Karoly Szuhai
email: k.szuhai@lumc.nl.

Ivy Jennes

Danielle De Jong

Judith V M G Bovee

Malgorzata Wiweger

Wim Wuyts

C W Pancras

Hogendoorn

Károly Szuhai

Judith V M G Bovée

# Pancras

C W Hogendoorn

MD Karoly Szuhai

Tiling resolution array-CGH shows that somatic mosaic deletion of the EXT gene is causative in EXT gene mutation negative multiple osteochondromas patients

Keywords: somatic mosaicism, array-CGH, multiple osteochondroma, bone tumors, EXT1, EXT2, bone, multiple exostosis, FISH Somatic mosaicism, multiple osteochondroma, EXT1, EXT2, array-CGH, FISH, bone tumors, bone

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INTRODUCTION

Multiple osteochondromas (MO) (previously known as hereditary multiple exostosis) is characterized by cartilage capped bony outgrowths that typically occur at diaphyseal region of a bone. MO is an autosomal dominant disorder caused by mutations of the EXT1 (MIM ID *608177) or EXT2 genes (MIM ID *608210) [START_REF] Fletcher | Pathology and Genetics of Tumours of Soft Tissue and Bone[END_REF][START_REF] Ahn | Cloning of the putative tumour suppressor gene for hereditary multiple exostoses (EXT1)[END_REF][START_REF] Stickens | The EXT2 multiple exostoses gene defines a family of putative tumour suppressor genes[END_REF][START_REF] Wuyts | Positional cloning of a gene involved in hereditary multiple exostoses[END_REF]. Similarly, mutations of the EXT1 gene were detected in the cartilage cap of sporadic osteochondroma cases [START_REF] Bernard | Diminished levels of the putative tumor suppressor proteins EXT1 and EXT2 in exostosis chondrocytes[END_REF]. Loss of the wild-type allele in hereditary cases [START_REF] Bovee | EXT-mutation analysis and loss of heterozygosity in sporadic and hereditary osteochondromas and secondary chondrosarcomas[END_REF] and homozygous loss of both alleles in sporadic cases [START_REF] Hameetman | The role of EXT1 in nonhereditary osteochondroma: identification of homozygous deletions[END_REF] indicates that inactivation of both EXT alleles is required for osteochondroma formation, confirming its tumor suppressor action resulting in a loss of polarization of chondrocytes (de Andrea et al., 2010). The complexity of osteochondroma formation, however, is not yet completely clarified by the bi-allelic inactivation as mosaic distribution of cells with retained chromosome 8 regions (presumably normal cells) was observed in the cartilage cap of both human osteochondromas and animal model systems (de Andrea et al., 2010; [START_REF] Jones | A mouse model of osteochondromagenesis from clonal inactivation of Ext1 in chondrocytes[END_REF][START_REF] Bovee | Cartilage tumours and bone development: molecular pathology and possible therapeutic targets[END_REF] The majority of the hereditary cases (70-75%) are caused by point mutations resulting in truncated proteins [START_REF] Wuyts | Molecular basis of multiple exostoses: mutations in the EXT1 and EXT2 genes[END_REF][START_REF] Signori | A combined analytical approach reveals novel EXT1/2 gene mutations in a large cohort of Italian multiple osteochondromas patients[END_REF][START_REF] Lonie | Determination of the mutation spectrum of the EXT1/EXT2 genes in British Formatted: Space After: 12 pt Caucasian patients with multiple osteochondromas, and exclusion of six candidate genes in EXT negative cases[END_REF][START_REF] Pedrini | Novel EXT1 and EXT2 mutations identified by DHPLC in Italian patients with multiple osteochondromas[END_REF][START_REF] Jennes | Multiple osteochondromas: mutation update and description of the multiple osteochondromas mutation database (MOdb)[END_REF]. Deletions involving single or multiple exons were found in about 10 % of all hereditary cases using Multiplex Ligation-dependent Probe Amplification (MLPA) [START_REF] Jennes | Mutation screening of EXT1 and EXT2 by denaturing high-performance liquid chromatography, direct sequencing analysis, fluorescence in situ hybridization, and a new multiplex ligation-dependent probe amplification probe set in patients with multiple osteochondromas[END_REF][START_REF] Fredman | Complex SNP-related sequence variation in segmental genome duplications[END_REF][START_REF] Vink | Mutation screening of EXT1 and EXT2 by direct sequence analysis and MLPA in patients with multiple osteochondromas: splice site mutations and exonic deletions account for more than half of the mutations[END_REF]. However, in about 10-15 % of the MO cases, genomic alterations can not be detected implying the potential role of other alterations such as inversions, translocations or somatic mosaicism [START_REF] Vink | Mutation screening of EXT1 and EXT2 by direct sequence analysis and MLPA in patients with multiple osteochondromas: splice site mutations and exonic deletions account for more than half of the mutations[END_REF]. The involvement of other genes or the identification of the putative EXT3 gene still awaits investigation. Our goal was to possibly identify a novel gene responsible for the MO phenotype that would be involved in the heparan and chondroitin sulfate synthesis pathways. In order to detect these genomic alterations a custom-made, high densityoligonucleotide microarray was designed. In addition, high density coverage of all exons of the EXT1 and EXT2 genes facilitates the detection of partial deletions that could be outside of the single probe per exonic region type of MLPA detection system. Ultimately, this design would allow detection of mosaic deletions of any of the probed regions.

