PV antigen
PV strain
Immune context
30661
kevpaltavetgat
VP1
PV3 Sabin
B
31814
klefftysRFDMEltfvvtan
VP1
PV1 Mahoney
T
46859
paltavetgatnpl
VP1
PV1 Mahoney
B-T
48785
ppgapvpeKWDDYTWQTssnp
VP1
PV1 Mahoney
T
55952
rsrsessiesf
VP1
PV1 Mahoney
B
58511
siFYTYGtaparisvpyvgi
VP1
PV1 Mahoney
T
59797
snAYSHFYDgfskvplkdqs
VP1
PV1 Mahoney
T
66978
tvdnsasttnkdklfavwk
Polyprotein
PV1 Mahoney
T
71769
vvndhnptkvtsKIRVYlkp
Polyprotein
PV1 Mahoney
T
79155
altlslpkqqdslpdtka
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
79160
atnplapsdtvqtrhvvq
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
79186
dneqpttraqklfam
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
79269
kevpaltavetgatnpla
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
79272
khvrVWCPRppravpyyg
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
79318
nghalnqvyqimyippga
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
79350
qklfamwritykdtv
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
79354
qpttraqklfamwri
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
79433
traqklfamwrityk
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
79434
traqklfamwritykdtv
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
79435
trhvvqrrsrsestiesf
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
79436
tskvriymkpkhvrvw
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
79443
vaiievdneqpttraqkl
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
79461
vrvvndhnptkvtskvri
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
79480
yippgaptpksWDDYTwq
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
80446
argacvtimtvdnpa
VP1
PV1 Mahoney
B
81394
easgpthskeipalt
VP1
PV1 Mahoney
B
82831
gpthskeipaltave
VP1
PV1 Mahoney
B
83234
hskeipaltavetga
VP1
PV1 Mahoney
B
88446
sdtvqtrhvvqhrsr
VP1
PV1 Mahoney
B
88495
sessiesffargacv
VP1
PV1 Mahoney
B
99863
aaparisvpyvgla
Polyprotein
PV3 Sabin
B
99886
ahskevpaltavet
Polyprotein
PV3 Sabin
B
99901
arisvpyvglanay
Polyprotein
PV3 Sabin
B
99910
AYSHFYDgfakvpl
Polyprotein
PV3 Sabin
B
99933
dfgvlavrvvndhn
Polyprotein
PV3 Sabin
B
99963
dtvqlrrkleffty
Polyprotein
PV3 Sabin
B
100029
famwritykdtvql
Polyprotein
PV3 Sabin
B
100101
hfydgfakvplktd
Polyprotein
PV3 Sabin
B
100117
hnptkvtskvriym
Polyprotein
PV3 Sabin
B
100138
iFYTYGaaparisv
Polyprotein
PV3 Sabin
B
100244
lanAYSHFYDgfak
Polyprotein
PV3 Sabin
B
100349
npsiFYTYGaapar
Polyprotein
PV3 Sabin
B
100382
pkhvrVWCPRppra
Polyprotein
PV3 Sabin
B
100391
psdtvqtrhvvqrr
Polyprotein
PV3 Sabin
B
100425
qklfamwritykdt
Polyprotein
PV3 Sabin
B
100430
qlrrklefftysrf
Polyprotein
PV3 Sabin
B
100439
qpttraqklfamwr
Polyprotein
PV3 Sabin
B
100482
rppravpyygpgvd
Polyprotein
PV3 Sabin
B
100492
samtvddfgvlavr
Polyprotein
PV3 Sabin
B
100504
sevaqgaltlslpk
Polyprotein
PV3 Sabin
B
100536
svpyvglanAYSHF
Polyprotein
PV3 Sabin
B
100559
tkvtskvriymkpk
Polyprotein
PV3 Sabin
B
100573
traqklfamwrity
Polyprotein
PV3 Sabin
B
100575
tskvriymkpkhvr
Polyprotein
PV3 Sabin
B
100576
tssnpsiFYTYGaa
Polyprotein
PV3 Sabin
B
100580
tvddfgvlavrvvn
Polyprotein
PV3 Sabin
B
100583
tvqtrhvvqrrsrs
Polyprotein
PV3 Sabin
B
100585
twqtssnpsifyty
Polyprotein
PV3 Sabin
B
100586
tygaaparisvpyv
Polyprotein
PV3 Sabin
B
100587
tykdtvqlrrklef
Polyprotein
PV3 Sabin
B
100613
vlavrvvndhnptk
Polyprotein
PV3 Sabin
B
100619
vndhnptkvtskvr
Polyprotein
PV3 Sabin
B
100628
vriymkpkhvrvwc
Polyprotein
PV3 Sabin
B
100630
vrvvndhnptkvts
Polyprotein
PV3 Sabin
B
100631
vrVWCPRppravpy
Polyprotein
