Analysis of the Amino Acid Sequence Variation of the 67–72p Protein and the Structural Pili Proteins of  for their Suitability as Potential Vaccine Antigens

MHC II
Target protein	Alleles	Start	End	Peptide	Percentile rank
67–72p	HLA-DRB3^*01:01	660	674	DGSVDLYEFDENDPV	0.01
67–72p	HLA-DRB3^*01:01	713	727	MLARYHVDDARDFFT	0.01
67–72p	HLA-DPA1^03:01/DPB1^04:02	15	29	PQRRLTWLIPLLMIL	0.01
67–72p	HLA-DPA1^03:01/DPB1^04:02	173	187	STFSVLLVVAFLIAL	0.01
67–72p	HLA-DRB1^*07:01	49	63	VDFRGVFNKVIATRI	0.01
67–72p	HLA-DPA1^01/DPB1^04:01	165	179	LPALRLVVSTFSVLL	0.01
67–72p	HLA-DPA1^01/DPB1^04:01	267	281	VISAVVAISFFSVIV	0.01
67–72p	HLA-DRB1^*09:01	100	114	PVVQYRAAVEKGVHR	0.02
67–72p	HLA-DRB3^*01:01	712	726	KMLARYHVDDARDFF	0.02
67–72p	HLA-DPA1^01:03/DPB1^02:01	172	186	VSTFSVLLVVAFLIA	0.02
SapD	HLA-DRB3^*01:01	181	195	GKDSIPEHLDKNMYF	0.01
SapD	HLA-DRB1^*03:01	531	545	PLHISYLVGDATIAR	0.03
SapD	HLA-DQA1^04:01/DQB1^04:02	535	549	SYLVGDATIARAKEI	0.09
SapD	HLA-DRB1^*03:01	444	458	PSDALLPDSKMTVSL	0.12
SapD	HLA-DQA1^03:01/DQB1^03:02	616	630	VQDEAVTTAAEWQEL	0.13
SapD	HLA-DRB1^*03:01	442	456	DLPSDALLPDSKMTV	0.13
SapD	HLA-DQA1^03:01/DQB1^03:02	615	629	DVQDEAVTTAAEWQE	0.16
SapD	HLA-DRB5^*01:01	640	654	LLGILGIVGAFVLFR	0.24
SapD	HLA-DQA1^04:01/DQB1^04:02	537	551	LVGDATIARAKEILA	0.27
SapD	HLA-DRB1^*03:01	346	360	TGTPKTIINDGHMDL	0.29
SpaC	HLA-DRB3^*01:01	157	171	NDIDRGIKYDAVYFI	0.01
SpaC	HLA-DRB3^*01:01	505	519	DNGTYRFKADTDAFK	0.01
SpaC	HLA-DRB3^*01:01	1427	1441	EHSVDPWLLNTTYSI	0.01
SpaC	HLA-DRB3^*01:01	1699	1713	VVINNVYTTDAEINI	0.01
SpaC	HLA-DRB3^*01:01	1804	1818	EVTLDNYDADSGLIT	0.01
SpaC	HLA-DRB3^*01:01	1450	1464	IKDRSYSNDVDIQAD	0.02
SpaC	HLA-DRB1^*03:01	769	783	AGDIVKVVVDNTAKR	0.03
SpaC	HLA-DRB1^*09:01	1845	1859	NGYLRWLLAGAAGLL	0.04
SpaC	HLA-DPA1^03:01/DPB1^04:02	21	35	LAMVMSIVLVPLIAA	0.05
SpaC	HLA-DRB3^*01:01	1432	1446	PWLLNTTYSITYKCD	0.07
SpaI	HLA-DRB3^*01:01	147	161	RPAEYRRTLDFLITV	0.07
SpaI	HLA-DRB1^*08:02	3	17	KTHLFRIPAATTAAV	0.18
SpaI	HLA-DRB3^*01:01	151	165	YRRTLDFLITVPAGM	0.19
SpaI	HLA-DPA1^01/DPB1^04:01	248	262	LGIALAIAGFLVQRR	0.28
SpaI	HLA-DRB3^*01:01	43	57	ISDIRCDTGSLTLIK	0.29
SpaI	HLA-DRB5^*01:01	155	169	LDFLITVPAGMRTAD	0.42
SpaI	HLA-DRB1^*09:01	95	109	AGWDAAKALTIQEAR	0.44
SpaI	HLA-DRB1^*11:01	50	64	TGSLTLIKRPPAAFE	0.44
SpaI	HLA-DQA1^01:02/DQB1^06:02	242	256	VLGIAALGIALAIAG	0.48
SpaI	HLA-DPA1^02:01/DPB1^05:01	150	164	EYRRTLDFLITVPAG	0.6