MATERIALS AND METHODS

Array-design

A custom-made Agilent oligonucleotide microarray containing 44000 reporter elements was (named further as: EXT-tiling array) designed using the Agilent E-array web tool service (named further as: EXT-tiling array). For oligonucleotide selection we used the probe pool provided by Agilent following recommended selection criteria for probe length, GC content and melting temperature (Tm). In addition to EXT1, EXT2, EXTL1, EXTL2, EXTL3 and several other genes involved in heparan sulfate and chondroitin sulfate biosynthesis pathways were selected. The list of selected gene is shown in Table1. For probe design genomic regions 5000 bps before the first and after last exon of each gene were selected. For each tested gene multiple probes were selected with the highest possible coverage of the coding exonic sequences. In case several genes of interest were clustered together in one chromosomal region adjacent genes were partially or in full length included (see the second and third part of the gene list in Table 1). For small exons exon-intron boundaries were also included for probe design. An average 500 bps interval was used for probe selection within intronic regions, with exceptions of large repeat blocks (such as LINE or SINE elements). For the EXT1 and EXT2 genes each probe set was printed in triplicate while for all other genes in duplicate. The reporter probe list and the array design are available upon request. ... [START_REF] Bernard | Diminished levels of the putative tumor suppressor proteins EXT1 and EXT2 in exostosis chondrocytes[END_REF] ... [START_REF] Bovee | Cartilage tumours and bone development: molecular pathology and possible therapeutic targets[END_REF] ... [START_REF] Ahn | Cloning of the putative tumour suppressor gene for hereditary multiple exostoses (EXT1)[END_REF] ... [START_REF] De Andrea | Primary cilia organization reflects polarity in the growth plate and implies loss of polarity and mosaicism in osteochondroma[END_REF] ... [25] ... [START_REF] Erickson | Somatic gene mutation and human disease other than cancer: an update[END_REF] ... [26] ... [START_REF] Fletcher | Pathology and Genetics of Tumours of Soft Tissue and Bone[END_REF] ... [27] ... [START_REF] Fredman | Complex SNP-related sequence variation in segmental genome duplications[END_REF] ... [28] ... [START_REF] Goobie | Molecular and clinical characterization of de novo and familial cases with microduplication 3q29: guidelines for copy number variation case reporting[END_REF] ... [29] ... [START_REF] Hameetman | The role of EXT1 in nonhereditary osteochondroma: identification of homozygous deletions[END_REF] ... [30] ... [START_REF] Jennes | Mutation screening of EXT1 and EXT2 by denaturing high-performance liquid chromatography, direct sequencing analysis, fluorescence in situ hybridization, and a new multiplex ligation-dependent probe amplification probe set in patients with multiple osteochondromas[END_REF] ... [31] ... [START_REF] Jennes | Multiple osteochondromas: mutation update and description of the multiple osteochondromas mutation database (MOdb)[END_REF] ... [32] ... [START_REF] Jones | A mouse model of osteochondromagenesis from clonal inactivation of Ext1 in chondrocytes[END_REF] ... [33] ... [START_REF] Lonie | Determination of the mutation spectrum of the EXT1/EXT2 genes in British Formatted: Space After: 12 pt Caucasian patients with multiple osteochondromas, and exclusion of six candidate genes in EXT negative cases[END_REF] ... [34] ... [START_REF] Male | Delineation of an estimated 6.7 MB candidate interval for an anophthalmia gene at 3q26.33-q28 and description of the syndrome associated with visible chromosome deletions of this region[END_REF] ... [35] ... [START_REF] Notini | Copy number variation and mosaicism[END_REF] ... [36] ... [START_REF] Pedrini | Novel EXT1 and EXT2 mutations identified by DHPLC in Italian patients with multiple osteochondromas[END_REF] ... [37] ... [START_REF] Signori | A combined analytical approach reveals novel EXT1/2 gene mutations in a large cohort of Italian multiple osteochondromas patients[END_REF] ... [38] ... [START_REF] Stickens | The EXT2 multiple exostoses gene defines a family of putative tumour suppressor genes[END_REF] ... [39] ... [START_REF] Vink | Mutation screening of EXT1 and EXT2 by direct sequence analysis and MLPA in patients with multiple osteochondromas: splice site mutations and exonic deletions account for more than half of the mutations[END_REF] . BOLD: genes related to extracellular matrix production/pathways with full coverage ITALIC: genes with full coverage located between the extracellular matrix related genes Other genes are with partial coverage at the border of the extracellular matrix related genes.

Samples:

Peripheral blood derived DNA samples of seventeen patients from a cohort of clinically well defined multiple osteochondroma patients (eight female, nine male). Mutation screening of the EXT1 or EXT2 genes using standard sequencing for all exons including exon-intron junction points and MLPA testing using exon dedicated probe set for each exon coding regions were normal as reported earlier [START_REF] Jennes | Mutation screening of EXT1 and EXT2 by denaturing high-performance liquid chromatography, direct sequencing analysis, fluorescence in situ hybridization, and a new multiplex ligation-dependent probe amplification probe set in patients with multiple osteochondromas[END_REF][START_REF] Wuyts | An optimized DHPLC protocol for molecular testing of the EXT1 and EXT2 genes in hereditary multiple osteochondromas[END_REF]. These mutation-negative patient samples were tested using the custom made EXT-tiling-array. As positive control, three samples with MLPA proven deletions in either EXT1 (54782: exon 2-exon 11 and 73585: exon 8) or EXT2 (84389: exon 8) were used (Table 2).