PV3 Sabin
B
100638
wcprppravpyygp
Polyprotein
PV3 Sabin
B
100644
writykdtvqlrrk
Polyprotein
PV3 Sabin
B
100667
ymkpkhvrVWCPRp
Polyprotein
PV3 Sabin
B
100672
yvglanAYSHFYDg
Polyprotein
PV3 Sabin
B
146178
ayappgaqpptsrk
Polyprotein
PV3 Sabin
B
146181
cgSMMATGkilvay
Polyprotein
PV3 Sabin
B
146248
flfcgSMMATGkil
Polyprotein
PV3 Sabin
B
146311
hqgalgvfaipeyc
Polyprotein
PV3 Sabin
B
146333
ilvayappgaqppt
Polyprotein
PV3 Sabin
B
146390
kftflfcgSMMATG
Polyprotein
PV3 Sabin
B
146494
phqiinlrtnnsat
Polyprotein
PV3 Sabin
B
146496
ppgaqpptsrkeam
Polyprotein
PV3 Sabin
B
146516
ravpyygpgvdyrn
Polyprotein
PV3 Sabin
B
aPV-derived epitopes are listed according to increasing IEDB ID number. Further details and reference(s) on each IEDB ID are reported at http://www.immuneepitope.org [17].
bOnly PV-derived epitopes that had been experimentally validated in the human host are reported.
cThe twenty PV-derived epitopes (and related IDs) containing PV-pentapeptide(s) absent in the human proteome are in bold, with the pentapeptide(s) absent in the human proteome given in capital.Moreover, it can be seen that the 12 unique PV1 pentapeptides are not distributed at random among the PV-derived epitopes. For example, three unique pentapeptides (KWDDY, WDDYT, and YTWQT) overlap each other in the epitope IEDB ID 48785, sequence ppgapvpeKWDDYTWQTssnp (with unique overlapping pentapeptides in capital); the heptapeptide AYSHFYD, formed by three overlapping unique pentapeptides shifted by one residue, characterizes four PV-derived epitopes (IEDB IDs: 59797, 99910, 100244, and 100672); the hexapeptide SMMATG formed by two overlapped 5-mers is present in three epitopes (IEDB IDs: 146181, 146248, and 146390).Theoretically, vaccines based on such PV epitopic peptides (i.e., KWDDYTWQT, AYSHFYD, and SMMATG) might evoke highly specific anti-PV immune responses exempt of possible collateral cross-reactions in the human host.


### 3.3. Identification of Consensus Pentapeptides Unique to PVs and Endowed with Immunologic Potential

We reasoned that using epitopic peptides unique to the virus and conserved among PV strains might help develop a global anti-PV peptide-based vaccination protocol. Such an approach would be of special importance in providing an effective and wide coverage to the human population worldwide, thus allowing reaching the goal of PV eradication [29, 30]. To this aim, we searched for unique conserved sequences by analysing a set of 43 PV polyproteins selected as described under Section 2 and comprehending also PV variants isolated from faeces of VAPP or AFP or from immunocompromised patient(s) with residual paralysis. The 43 PV polyprotein sequences were aligned using multialignment ClustalW program [24] and the peptide sequences present in PV-derived epitopes (see Table 2) and absent in the human proteome were localized.Table3 shows that seven potentially immunogenic peptides (in the order PV465–469MMATG, PV708–712RFDME, PV752–756DYTWQT, PV763–767FYTYG, PV783–789AYSHFYD, PV835–839KIRVY, and PV847–851VWCPR), derived from epitopes corresponding to (or formed of) pentapeptides unique to PV1 (see Tables 1 and 2), have 100% conservation among the 43 PV strains/variants under analysis. The seven potentially immunogenic unique peptide sequences had the same level of conservativeness in PV2 derived strains (data not shown).Table 3
Conservativeness  of epitopic PV peptide regions among 43 PV strains and variants.