Table II Primers used in this study.

Gene	Primer	Sequence	Length of the amplified fragment
67–72p	6772p1L	TGAAAAATAATTTAAGGAGTTCCAA	695 bp
	6772p1R	CAACCCACCAGTAACAGCAA	695 bp
	6772p2L	CTGTTTTGCTGGTCGTAGCA	841 bp
	6772p2R	ACCTCATCAACCTGGTTTGC	841 bp
	6772p3L	GAATCGTTGCAGCCCAAG	699 bp
	6772p3R	CCTTAAGCACTGGGTCGTTT	699 bp
	6772p4L	CACCGACAACGTTGGTTACA	844 bp
	6772p4R	TTCTGGCTTGTCCCTGTTCT	844 bp
	6772p5L	TCAAGCCGGAGTCCCAGA	692 bp
	6772p5R	TCAGTTGTGTCTGGTGAAAGG	692 bp
spaI	SpaI1L	GCGGAATCAACACCAACAC	600 bp
	SpaI1R	AAGCGCTTACGATCCAAGAA	600 bp
	SpaI2L	ACACGGCCTTCCAAACTTC	482 bp
	SpaI2R	TGATATTGAGGCGTCGCTAA	482 bp
sapD	SapD1L	TCGCGAAGGTAAGAAATACTCA	698 bp
	SapD1R	CGTTTGTATCCGAGCCACTT	698 bp
	SapD2L	GTCCAAAACAAGAGCGGAAA	814 bp
	SapD2R	GGTTCAGTGAAAACCCAGTTG	814 bp
spaC	SpaC1L	GCCTACTCTCACTGGCAAGG	824 bp
	SpaC1R	ACATGGCGATCTCCTGAAGT	824 bp
	SpaC2L	TCGTGCAGGACGTACCAATA	838 bp
	SpaC2R	AACTGCACTGTGACCGAAAA	838 bp
	SpaC3L	GGCATCATAAAGTGCAATCG	808 bp
	SpaC3R	TCACGTTGAGTTCTTCGTTCA	808 bp
	SpaC4L	CATTCGTTTTTGTTCCGTGA	850 bp
	SpaC4R	GGTGTAGAAACGCCTCGAAA	850 bp
	SpaC5L	CCAAATTCAACAGTTTGATTATCACT	850 bp
	SpaC5R	TTCCTGTCACTTACACCTGTCG	850 bp
	SpaC6L	CAAAATACGGATTGGTTTCTGG	845 bp
	SpaC6R	AGCTGGCTGGAATTTCGAT	845 bp
	SpaC7L	CAAAGGTGTCTTGGCCATTT	687 bp
	SpaC7R	TCACGCCAGTAAGTCTTGCTAA	687 bp
	SpaC8L	CTGGCATCTGGATGTCATTG	578 bp
	SpaC8R	ACCGAACGTGCCTAGCGTA	578 bp

B-cell epitopes predicted from the target proteins.