Labeling and hybridization protocols:

Labeling of the DNA samples was performed using a BioPrime total arrayCGH labeling kit (Invitrogen, Breda, The Netherlands) following the manufacturer's protocol. Array hybridizations, scanning, image and data processing were performed according to standard protocols recommended by Agilent (Agilent, Amstelveen, The Netherlands).

Data processing:

Scan-images were loaded into Feature extraction software package (Agilent). The generated raw data files were further processed using Genomic Workbench Standard Edition 5.0. In short, the mean of the background corrected, Lowess normalized log2 ratio values of identical features were calculated. Normalization of the log ratio values was performed using the overall values as well as the build in control reporter probe set values using factory defaults. Furthermore, reporters with <1.5x signal-to-background ratio were excluded from further analysis. If triplicate probes was available for calculation, reporter probes with >1SD of the mean was removed and the mean of the remaining two reporter elements was calculated. Values were then displayed by Moving average window of 3 points and aberrations were calculated using the ADM-2 algorithm with a threshold value of 11.

Confirmatory Fluorescence in situ Hybridization (FISH):

FISH analysis to confirm mosaic EXT1 deletion in sample 59872 was performed with EXT1 probes 46F10 and 90D8 [START_REF] Ahn | Cloning of the putative tumour suppressor gene for hereditary multiple exostoses (EXT1)[END_REF].

RESULTS:

We have designed a custom-made Agilent oligonucleotide-based microarray containing 44000 probes with a tiling coverage of EXT1/2 genes and additional 68 genes involved in heparan and chondroitin sulfate biosynthesis. This array platform has the potential to detect novel gene alterations involved in cartilaginous matrix formation as well as deletions involving any part of the EXT1 or EXT2 genes. The genomic DNA of 17 patient samples with confirmed clinico-pathological appearance of MO and no mutations in EXT1 and EXT2 when sequenced and MLPA tested were used in this study. By using the custom-made targeted array approach, deletions with variable sizes involving the EXT1 and the EXT2 genes were readily detected in the three control samples tested previously positive (Figure 1A and 1B)(Table 3). 1A) and low lever of deletion of EXT2 was detected in one patient (Figure 1B). Based on the level of deletion observed in the array-CGH profile the presence of a mosaic deletion was implicated and estimated to be present in about 10-15% of the blood cells for the patients with both EXT1 (83350: exon 2 to exon 3; 59872: exon 2 to exon 11) and EXT2 (84458: exon 2) mosaic deletions (Figure 1A andB). For sample 59872 material was available for FISH confirmation which showed presence of the EXT1 exon 1 probe 90D8 in all cells but a deletion of the exon 6-11 probe 46F10 in 7 out of 50 cells (14%). The percentage of cells, carrying these EXT gene deletions were below the detection threshold of MLPA testing and therefore were not detected previously.

No copy number alterations, involving the promoter region of EXT1 or EXT2, were detected in any of the tested samples.

By analyzing all other probes, targeted to members of the cartilaginous matrix synthesis pathway, genomic copy number alterations were observed neither in the translated coding sequences (exons) nor in the promoter regions. However, alterations of other genomic regions were observed involving 9 different genomic regions. One non-coding region of chromosome 6 showed a recurrent 3kb deletion in 4 of the 17 analyzed samples (1 in EXTarray negative, 1 mosaic deletion carrier, 2 positive control samples with different deletions involving the EXT1 gene). This region has been reported in the Database of Genomic Variants (DGV) (http://projects.tcag.ca/variation/). Three genomic regions have been detected with the involvement of intronic parts for NDST3, SUPT3H and HS6ST2. The NDST3 region has been reported in the DGV database while the other two have not (data not shown). The role of these detected alterations in the development of MO is questionable as they were present both in the EXT-deletion carriers positive control samples as well as EXT negative patients.

DISCUSSION

A disease causing germline mutation of either the EXT1 or EXT2 gene can be identified in more than 85% of the analyzed de novo MO cases and MO families (Wuyts et , 2009). For the remaining mutation or deletion negative cases several possibilities were proposed. Either the involvement of i. (an) additional gene(s) ii. partial exonic deletions that would not be detected by MLPA probes iii. positional changes like inversion, insertion or cryptic translocations causing neither copy number alterations nor sequence substitutions, iv. (epi)genetic changes influencing EXT1/2 promoter function, v. presence of somatic mosaic deletions were proposed [START_REF] Vink | Mutation screening of EXT1 and EXT2 by direct sequence analysis and MLPA in patients with multiple osteochondromas: splice site mutations and exonic deletions account for more than half of the mutations[END_REF].