PV strain
UniProt/Swiss-Prot entry
aa Pos 464
aa Pos 708
aa Pos  748
aa Pos 763
aa Pos783
aa Pos 835
aa Pos 847
PV1 Mahoney
P03300
FMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1 Sabin
P03301
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV3 P3/Leon/37
P03302
SMMATG
RFDME
sWDDYTWQT
FYTYG
AYSHFYD
KvRiY
VWCPR
PV3 23127
P06209
SMMATG
RFDME
sWDDYTWQT
FYTYG
AYSHFYD
KvRvY
VWCPR
PV1 isolated
Q9Q281
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KvRVY
VWCPR
PV1 isolated
Q9Q280
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1 isolated
Q71AZ9
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1 isolated
Q5TLH5
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-HAI01008C2
D1YSI9
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-HAI01009
D1YSJ1
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-HAI01008
D1YSJ2
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-HAI01002
D1YSJ3
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-HAI01001
D1YSJ4
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR01012
D1YSJ5
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR01002C
D1YSJ6
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR01002
D1YSJ7
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR01001C1
D1YSJ9
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR00042C2
D1YSK1
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR00042C1
D1YSK2
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR00042
D1YSK3
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR00041C3
D1YSK4
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR00041C2
D1YSK5
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR00044
D1YSK6
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR00028C
D1YSK7
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR00028
D1YSK8
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR00023C
D1YSK9
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR00025
D1YSL0
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOOR24
D1YSL1
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR00015
D1YSL2
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR00016
D1YSL3
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-HAI01013all
D1YSL4
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-HAI01015
D1YSL5
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-S302
D2X673
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
CHN8184/GZ/CHN/2004
D8L541
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV3 isolated
B4YUL3
SMMATG
RFDME
sWDDYTWQT
FYTYG
AYSHFYD
KvRVY
VWCPR
PV3 isolated
B4YUL4
SMMATG
RFDME
sWDDYTWQT
FYTYG
AYSHFYD
KvRVY
VWCPR
PV3 (vacc.StrainSabin3(Leon 12a1b))
Q84792
SMMATG
RFDME
sWDDYTWQT
FYTYG
AYSHFYD
KvRiY
VWCPR
PV3-33239
C5HJY2
SMMATG
RFDME
sWDDYTWQT
FYTYG
AYSHFYD
KvRiY
VWCPR
PV3-31974
C5HJY3
SMMATG
RFDME
sWDDYTWQT
FYTYG
AYSHFYD
KvRiY
VWCPR
PV3-FIN84-60212
D1GE40
SMMATG
RFDME
sWDDYTWQT
FYTYG
AYSHFYD
KvRiY
VWCPR
PV3-SWI10947
D2E679
SMMATG
RFDME
sWDDYTWQT
FYTYG
AYSHFYD
KvRiY
VWCPR
PV3-FIN84-2493
D1GE41
SMMATG
RFDME
sWDDYTWQT
FYTYG
AYSHFYD
KvRiY
VWCPR
PV3-P3/Jinan/1/09
D2XUS9
SMMATG
RFDME
sWDDYTWQT
FYTYG
AYSHFYD
KvRiY
VWCPR
The 43 PV sequences were aligned using ClustalW multialignment program (http://www.uniprot.org/align/) [24]. The analyzed PV sequences and related references are described at http://www.uniprot.org/ and reported by Swiss-Prot/UniProtKB accession number. Peptide sequences present in PV-derived epitopes (see Table 2) and absent in the human proteome were localized along the 43 aligned PV sequences and analyzed for conservativeness. Peptide sequences are indicated by their position along the PV polyprotein. Mutated aa residues are in lower case.