Target protein	No.	Start	End	Peptide	Length
6772p	1	4	20	GFTRPAAAPKRPQRRLT	17
6772p	2	48	53	EVDFRG	6
6772p	3	87	111	GRPDELEFFDPDSPVVQYRAAVEKG	25
6772p	4	143	156	NRQDFGVSDQQFGM	14
6772p	5	194	211	GGIRAGNQAAGVKGSITN	18
6772p	6	281	287	VTKDLRI	7
6772p	7	316	329	SPNRAEKESEYISR	14
6772p	8	337	369	AYGITDDAVTYKDNWGAKGASSEKVASDSATVS	33
6772p	9	381	410	PTFTQQQQLRNFYGFPKSLAMDRYVIDGEL	30
6772p	10	421	434	DPNALKENQRDWIN	14
6772p	11	452	467	QVDEVARDVGSARGGY	16
6772p	12	474	490	DLQTTDKEAQELGIVVK	17
6772p	13	498	507	PVIASATDGA	10
6772p	14	514	541	SENDSSVEYDTDSSTYTYQGKGGVNIGN	28
6772p	15	562	566	RVNGN	5
6772p	16	573	581	RDPRERVHN	9
6772p	17	612	645	TSLPYAERTSLSEATNDTTAQVGNSAQRLVTDNV	34
6772p	18	680	705	GVFPGTVKAKSEISEELMNHLRYPED	26
6772p	19	714	749	LARYHVDDARDFFTNDRFWSVPSDPSATEGQKDVAQ	36
6772p	20	760	763	DTGK	4
6772p	21	773	777	RGLQR	5
6772p	22	803	837	TDTLTQGPKQAQDTMMSSDQIASDRTLWKDTNDLF	35
6772p	23	861	868	RKNQASAF	8
6772p	24	896	944	GIDPKEAQDLGEAKGLKPESQNRDKPEDKEGKAPSTPSAPASGSGTTGE	49
6772p	25	956	976	LQSAKNGSNEEYGRALDELDK	21
SpaC	1	38	49	ANAEPLPKKEFE	12
SpaC	2	64	69	SLSASD	6
SpaC	3	100	113	SPAAGNKNFTPVSL	14
SpaC	4	131	146	MPAIRENKKGSPNGGT	16
SpaC	5	176	184	PTWDNNGRN	9
SpaC	6	225	228	PFDL	4
SpaC	7	231	234	PILP	4
SpaC	8	246	254	WKIDRSLTG	9
SpaC	9	324	332	PSIETDKNG	9
SpaC	10	355	358	TGDQ	4
SpaC	11	371	387	RYSYGQAPTDIPIKTSD	17
SpaC	12	417	432	KVNVNTPQLLEELNNQ	16
SpaC	13	455	468	GVHNGESKEIGKVA	14
SpaC	14	478	507	VTPKVDDSRMKLSTTWSSENTTADANQDNG	30
SpaC	15	512	522	KADTDAFKNKK	11
SpaC	16	531	537	NYEAQTA	7
SpaC	17	545	561	IINRDKIPATKLPEKFP	17
SpaC	18	569	591	VPHPNARPEHGGLPETNPYFVDS	23
SpaC	19	601	610	SIEIGPFPVG	10
SpaC	20	619	659	ARLDPNVQADAKIPGFSLKTEWNSNICFGNTIDNNSQDCST	41
SpaC	21	664	672	IPKPGQYSL	9
SpaC	22	676	684	NTYTRELAS	9
SpaC	23	690	702	TVSGDASDLTNSH	13
SpaC	24	712	731	DSGVEVYSQDNIVVKKDGRQ	20
SpaC	25	746	754	EKQPEQKGV	9
SpaC	26	761	769	PFHLRASTA	9
SpaC	27	779	786	NTAKRQVA	8
SpaC	28	792	812	KKVHKKDTFSPEISASIDALT	21
SpaC	29	819	846	CTVPGVETPRKVLKTVSDNQTVEFGNFP	28
SpaC	30	857	861	TEAPA	5
SpaC	31	881	885	TPINK	5
SpaC	32	891	895	FENAR	5
SpaC	33	904	948	VLDGDMPQALVDQIPSSFTVNVACSITGNHSITLQKDEQKAVPGV	45
SpaC	34	957	968	SEEVTPITGATH	12