The aim of this study was to develop a targeted custom-made oligonucleotide-based microarray containing ~44 thousand 60mer oligonucleotide reporters to detect hitherto undetected genomic alterations possibly responsible for the development of MO. For the design we used tiling resolution detection on both EXT1 and EXT2 genes including as many probes as possible to cover all exons and introns. This set up facilitates the detection of a partial exonic deletion that could have been unrevealed using the single MLPA probe per exon approach. To facilitate the detection of a candidate EXT3 gene alterations, additional genomic regions were interrogated by tiling probe resolution containing 68 genes that were selected based on the involvement in heparan or chondroitin sulfate biosynthesis.

By using a highly sensitive, custom-made chip we identified mosaic deletions of the EXT1 gene in two and of the EXT2 gene in one out of 17 analyzed MO patients with otherwise negative test results. For the first time somatic mosaicism with large genomic deletions as the underlying mechanism in MO formation was confirmed. As anticipated, none of the mosaic deletion cases were ascertained from the five familial cases (table 1). The presence of somatic and germ-line mosaicism was proved for case 83350, a patient with clinically established MO phenotype without detectable changes who had a daughter with clinically established MO with deletion of exon 2-3 of the EXT1 gene detected by MLPA and EXT-tiling array. Side-by-side comparison these two cases 83350 (father) and 83349 (daughter) showed identical breakpoint regions and the expected differences between the hemizygous and mosaic state (Figure 2). For the first time, we showed that the existence of mosaic mutations (large deletions) may play a significant role in the development of MO in patients who are negative for mutations in Exostosins when analyzed by sequencing and MLPA. In addition, the high probe coverage of the EXT1 and EXT2 genes permits a more precise detection of the size of a deletion in cases with similar exonic deletion features as detected by MLPA only (Table 3 and Figure 1).

In theory, partial gene duplication may result in a similar non-sense transcript, however, in our study none of the investigated genomic regions showed duplication of the EXT1/2 genes or in other genes. Based on this observation, it is unlikely that copy number neutral inversions could play a role in the other cases, especially as genomic inversions are often found to be associated with both by deletions and duplications [START_REF] Goobie | Molecular and clinical characterization of de novo and familial cases with microduplication 3q29: guidelines for copy number variation case reporting[END_REF][START_REF] Male | Delineation of an estimated 6.7 MB candidate interval for an anophthalmia gene at 3q26.33-q28 and description of the syndrome associated with visible chromosome deletions of this region[END_REF]. For the remaining deletion-and mutation-negative cases there are several possible explanations why pathogenic alterations could not be detected. Other genetic or epigenetic alterations without detectable copy number change or sequence alterations, like translocations and insertions that might change the structure of the gene resulting in the loss of function at the protein level, should be also considered. Our tiling array design would not detect small genomic changes (i.e. point mutations) and as sequencing of the other candidate genomic regions was not performed the involvement of the candidate EXT3 gene could not be ruled out with absolute certainty. However, our results eliminate the necessity of the involvement of a potential third gene responsible for MO. For the remaining sporadic cases, the presence of mosaic point mutation can be predicted based on the fact that in the vast majority (75-80%) of MO cases point mutations are detected and only about 10% of the cases showed the presence of larger deletions involving one or more exons in EXT1 or EXT2 genes (Vink et The high sensitivity of the tiling-oligonucleotide array makes this platform as a versatile tool for studies dealing with genetic mosaicism seen in acquired genomic changes in neoplasia such as solitary osteochondroma or chondrosarcoma cases.

Taken together, we have developed a robust tool for the detection and delineation of mosaic deletions in EXT1, EXT2 and additional genes involved in heparan and chondroitin sulfate biosynthesis. For the first time we prove that somatic mosaicism rather than the involvement of a third predicted gene is responsible for the MO development in sporadic and index cases of familial forms.

AUTHORS' CONTRIBUTION:

KS: Designed the study and the array, selected the genes, analyzed the data, interpreted the results and drafted the manuscript IJ: provided samples, performed EXT mutation studies, performed FISH verification experiments DJ: Designed the array, validated the data, analyzed the results and partly drafted the manuscript JVMGB; designed the array, commented on the manuscript MW selected genes for the array WW selected genes for the array, provided patient samples, performed EXT mutation studies PCWH designed the study, reviewed the data and the manuscript

FIGURE LEGEND

Figure1:

Array-CGH result overview showing the log2 ratio profile of samples with positive results only.

A: An overview of about 1Mb genomic region on chromosome 8 including the EXT1, MED30 and SLC30A8 gene area. Upper two samples (54782 and 73585) are positive controls with known EXT1 deletion. Middle two samples (84389 and 84458) with EXT2 alteration serve as negative control for this view. The bottom two samples (83350 and 59872) show mosaic deletions involving the region exon 2 to exon 3 and exon 2 to exon 11, respectively. For all cases, vertical double headed arrows indicate the borders of the detected alterations. B: An overview of about 240 kb genomic region on chromosome 11 including the EXT2, ACCS and ACCSL gene area. Upper two samples (54782 and 73585) and bottom two samples (83350 and 59872) are with EXT1 alterations and serving as negative controls for this genome segment. 84389is a positive control with MLPA detected deletion of exon 8, and 84458 shows the mosaic deletion of exon 2 of the EXT2 gene. Vertical double headed arrows indicate the borders of the detected alterations. Inset of the right top part in both panels shows the exon localization of the EXT1 and EXT2 loci; arrows in inset indicate the direction of transcription, note that EXT1 gene is transcribed from the minus strand. C: Overlay of array-CGH results of a sample with hemizygous deletion (54782, red) and one with a mosaic deletion (59872, blue). Based on the array-profile, the proportion of the deletion containing cell fraction was ~15%. In both cases the deletion involved exon2-11 of the coding sequence of the EXT1 gene. The array clearly depict that the breakpoints were at different position between the two different patients.