## 3.1. Identification of Pentapeptides Unique to PV Type 1, Strain Mahoney

Using the procedure described under Section2, we searched the human PV1, strain Mahoney, primary sequence for pentapeptides not shared with human proteins. We used pentapeptides as probes since a pentapeptide is a minimal functional unit in immunology [27], thus representing an appropriate length unit in measuring the qualitative/quantitative parameters of immunological phenomena [28].Table1 reports the pentapeptide platform that characterizes PV1 Mahoney polyprotein when compared to theHomo sapiens proteome. We find that 164 pentapeptides are unique to the viral polyprotein and absent in human proteins. In a few instances, viral 5-mers consecutively overlap (Table 1, pentapeptides in bold), thus forming 6-, 7-, and 8-mer stretches (e.g., PV163–169QNMYYHY, PV446–453NYYTHWAG, PV783–789AYSHFYD, PV1496–1498KGWIVNI, PV1594–1600GIHDNVA, PV1699–1706RTLMYNFP, and PV1750–1756EIQWMRP).Table 1
Pentapeptides unique to PV type 1, strain Mahoney, and absent in the human proteome.
Posa
Sequenceb,c
Posa
Sequenceb,c
Posa
Sequenceb,c
Posa
Sequenceb,c
Posa
Sequenceb,c
Posa
Sequenceb,c
11
GAHEN
393
MIPLN
746
PAKWD
969
YYPAR
1399
CHQPA
1750
EIQWM
29
TINYY
403
KNTMD
748
KWDDY
974
YQSHI
1403
ANFKR
1751
IQWMR
72
NIEAC
404
NTMDM
749
WDDYT
996
CHHGV
1413
CGKAI
1752
QWMRP
73
IEACG
408
MYRVQ
752
YTWQT
1014
FADIR
1418
QLMDK
1762
YPIIN
104
YGRWP
414
NDNPH
763
FYTYG
1021
YAYEE
1437
IVNER
1810
YVGNK
106
RWPEY
436
LSHTM
783
AYSHF
1084
ITRNY
1458
PIQYK
1840
TEQMC
107
WPEYL
446
NYYTH
784
YSHFY
1105
VSPWQ
1475
ECIND
1849
MYGTD
130
CRFYT
447
YYTHW
785
SHFYD
1136
TEACN
1489
VRNYC
1933
VAMRM
145
RGWWW
448
YTHWA
835
KIRVY
1139
CNAAK
1490
RNYCE
1935
MRMAF
146
GWWWK
449
THWAG
841
KPKHI
1188
STIHQ
1496
KGWIV
1946
FHKNP
148
WWKLP
464
SMMAT
847
VWCPR
1190
IHQSC
1497
GWIVN
1947
HKNPG
149
WKLPD
465
MMATG
857
AYYGP
1204
FNNVR
1498
WIVNI
1966
WSKIP
163
QNMYY
494
HVIWD
863
VDYKD
1205
NNVRW
1513
NRAMT
1979
AFDYT
164
NMYYH
495
VIWDI
880
TYGFG
1209
WLSIQ
1532
VYVMY
1983
TGYDA
165
MYYHY
497
WDIGL
881
YGFGH
1231
LEHTI
1534
VMYKL
1993
WFEAL
179
VQCNA
507
MVVPW
883
FGHQN
1291
FDGYK
1536
YKLFA
2028
YCVKG
199
MCLAG
510
PWISN
884
GHQNK
1311
GADMK
1588
GEFTM
2070
KMIAY
242
RFCPV
518
RQTTN
894
GYKIC
1314
MKLFC
1592
MLGIH
2073
AYGDD
244
CPVDY
534
FYQTR
895
YKICN
1326
FIPPM
1594
GIHDN
2115
TWENV
271
TNNCA
556
SACND
897
ICNYH
1340
FTSNY
1595
IHDNV
2131
KYPFL
292
KHNNW
557
ACNDF
912
VSTMW
1341
TSNYV
1596
HDNVA
2148
SIRWT
293
HNNWG
558
CNDFS
914
TMWDR
1368
RFAFD
1660
TETND
2158
TQDHV
295
NWGIA
568
DTTHI
934
ARCNC
1369
FAFDM
1677
MFVPV
2168
LAWHN
323
PMCC
569
TTHIG
942
VYYCE
1371
FDMDI
1699
RTLMY
2201
LYRRW
324
PMCCE
588
MIDNT
948
RRKYY
1388
DMTMA
1700
TLMYN
326
CCEFN
665
CVSII
959
PTFQY
1389
MTMAT
1701
LMYNF
327
CEFNG
708
RFDME
961
FQYME
1394
EMCKN
1702
MYNFP
391
DTMIP
735
QIMYV
962
QYMEA
1398
NCHQP
1742
SYFTQ
aaa position along the human PV type 1, strain Mahoney, primary sequence.
baa sequences given in one-letter code.
cConsecutively overlapping pentapeptides forming 7- and 8-mer stretches unique to PV are given in bold.