SpaC	35	971	991	HWIKGELLEVADSTDITINPN	21
SpaC	36	1001	1007	HYETDAV	7
SpaC	37	1012	1037	TKRVRVIDQVGNDVNSELKNAVVRPE	26
SpaC	38	1043	1052	RYRCEINGQV	10
SpaC	39	1059	1073	SADAINTGATKVPRG	15
SpaC	40	1079	1131	EEDSSSVELSNATLSHVEFFVHGTKTNDKASVAINSDHNRLDATNTFTLKTGS	53
SpaC	41	1135	1146	KKKVDGEGVSTI	12
SpaC	42	1157	1164	RCTLGDWK	8
SpaC	43	1174	1188	FDSAESHSVKDIPVG	15
SpaC	44	1195	1204	EDSEKAQEPN	10
SpaC	45	1210	1240	RWTHTDSTNGWGDTEAACENHAACEVDPKNE	31
SpaC	46	1250	1255	NEKENF	6
SpaC	47	1276	1288	KVLTNDGPELAGK	13
SpaC	48	1298	1346	TDPRFAGSDLADKHSIPDPTITVALNAKGQSRASYQVADERHDSVEVPV	49
SpaC	49	1357	1360	IALY	4
SpaC	50	1378	1401	AVQRTSSNSASARFVTEKQENNGT	24
SpaC	51	1409	1413	DYIRP	5
SpaC	52	1424	1437	AKPEHSVDPWLLNT	14
SpaC	53	1443	1483	YKCDDPYIKDRSYSNDVDIQADAEKPTPIFADPTAHVKIPA	41
SpaC	54	1492	1498	NTEGHLP	7
SpaC	55	1506	1555	DETNKVAEFAGEHEKRSYFTPEIKDVVLSESEPTRIEFTNSYVMPQRILS	50
SpaC	56	1560	1569	VEGDPGHAVI	10
SpaC	57	1582	1605	TMPHLFPNQPNPMSQEVGNKVARG	24
SpaC	58	1614	1622	TWRSPEVPI	9
SpaC	59	1630	1643	EEDDPALRTKLENN	14
SpaC	60	1645	1687	LRMVPTYLFPTERAGAASAPVIPPLTDRPIYNGTEPRLQMPES	43
SpaC	61	1718	1723	ADNSPL	6
SpaC	62	1734	1755	GENGQRKELPEVADAPAKSAKS	22
SpaC	63	1808	1825	DNYDADSGLITVEHPQGK	18
SpaC	64	1837	1842	STLPLT	6
SapD	1	23	72	PVSASEDAALDATGHKKGEPAFGVTIPKGTTYRDSDGKEVPHPCVDRKIG	50
SapD	2	86	96	YSVKEPATDLP	11
SapD	3	104	113	DGQQVVPQES	10
SapD	4	122	145	AGEDGEELSRIRIPDDEEFSFLGK	24
SapD	5	157	162	IPFANG	6
SapD	6	174	190	DPHHEPKGKDSIPEHLD	17
SapD	7	224	234	SNDEELKTIEY	11
SapD	8	264	269	AFKVKT	6
SapD	9	281	350	DEEVGLPEGTTTNLNKITKPLDKDATNEPPTDPSEKKKPPRPEKGHSETSSPSA LDDSIERAWKLTGTPK	70
SapD	10	371	380	TVINREGKKY	10
SapD	11	392	418	SGGDQGGPLVKTDSWKDRIEAQISGSL	27
SapD	12	441	451	EDLPSDALLPD	11
SapD	13	525	529	GKQES	5
SapD	14	542	606	TIARAKEILAGEKLGGSLKKKPQEKETKKPASVQNKSGKHNKDTVGSESARK RQQLAATSGSDTN	65
SapD	15	624	632	AAEWQELDT	9
SpaI	1	22	50	ASGPIASADSRTITGATDGLNISDIRCDT	29
SpaI	2	55	75	LIKRPPAAFEGVDKADLPAGT	21
SpaI	3	86	124	IEGIDLTKQAGWDAAKALTIQEARSRLSDEVWKAVSGRD	39
SpaI	4	144	153	PAKRPAEYRR	10
SpaI	5	166	174	RTADGNVAS	9
SpaI	6	186	242	TDDLPPTVPVFPPVESSVTLTPSSPVPGTPKTPGKPDLPEKFRKEVTDRLGNT GANV	57
SpaI	7	263	266	KKNE	4