Figure 2

Side-by-side comparison of mosaic EXT1 exon 2-3 deletion carrier father (83350, red line) and daughter (83349, blue line) with the inherited paternal deleted allele resulting in a hemizygous deletion with identical breakpoints.

F

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 2 Figure 2

  al., 2005; Jones et al., 2010; Lonie et al., 2006). Copy number neutral, structural changes such as inversions involving the EXT genes could be expected in mutation negative familial cases. Novel approaches, such as next generation sequencing might provide the required sensitivity to detect mosaic point mutations in these remaining cases.

Formatted 2 Deleted:

 2 The presence of mosaic point mutation can be speculated based on the fact that in the vast majority (75-80%) of MO cases point mutations are detected and only about 10% of the cases showed the presence of larger deletions involving one or more exons in EXT1 or EXT2 genes(Vink et al., 2005; Jones et al., 2010; Lonie et al., 2006). Novel approaches, such as next generation sequencing could have the required sensitivity to detect mosaic point mutations in these remaining cases.

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Table 1

 1 List of genes for the custom made oligonucelotide array design

				Fully
		Number	Probes on	Covere
	GeneSymbol	of Probes	Exons	d	Gene name
	ADAM12	1177	459	Yes	ADAM metallopeptidase domain 12 (meltrin alpha)
					UDP-Gal:betaGal beta 1,3-galactosyltransferase polypeptide
	B3GALT6	74	68	Yes	6
	B3GAT1	213	152	Yes	beta-1,3-glucuronyltransferase 1 (glucuronosyltransferase P)
	B4GALNT1	147	143	Yes	beta-1,4-N-acetyl-galactosaminyl transferase 1
					xylosylprotein beta 1,4-galactosyltransferase, polypeptide 7
	B4GALT7	61	51	Yes	(galactosyltransferase I)
	CDCP1	435	313	Yes	CUB domain containing protein 1
	CHST11	688	109	Yes	carbohydrate (chondroitin 4) sulfotransferase 11
	CHST3	331	243	Yes	carbohydrate (chondroitin 6) sulfotransferase 3
	CHSY1	318	160	Yes	chondroitin sulfate synthase 1
	CSGALNACT1 713	341	Yes	chondroitin sulfate N-acetylgalactosaminyltransferase 1
	DSE	475	181	Yes	dermatan sulfate epimerase
	EXT1	724	99	Yes	exostoses (multiple) 1
	EXT2	314	85	Yes	exostoses (multiple) 2
	EXTL1	59	22	Yes	exostoses (multiple)-like 1
	EXTL2	187	147	Yes	exostoses (multiple)-like 2
	EXTL3	195	47	Yes	exostoses (multiple)-like 3
	GALNAC4S-
	6ST	320	252	Yes	B cell RAG associated protein
	GIPC1	87	70	Yes	GIPC PDZ domain containing family, member 1
	HAPLN4	16	9	Yes	hyaluronan and proteoglycan link protein 4
	HS2ST1	668	44	Yes	heparan sulfate 2-O-sulfotransferase 1
	HS3ST1	120	7	Yes	heparan sulfate (

glucosamine) 3-O-sulfotransferase 1 HS3ST2 309 6 Yes heparan sulfate (glucosamine) 3-O-sulfotransferase 2 HS3ST3A1 395 8 Yes heparan sulfate (glucosamine) 3-O-sulfotransferase 3A1 HS3ST3B1 156 22 Yes heparan sulfate (glucosamine) 3-O-sulfotransferase 3B1 HS3ST4 1342 17 Yes heparan sulfate (glucosamine) 3-O-sulfotransferase 4 HS3ST5 26 3 Yes heparan sulfate (glucosamine) 3-O-sulfotransferase 5

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	HS3ST6	22	1	Yes	heparan sulfate (glucosamine) 3-O-sulfotransferase 6	Formatted
	HS6ST1 HS6ST2 HS6ST3	157 1142 2591	4 26 35	Yes Yes Yes	heparan sulfate 6-O-sulfotransferase 1 heparan sulfate 6-O-sulfotransferase 2 heparan sulfate 6-O-sulfotransferase 3	Formatted Formatted
	IL5RA	261	181	Yes	interleukin 5 receptor, alpha	Formatted
	MED30	73	7	Yes	mediator complex subunit 30	Formatted
	NCAN	101	40	Yes	Neurocan	Formatted
	NDST1	131	50	Yes	N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 1
	NDST2	41	29	Yes	N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 2
	NDST3	704	34	Yes	N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 3
	NDST4	1072	32	Yes	N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 4
					nudix (nucleoside diphosphate linked moiety X)-type motif
	NUDT16L1	25	24	Yes	16-like 1

Table 2 :