## 3.2. Analysis of the Immunologic Potential of Peptides Unique to PV

Next, the pentapeptides described in Table1 were analyzed for their immunologic potential as follows. PV-derived epitopes were retrieved from IEDB, and the epitopes that had been experimentally validated in the human host were analyzed for the presence of pentapeptides unique to PV1 (see Table 1).As reported in Table2, the search through IEDB produced a final list of 78 viral epitopes derived from PV1 Mahoney, PV3 Sabin, and PV3 (P3/LEON/37 and P3/LEON 12A (1)B). Epitopes derived from PV2 strains were not considered since only data from immunoassays in mice, rats, and/or rabbits were available in IEDB for PV2 strains at the time of this study. Following sequence analysis, it was found that 20 of the 78 epitopes (i.e., IEDB IDs: 31814, 48785, 58511, 59797, 71769, 79272, 79480, 99910, 100138, 100244, 100349, 100382, 100536, 100576, 100631, 100667, 100672, 146181, 146248, and 146390; see IEDB IDs in bold in Table 2) have a total of 12 viral pentapeptides that are absent in the human proteome (pentapeptides in capital letters in Table 2). That is, a first conclusion from Table 2 is that 12 out of the 164 pentapeptides unique to PV1, strain Mahoney, are part of 20 PV epitopic sequences endowed with an immunologic potential in the human host.Table 2
Twelve pentapeptides unique to PV1, Mahoney strain, and absent in the human proteome are distributed among twenty PV-derived epitopes recognized by human sera and/or T cells.
IEDB IDa
Epitope sequenceb,c
PV antigen
PV strain
Immune context
30661
kevpaltavetgat
VP1
PV3 Sabin
B
31814
klefftysRFDMEltfvvtan
VP1
PV1 Mahoney
T
46859
paltavetgatnpl
VP1
PV1 Mahoney
B-T
48785
ppgapvpeKWDDYTWQTssnp
VP1
PV1 Mahoney
T
55952
rsrsessiesf
VP1
PV1 Mahoney
B
58511
siFYTYGtaparisvpyvgi
VP1
PV1 Mahoney
T
59797
snAYSHFYDgfskvplkdqs
VP1
PV1 Mahoney
T
66978
tvdnsasttnkdklfavwk
Polyprotein
PV1 Mahoney
T
71769
vvndhnptkvtsKIRVYlkp
Polyprotein
PV1 Mahoney
T
79155
altlslpkqqdslpdtka
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
79160
atnplapsdtvqtrhvvq
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
79186
dneqpttraqklfam
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
79269
kevpaltavetgatnpla
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
79272
khvrVWCPRppravpyyg
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
79318
nghalnqvyqimyippga
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
79350
qklfamwritykdtv
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
79354
qpttraqklfamwri
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
79433
traqklfamwrityk
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
79434
traqklfamwritykdtv
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
79435
trhvvqrrsrsestiesf
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
79436
tskvriymkpkhvrvw
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
79443
vaiievdneqpttraqkl