Comparison of amino acid sequences of all strains tested against the reference strain, given in percent (%).

Target protein	27E	40E	68E	71E	73E	77E	78E	79E	86E	89E
67–72p	99	100	100	100	100	100	100	98	98	100
SapD	97	99	100	100	100	100	100	100	99	99
SpaI	−	100	100	100	100	100	100	84	100	100
SpaC	48	99	99	99	69	99	69	69	69	51

Number of alleles for which epitopes were found in the proteins tested.

Target protein	Number of MHC alleles of class I (per 40)	% Bound alleles MHC class I	Number of MHC alleles of class II (per 51)	% Bound alleles MHC class II
67–72p	40	100	51	100
SpaC	37	92.5	51	100
SpaI	34	85	50	98
SapD	33	82.5	51	100

C. diphtheriae strains used in this study.

Strain	Biotype	Site of isolation	Year of isolation
27/E	mitis	Serous cyst contents	2010
40/E	gravis	Blood	2014
68/E	gravis	Endocarditis	2015
71/E	gravis	Wound	2015
73/E	gravis	Blood	2016
77/E	gravis	Wound	2016
78/E	gravis	Blood	2016
79/E	gravis	Blood and joint fluid	2016
86/E	gravis	Blood	2017
89/E	gravis	Wound	2017

Comparison of the nucleotide sequences of all strains tested against the reference strain, given in percent (%). The sequences are presented according to the analysed fragments.

Target protein	Fragment	27E	40E	68E	71E	73E	77E	78E	79E	86E	89E
67–72p	1	98.35	100	100	100	100	100	100	100	100	100
	2	99.5	100	100	100	100	100	100	100	100	99.87
	3	100	100	100	100	100	100	100	100	100	100
	4	99.01	100	100	100	100	100	100	100	97.90	97.90
	5	100	100	100	100	100	100	100	100	100	100
SapD	1	98.91	100	100	100	100	100	100	100	100	100
SapD	2	98.37	27.52	100	100	100	100	100	100	99.86	99.86
SpaI	1	−	100	100	100	100	100	100	62.77	100	100
SpaI	2	−	100	100	100	100	100	100	100	100	100
SpaC	1	72.78	99.87	99.87	99.87	99.87	99.87	99.87	99.87	99.87	99.87
	2	97.11	100	100	100	100	100	100	100	100	100
	3	−	100	100	100	100	100	100	100	100	100
	4	−	100	100	100	100	100	100	100	99.75	IS^*
	5	91.47	99.21	100	100	99.87	100	100	100	100	100
	6	−	100	100	100	100	100	99.87	99.87	100	100
	7	50.16	100	100	100	100	100	100	100	100	100
	8	−	100	100	100	100	100	100	100	100	100

Prediction of the protective antigens from the VaxiJen server.

Protein	Overall Prediction for the Antigen
6772p	0.5123
SpaC	0.6757
SpaI	0.5504
SapD	0.5544

Extracellular regions of individual proteins.

Region^*	67–72p				SpaC	SpaI	SapD
Start	44	138	235	310	36	1	1
Stop	57	169	257	987	1845	236	631

MHC class I epitopes predicted from the target proteins.