 2 Patient characteristics

	MGRN1	37	36	No	mahogunin, ring finger 1		Formatted: Font: (Default) Times
					nuclear factor of kappa light polypeptide gene enhancer in B-	New Roman
	NFKBIE	17	16	No	cells inhibitor, epsilon		Formatted: Font: (Default) Times
	NSMAF	123	115	No	neutral sphingomyelinase (N-SMase) activation associated factor	New Roman
	PRICKLE4	9	7	No	prickle homolog 4 (Drosophila)	Formatted: Font: (Default) Times
	PRRG1	28	27	No	proline rich Gla (G-carboxyglutamic acid) 1	New Roman
	PRSS12	86	13	No	protease, serine, 12 (neurotrypsin, motopsin)	Formatted: Font: (Default) Times
					pleckstrin homology, Sec7 and coiled-coil domains, binding	New Roman
	PSCDBP	111	10	No	protein			Formatted: Font: (Default) Times
	PSMC2	70	22	No	proteasome (prosome, macropain) 26S subunit, ATPase, 2	New Roman
	PTGER1	12	2	No	prostaglandin E receptor 1 (subtype EP1), 42kDa	Formatted: Font: (Default) Times
	SDF4	27	16	No	stromal cell derived factor 4		New Roman
	SE15(sep15) SGSH SLC30A7	19 7 10	1 0 1	No No No	15 kDa selenoprotein isoform 1 precursor N-sulfoglucosamine sulfohydrolase (sulfamidase) solute carrier family 30 (zinc transporter), member 7	Formatted: Font: (Default) Times New Roman Formatted: Font: (Default) Times New Roman
	SNPH SRPX	130 17	129 16	No No	Syntaphilin sushi-repeat-containing protein, X-linked	Formatted: Font: (Default) Times New Roman
	TMED9	46	45	No	transmembrane emp24 protein transport domain containing 9	Formatted: Font: (Default) Times
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		Sample Nr.	Gender	Clinical diagnosis	EXT1/EXT2 mutation or MLPA testing	Age (years)	Familial**	Formatted Formatted: Centered Formatted
		33191	m	MO	-	51	No	Formatted
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		33193	f	MO	-	43	No	Formatted
		53128	m	MO	-	44	No	Formatted
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		59872	m	MO	-	3	No	Formatted
		60518	m	MO	-	n.a.	n.a.	Formatted
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Deleted: tested negative for a m…in Deleted: were selected ... [144] ... [137] ... [147] ... [146] ... [148] ... [139] ... [149] ... [138] ... [150] ... [141] ... [151] ... [145] ... [152] ... [142] ... [153] ... [143] ... [154] ... [140] *MLPA confirmed EXT deletion samples used as positive control **Familial: samples with clinically established MO with a positive family history

Table 3 :

 3 Overview of tile-resolution array-CGH results with detected deletions both mosaic and hemizygous of the analyzed mutation and MLPA negative patient samples and positive control, respectively

	Sample Nr.	Gender	EXT1***	EXT2***	Deletion (kb) size	Familial
			chr8:EXT1.(118,808,395_
	59872*	m	118,808,900)_(119,004,41 3_119,004,064)del(NCBI3	-	196	No
			6, hg18)
			chr8:EXT1.(118,914,729_
	83350*	m	118,915,174)_(119,064,40 8_119,062,731)del(NCBI3	-	149.7	No
			6, hg18)
				chr11:EXT2.(44,084,506_
	84458*	m	-	44,086,611)_(44,085,734 _44,086,166)del(NCBI36,	10.6	No
				hg18)
			chr8:EXT1.(118,889,693_
	73585**	m	118,891,168)_(118,897,37 1_118,896,538)del(NCBI3	-	43	No
			6, hg18)
			chr8:EXT1.(118,829,257_
	54782**	m	118,830,034)_(119,089,19 7_119,087,204)del(NCBI3	-	259.9	Familial
			6, hg18)

  al., 1998; Wuyts et al., 2005; Lonie et al., 2006; Pedrini et al., 2005; Jennes et al., 2009). Intragenic deletions involving single or multiple exons of the EXT1 or EXT2 genes were found in about 10 % of all tested cases (Vink et al., 2005; Jennes et al.

John Wiley & Sons, Inc.Human Mutation

These authors contributed equally to this study. Contract grant sponsor: EuroBoNeT, a European Commission-granted European Network of excellence for studying the pathology and genetics of bone tumours. Contract grant number: LSHC-CT-2006-018814. This study is supported by the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen) (grant to I.J.) and the Netherlands Organization for Scientific Research (917-76-315)(grant to J.V.M.G.B.).