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
79461
vrvvndhnptkvtskvri
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
79480
yippgaptpksWDDYTwq
Polyprotein
PV3 (P3/LEON/37 and P3/LEON 12A (1)B)
T
80446
argacvtimtvdnpa
VP1
PV1 Mahoney
B
81394
easgpthskeipalt
VP1
PV1 Mahoney
B
82831
gpthskeipaltave
VP1
PV1 Mahoney
B
83234
hskeipaltavetga
VP1
PV1 Mahoney
B
88446
sdtvqtrhvvqhrsr
VP1
PV1 Mahoney
B
88495
sessiesffargacv
VP1
PV1 Mahoney
B
99863
aaparisvpyvgla
Polyprotein
PV3 Sabin
B
99886
ahskevpaltavet
Polyprotein
PV3 Sabin
B
99901
arisvpyvglanay
Polyprotein
PV3 Sabin
B
99910
AYSHFYDgfakvpl
Polyprotein
PV3 Sabin
B
99933
dfgvlavrvvndhn
Polyprotein
PV3 Sabin
B
99963
dtvqlrrkleffty
Polyprotein
PV3 Sabin
B
100029
famwritykdtvql
Polyprotein
PV3 Sabin
B
100101
hfydgfakvplktd
Polyprotein
PV3 Sabin
B
100117
hnptkvtskvriym
Polyprotein
PV3 Sabin
B
100138
iFYTYGaaparisv
Polyprotein
PV3 Sabin
B
100244
lanAYSHFYDgfak
Polyprotein
PV3 Sabin
B
100349
npsiFYTYGaapar
Polyprotein
PV3 Sabin
B
100382
pkhvrVWCPRppra
Polyprotein
PV3 Sabin
B
100391
psdtvqtrhvvqrr
Polyprotein
PV3 Sabin
B
100425
qklfamwritykdt
Polyprotein
PV3 Sabin
B
100430
qlrrklefftysrf
Polyprotein
PV3 Sabin
B
100439
qpttraqklfamwr
Polyprotein
PV3 Sabin
B
100482
rppravpyygpgvd
Polyprotein
PV3 Sabin
B
100492
samtvddfgvlavr
Polyprotein
PV3 Sabin
B
100504
sevaqgaltlslpk
Polyprotein
PV3 Sabin
B
100536
svpyvglanAYSHF
Polyprotein
PV3 Sabin
B
100559
tkvtskvriymkpk
Polyprotein
PV3 Sabin
B
100573
traqklfamwrity
Polyprotein
PV3 Sabin
B
100575
tskvriymkpkhvr
Polyprotein
PV3 Sabin
B
100576
tssnpsiFYTYGaa
Polyprotein
PV3 Sabin
B
100580
tvddfgvlavrvvn
Polyprotein
PV3 Sabin
B
100583
tvqtrhvvqrrsrs
Polyprotein
PV3 Sabin
B
100585
twqtssnpsifyty
Polyprotein
PV3 Sabin
B
100586
tygaaparisvpyv
Polyprotein
PV3 Sabin
B
100587
tykdtvqlrrklef
Polyprotein
PV3 Sabin
B
100613
vlavrvvndhnptk
Polyprotein
PV3 Sabin
B
100619
vndhnptkvtskvr
Polyprotein
PV3 Sabin
B
100628
vriymkpkhvrvwc
Polyprotein
PV3 Sabin
B
100630
vrvvndhnptkvts
Polyprotein
PV3 Sabin
B
100631
vrVWCPRppravpy
Polyprotein
PV3 Sabin
B
100638
wcprppravpyygp
Polyprotein
PV3 Sabin
B
100644
writykdtvqlrrk
Polyprotein
PV3 Sabin
B
100667
ymkpkhvrVWCPRp
Polyprotein
PV3 Sabin
B
100672
yvglanAYSHFYDg
Polyprotein
PV3 Sabin
B
146178
ayappgaqpptsrk
Polyprotein
PV3 Sabin
B
146181
cgSMMATGkilvay
Polyprotein
PV3 Sabin
B
146248
flfcgSMMATGkil
Polyprotein
PV3 Sabin
B
146311
hqgalgvfaipeyc
Polyprotein
PV3 Sabin
B
146333
ilvayappgaqppt
Polyprotein
PV3 Sabin
B
146390
kftflfcgSMMATG
Polyprotein
PV3 Sabin
B
146494
phqiinlrtnnsat
Polyprotein
PV3 Sabin
B
146496
ppgaqpptsrkeam
Polyprotein
PV3 Sabin
B
146516
ravpyygpgvdyrn
Polyprotein
PV3 Sabin
B
aPV-derived epitopes are listed according to increasing IEDB ID number. Further details and reference(s) on each IEDB ID are reported at http://www.immuneepitope.org [17].
bOnly PV-derived epitopes that had been experimentally validated in the human host are reported.