MHC I
Target protein	Alleles	Start	End	Peptide	Percentile rank
67–72p	HLA-A^*02:06	6	14	FTNDRFWSV	0.06
67–72p	HLA-B^*44:02	34	42	SENDSSVEY	0.06
67–72p	HLA-A^*30:02	68	77	RMASYWLDRY	0.06
67–72p	HLA-B^*44:02	9	17	AEALSQVGI	0.07
67–72p	HLA-A^*02:06	27	36	MILGALVPTV	0.07
67–72p	HLA-A^*68:01	1	9	YAFTLPALR	0.11
67–72p	HLA-A^*01:01	43	51	DTDSSTYTY	0.11
67–72p	HLA-A^*01:01	48	56	YTTLTSLPY	0.11
67–72p	HLA-B^*44:03	34	42	SENDSSVEY	0.11
67–72p	HLA-B^*57:01	58	66	SSLAIGNAW	0.12
SapD	HLA-B^*44:03	65	74	AEWQELDTWW	0.06
SapD	HLA-B^*07:02	4	13	RPIWAGIGAF	0.11
SapD	HLA-B^*44:03	28	36	KEGAYGLEY	0.11
SapD	HLA-A^*68:01	47	55	NVFFKNNSR	0.12
SapD	HLA-B^*40:01	10	18	IEAQISGSL	0.17
SapD	HLA-A^*24:02	70	79	VWYAPQNIPF	0.18
SapD	HLA-A^*68:01	24	32	DTVGSESAR	0.2
SapD	HLA-B^*51:01	50	58	YPLHISYLV	0.2
SapD	HLA-A^*68:01	41	50	EPAFGVTIPK	0.22
SapD	HLA-A^*68:01	17	26	EAYVKNGAFK	0.26
SpaC	HLA-A^*11:01	658	666	STNSVWIPK	0.06
SpaC	HLA-A^*01:01	220	229	LSDDKPFDLY	0.07
SpaC	HLA-B^*53:01	233	241	LPSEDDYYW	0.1
SpaC	HLA-A^*68:02	191	199	EVVELENAV	0.1
SpaC	HLA-A^*02:06	1859	1867	LVAAALWLV	0.11
SpaC	HLA-A^*23:01	88	96	PYRFGIYTF	0.11
SpaC	HLA-A^*68:01	1436	1444	NTTYSITYK	0.11
SpaC	HLA-A^*31:01	363	371	RFKNARCQR	0.11
SpaC	HLA-B^*44:02	1054	1063	AENTLSADAI	0.11
SpaC	HLA-A^*23:01	1578	1587	SYTCTMPHLF	0.12
SpaI	HLA-A^*30:01	2	11	KKTHLFRIPA	0.08
SpaI	HLA-B^*07:02	9	17	IPAATTAAV	0.1
SpaI	HLA-B^*07:02	147	155	RPAEYRRTL	0.1
SpaI	HLA-B^*57:01	109	117	RSRLSDEVW	0.12
SpaI	HLA-A^*30:02	129	137	VTGLPMGVY	0.18
SpaI	HLA-A^*02:01	137	145	YLVSETPPA	0.2
SpaI	HLA-A^*02:03	20	29	LLASGPIASA	0.2
SpaI	HLA-A^*02:06	153	161	RTLDFLITV	0.21
SpaI	HLA-B^*51:01	196	205	FPPVESSVTL	0.24
SpaI	HLA-A^*68:01	252	261	LAIAGFLVQR	0.32

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Analysis of the Amino Acid Sequence Variation of the 67–72p Protein and the Structural Pili Proteins of Corynebacterium diphtheriae for their Suitability as Potential Vaccine Antigens

Article Category: original-paper

Online veröffentlicht: 28. Juni 2019

Seitenbereich: 233 - 246

Eingereicht: 04. Dez. 2018

Akzeptiert: 09. März 2019

DOI: https://doi.org/10.33073/pjm-2019-025

Schlüsselwörternon-toxigenic, pili, 67–72p protein, vaccine

© 2019 Klaudia Brodzik et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

MHC class II epitopes predicted from the target proteins.

Table II Primers used in this study.

B-cell epitopes predicted from the target proteins.

Comparison of amino acid sequences of all strains tested against the reference strain, given in percent (%).

Number of alleles for which epitopes were found in the proteins tested.

C. diphtheriae strains used in this study.

Comparison of the nucleotide sequences of all strains tested against the reference strain, given in percent (%). The sequences are presented according to the analysed fragments.

Prediction of the protective antigens from the VaxiJen server.

Extracellular regions of individual proteins.

MHC class I epitopes predicted from the target proteins.

Schlüsselwörter
non-toxigenic, pili, 67–72p protein, vaccine