Laila M R El-Attar

Charalambos D Partidos

Colin R Howard
email: choward@rvc.ac.uk

A PEPTIDE MIMOTOPE OF HEPATITIC C VIRUS E2 PROTEIN IS IMMUNOGENIC IN MICE AND BLOCK HUMAN ANTI-HCV SERA

Keywords: HCV, mimotope, therapeutic vaccine

HAL is a

Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Twenty percent 5 of chronically infected individuals develop liver cirrhosis that is linked to hepatocellular 6 carcinoma in many regions [START_REF] Kuo | An assay for circulating antibodies to a 28 major etiologic virus of human non-A, non-B hepatitis[END_REF][START_REF] Saito | Hepatitis C virus infection is associated with the 29 development of hepatocellular carcinoma[END_REF]]. However, there is no 7 protective vaccine against HCV infection [START_REF] Houghton | Prospects for a vaccine against the hepatitis C virus[END_REF]. Gamma 8 interferon and ribavirin, are currently the only antiviral agents for therapy that are 9 successful in 50% of treated patients [START_REF] Ahn | Peginterferon-alpha(2b) and ribavirin[END_REF]. Therefore, the development 10 of new antiviral agents or therapeutic vaccines is essential. 11 HCV contains a 9.6kb positive-stranded RNA genome with considerable homology to 12 other members of the family flaviviridae [START_REF] Miller | Hepatitis C virus shares amino acid sequence similarity with 43 pestiviruses and flaviviruses as well as members of two plant virus supergroups[END_REF]]. The genome encodes 13 two envelope proteins E1 and E2 which are important candidate antigens for a vaccine 14 against HCV. Both glycoproteins show substantial amino acid sequence variation with a 15 hypervariable region (HVR1) located at the N-terminal region of E2 [START_REF] Hijikata | 9 Hypervariable regions in the putative glycoprotein of hepatitis C virus[END_REF]16 Kato et al., 1992;[START_REF] Weiner | Variable and hypervariable 41 domains are found in the regions of HCV corresponding to the flavivirus envelope 42 and NS1 proteins and the pestivirus envelope glycoproteins[END_REF]. 17

18

In chimpanzees, immunisation with recombinant E1/E2 partially protects against 19 homologous virus challenge [START_REF] Choo | Vaccination of chimpanzees against infection by the 30 hepatitis C virus[END_REF] and antibodies to the E2 protein correlated 20 with reduction in virus load but not from reinfection [START_REF] Farci | Lack of protective immunity against 42 reinfection with hepatitis C virus[END_REF]Lanford et al., 21 2004;[START_REF] Youn | Sustained E2 antibody 49 Page 35 of[END_REF]. In persons infected with HCV, the presence of neutralising 22 antibodies against HCV E2 correlates with virus clearance [START_REF] Lavillette | Characterization of host-range and cell entry properties 34 of the major genotypes and subtypes of hepatitis C virus[END_REF] (LEL) of CD81 on human cells [START_REF] Evans | Claudin-1 is a hepatitis C virus co-39 receptor required for a late step in entry[END_REF][START_REF] Pileri | Binding of hepatitis C virus to CD81[END_REF]Scarselli et al., 8 2002]. The capacity of antibodies to inhibit E2 binding to CD81 had been used previously 9 for detecting the neutralising capacity of antibodies against (Nt Ab) HCV [Flint et al., 10 1999]. These neutralising antibody titres were reported to correlate with spontaneous 11 resolution of chronic infection [START_REF] Abrignani | Immune responses throughout hepatitis C virus (HCV) infection: HCV 3 from the immune system point of view[END_REF]Bartosch et al., 2003a]. Such Nt Ab is 12 likely to be directed against conformational B-cell epitopes of E2, which appear to be 13 conserved between different genotypes [START_REF] Habersetzer | Characterization of human 2 monoclonal antibodies specific to the hepatitis C virus glycoprotein E2 with in 3 vitro binding neutralization properties[END_REF][START_REF] Hadlock | Human monoclonal antibodies that inhibit binding of hepatitis C 6 virus E2 protein to CD81 and recognize conserved conformational epitopes[END_REF]14 Owsianka et al., 2006]. 15 16 The E2 protein model based on the envelope protein of tick borne encephalitis virus 17 proposed two CD81 binding regions 1 and 2 on E2 protein [START_REF] Yagnik | A model for the hepatitis C virus envelope glycoprotein E2[END_REF]. 18

Therefore, blocking the binding of HCV to CD81 would be of great value in the quest for 19 a vaccine development. In this study two anti-E2 mAbs, 3e5-1 and 5E5/H7, were 20 employed to select mimotopes that are involved in protection, neutralising activityusing a 21 15-mer random phage displayed peptide library. mAb 3e5-1 and 5E5/H7 were of 22 considerable interest. The conformational mAb 5E5/H7 was produced initially to 23 intracellularly envelope protein hence epitopes(s) recognised by this antibody would 24 display enhanced receptor-binding ability as would the E2 native form. The anti-linear 25 mAb3e5-1, however, was produced to E2 truncated form and recognised secreted and 1 intracellularly forms of E2, suggesting possible shared epitope recognition as mAb 2 5E5/H7 (Houghton M, personnel communication; invention EP0980434B1 3 (http://www.freepatentsonline.com/EP098043B14.html)). Additionally, mAb 5E5/H7 was 4 initially used to affinity purify the E1/E2 proteins used in the protection study in 5 chimpanzees where considerable protection was achieved against homologous challenge 6

[ [START_REF] Choo | Vaccination of chimpanzees against infection by the 30 hepatitis C virus[END_REF][START_REF] Rosa | A 24 quantitative test to estimate neutralizing antibodies to the hepatitis C virus: 25 cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells[END_REF]. As such, epitope recognised by this antibody could 7 block viral infectivity. The mimotope was tested for its ability to bind to human antisera to 8 HCV, to block E2-CD81 interaction and to raise antibodies to HCV in mice. The 9 identified mimotope could have potential applications for the design of an 10 immunotherapeutic vaccine that would target the CD81-HCV E2 interaction site(s). 11

12

MATERIALS AND METHODS

14

Monoclonal Antibodies 15

Two HCV E2 specific mAbs, 3e5-1 and 5E5/H7, were used in this study. Both were 16 kindly provided by Dr. M. Houghton, Chiron Corporation, USA. mAb 3e5-1(immunogen 17 Insect E2, aa 404-661) is directed against a linear epitope on the sectred and intracellular 18 E2, whereas, the conformational mAb 5E5/H7 (Hella E1/E2, aa 1-967) is thought to 19 recognises intracllulary expressed conformational epitope [Houghton M Invention 20 EP0980434B1(http://www.freepatentsonline.com/EP0980434.html)).