cThe twenty PV-derived epitopes (and related IDs) containing PV-pentapeptide(s) absent in the human proteome are in bold, with the pentapeptide(s) absent in the human proteome given in capital.Moreover, it can be seen that the 12 unique PV1 pentapeptides are not distributed at random among the PV-derived epitopes. For example, three unique pentapeptides (KWDDY, WDDYT, and YTWQT) overlap each other in the epitope IEDB ID 48785, sequence ppgapvpeKWDDYTWQTssnp (with unique overlapping pentapeptides in capital); the heptapeptide AYSHFYD, formed by three overlapping unique pentapeptides shifted by one residue, characterizes four PV-derived epitopes (IEDB IDs: 59797, 99910, 100244, and 100672); the hexapeptide SMMATG formed by two overlapped 5-mers is present in three epitopes (IEDB IDs: 146181, 146248, and 146390).Theoretically, vaccines based on such PV epitopic peptides (i.e., KWDDYTWQT, AYSHFYD, and SMMATG) might evoke highly specific anti-PV immune responses exempt of possible collateral cross-reactions in the human host.


## 3.3. Identification of Consensus Pentapeptides Unique to PVs and Endowed with Immunologic Potential

We reasoned that using epitopic peptides unique to the virus and conserved among PV strains might help develop a global anti-PV peptide-based vaccination protocol. Such an approach would be of special importance in providing an effective and wide coverage to the human population worldwide, thus allowing reaching the goal of PV eradication [29, 30]. To this aim, we searched for unique conserved sequences by analysing a set of 43 PV polyproteins selected as described under Section 2 and comprehending also PV variants isolated from faeces of VAPP or AFP or from immunocompromised patient(s) with residual paralysis. The 43 PV polyprotein sequences were aligned using multialignment ClustalW program [24] and the peptide sequences present in PV-derived epitopes (see Table 2) and absent in the human proteome were localized.Table3 shows that seven potentially immunogenic peptides (in the order PV465–469MMATG, PV708–712RFDME, PV752–756DYTWQT, PV763–767FYTYG, PV783–789AYSHFYD, PV835–839KIRVY, and PV847–851VWCPR), derived from epitopes corresponding to (or formed of) pentapeptides unique to PV1 (see Tables 1 and 2), have 100% conservation among the 43 PV strains/variants under analysis. The seven potentially immunogenic unique peptide sequences had the same level of conservativeness in PV2 derived strains (data not shown).Table 3
Conservativeness  of epitopic PV peptide regions among 43 PV strains and variants.
PV strain
UniProt/Swiss-Prot entry
aa Pos 464
aa Pos 708
aa Pos  748
aa Pos 763
aa Pos783
aa Pos 835
aa Pos 847
PV1 Mahoney
P03300
FMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1 Sabin
P03301
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV3 P3/Leon/37
P03302
SMMATG
RFDME
sWDDYTWQT
FYTYG
AYSHFYD
KvRiY
VWCPR
PV3 23127
P06209
SMMATG
RFDME
sWDDYTWQT
FYTYG
AYSHFYD
KvRvY
VWCPR
PV1 isolated
Q9Q281
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KvRVY
VWCPR
PV1 isolated
Q9Q280
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1 isolated
Q71AZ9
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1 isolated
Q5TLH5
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-HAI01008C2
D1YSI9
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-HAI01009
D1YSJ1
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-HAI01008
D1YSJ2
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-HAI01002
D1YSJ3
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-HAI01001
D1YSJ4
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR01012
D1YSJ5
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR01002C
D1YSJ6
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR01002
D1YSJ7
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR01001C1
D1YSJ9
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR00042C2
D1YSK1
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR00042C1
D1YSK2
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR00042
D1YSK3
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR00041C3
D1YSK4
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR00041C2
D1YSK5
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR00044
D1YSK6
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR00028C
D1YSK7
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR00028
D1YSK8
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR00023C
D1YSK9
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR00025
D1YSL0
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOOR24
D1YSL1
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR00015
D1YSL2
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-DOR00016
D1YSL3
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-HAI01013all
D1YSL4
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-HAI01015
D1YSL5
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV1-S302
D2X673
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
CHN8184/GZ/CHN/2004
D8L541
SMMATG
RFDME
KWDDYTWQT
FYTYG
AYSHFYD
KIRVY
VWCPR
PV3 isolated
B4YUL3
SMMATG
RFDME
sWDDYTWQT
FYTYG
AYSHFYD
KvRVY
VWCPR
PV3 isolated
B4YUL4
SMMATG
RFDME
sWDDYTWQT
FYTYG
AYSHFYD
KvRVY
VWCPR
PV3 (vacc.StrainSabin3(Leon 12a1b))
Q84792
SMMATG
RFDME
sWDDYTWQT
FYTYG
AYSHFYD
KvRiY
VWCPR
PV3-33239
C5HJY2
SMMATG
RFDME
sWDDYTWQT
FYTYG
AYSHFYD
KvRiY
VWCPR
PV3-31974
C5HJY3
SMMATG
RFDME
sWDDYTWQT
FYTYG
AYSHFYD
KvRiY
VWCPR
PV3-FIN84-60212
D1GE40
SMMATG
RFDME
sWDDYTWQT
FYTYG
AYSHFYD
KvRiY
VWCPR
PV3-SWI10947
D2E679
SMMATG
RFDME
sWDDYTWQT
FYTYG
AYSHFYD
KvRiY
VWCPR
PV3-FIN84-2493
D1GE41
SMMATG
RFDME
sWDDYTWQT
FYTYG
AYSHFYD
KvRiY
VWCPR
PV3-P3/Jinan/1/09
D2XUS9
SMMATG
RFDME
sWDDYTWQT
FYTYG
AYSHFYD
KvRiY
VWCPR
The 43 PV sequences were aligned using ClustalW multialignment program (http://www.uniprot.org/align/) [24]. The analyzed PV sequences and related references are described at http://www.uniprot.org/ and reported by Swiss-Prot/UniProtKB accession number. Peptide sequences present in PV-derived epitopes (see Table 2) and absent in the human proteome were localized along the 43 aligned PV sequences and analyzed for conservativeness. Peptide sequences are indicated by their position along the PV polyprotein. Mutated aa residues are in lower case.


## 4. Conclusion

Anti-PV immunization has been one of the major public health measures of the last century. International campaigns to eliminate polio reduced the incidence of this disease in the world. However, problematic issues remain, such as the tendency of the PV genome to mutate, the potential risk to develop postvaccine paralytic poliomyelitis, and the difficulty to completely eradicate PV infection in the world. In fact, according to the World Health Organization, in 2012, still three countries in the world remain polio-endemic: Nigeria, Pakistan, and Afghanistan [31].The present data propose the concept of sequence uniqueness as a tool to define specific immunotherapies exempt of collateral effects [22] and describe a methodology to identify PV peptides that have zero percent identity to human proteins, are endowed with an immunologic potential, and are highly conserved among PV strains (e.g., PV465–469MMATG, PV708–712RFDME, PV752–756DYTWQT, PV763–767FYTYG, PV783–789AYSHFYD, PV835–839KIRVY, and PV847–851VWCPR; see Table 3). Importantly, polio peptide sequences alternate through the human proteome with a frequency versus rarity pattern that characterizes other pathogens too [32–35].Theoretically, such viral consensus epitopic peptides appear to be ideal tools to generate anti-PV immune responses promising of high specificity, thus avoiding serological cross-reactivity between human polyomaviruses [23], as well as possible cross-reactions with the human host [22]. As an example, a construct composed of the coding frames corresponding to the immunogenic consensus sequences described above (Table 2) might determine a specific anti-PV immune response and, at the same time, by being based on peptides, might eliminate the issues inherent to the tendency of the PV genome to mutate (i.e., VAPP). Such viral peptide sequences might also be used in passive anti-PV immunotherapies, that is, to produce specific antibodies capable of reacting with intact viral protein antigens.Actually, the present report is intended to represent a first approach to preclinical and animal studies. As a matter of fact, the solidity of a large body of theoretical andin silico data is the mandatory basis to designin vivo experimentation and validation protocols especially when considering that (i) although monkeys can be experimentally infected, humans are the only known natural hosts of poliovirus; (ii) small animal models for testing polio pathogenesis mainly relate to transgenic mice to express a human receptor to poliovirus [36]; and, moreover, (iii) current laws onin vivo experimentation are increasingly restrictive.



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*Source: 541282-2015-10-19.xml*