The anti-E2 21 conformational mAb 12D11 was generously given by Dr. Geert Maertens (Innogenetics,22 Gent, Belgium (http://www.freepatentsonline.com/EP1845108A2.html )) and used as a 23 positive control in some ELISAs. A mAb Tac specific for Tacaribe virus was used as an 24 unrelated negative control. Two human mAbs the CBH 7 and CBH 9 with characterised 25 Mello et al., 1997] except that all phage clones were harvested by two 2 successive precepitations in 16.7% PEG-8000/3.3M NaCL. The input and output phage 3 were titrated using Luria Broth (LB) media after each round of biopanning, so that the 4 percent enrichment of binding the phage could be calculated. Percent enrichment was 5 calculated as output/input x 100. 6 7

DNA Purification And Sequencing Of Phage Clones 8

The DNA was extracted from the purified phage using the miniprep DNA purification kit 9 (Promega) following the manufacture's procedure. Sequencing of the insert DNA was 10 performed using the AutoRead sequencing Kit (Pharmacia, UK) and following the 11 conditions recommended by the manufacturers. The fluorescence labelled sequencing 12 primer was 5'-FCCCTCATAGTTAGCGTAACG-3' downstream of the oligo insert site of 13 phage DNA. The sequencing reactions were analysed on the ALF automated DNA 14 sequencer (Pharmacia, UK) using a 6% Sequa gel XR (National Diagnostics, UK). [START_REF] Pestka | 17 Rapid induction of virus-neutralizing antibodies and viral clearance in a single-18 source outbreak of hepatitis C[END_REF] 16

Synthetic Peptides 17

The peptide mimotope K1 and the chimeric peptide (K1-MVF) containing a T-helper cell 18 epitope from the fusion protein of measles virus [START_REF] Partidos | Prediction and identification of a T cell epitope in the 8 fusion protein of measles virus immunodominant in mice and humans[END_REF] Dulbecco's medium respectively. Growth medium was supplemented with 10% foetal calf 14 serum, 100 I.U./ml penicillin, 100µg/ml streptomycin and 1% non-essential amino acids 15 (Gibco, BRL). The expression of E2 protein in COS-7 cells, was detected intracellular by 16 immunofluorescent staining using the mAb 5E5/H7 and extracellular by indirect ELISA 17 using the mAb 12D11. 10uM/ml forskolin was added to the maintenance medium to 18 enhance the expression of the E2 protein and the cell monolayers were incubated for three 19 days. The maintenance medium was replaced by serum-free medium and the secreted E2 20 protein was collected after six days. Supernatants from uninfected COS-7 cells were used 21 as a mock antigen to eliminate any irrelevant antibody binding. 22 Partial purification of E2 protein was achieved by first concentrating the cell supernatants 23 approximately 15-fold by ultra filtration using a 10KDa cut-off membrane (YM10, 24 Amicon) followed by successive gel-purification on S-Sepharose in fractionation range of 25 29 April 2010 $ASQ7726578_File000003_127231041.doc 9 5x10 3 -2.5x10 5 (Pharmacia Biotech) according to the manufacture's protocol. Collected 1 fractions (at a flow rate 4ml/min) were monitored at 280nm and assayed for the presence 2 of E2 protein by employing the mAb 12D11 at 1/800 dilution. The identity of the 3 produced E2 protein was confirmed by western blot using mAb 12D11 and by 4 deglycosylation using the peptide-N-Glycosidase F (Oxford GlcoScience, UK) and 5 following the manufacture protocol. Fractions containing E2 protein were pooled and 6 dialysed versus Phosphate-buffered saline pH 7.4, and stored at -20 o C. In some 7 experiments, lysate of COS-7 cells infected with RAd-79 was used as antigen in ELISA. 8

This will be referred to in the text as E2 cell lysate. Cell lysate from uninfected COS-7 9 cells were used as a mock cell lysate to eliminate any irrelevant antibody binding. [START_REF] Partidos | The effects of a flanking sequence on the immune 11 response to a B and a T cell epitope from the fusion protein of measles virus[END_REF] 11

Immunisation Of Mice 12

Female BALB/c mice, 6 to 8 weeks old, were injected subcutaneously with either 13 50µg/mouse mimotope K1 peptide or the chimeric peptide (K1-MVF) in Freund's 14 complete adjuvant (FCA) or without adjuvant (in PBS). Mice were boosted by the same 15 route and with the same amount of peptides in an emulsion (1:1 ratio) with incomplete 16

Freund's adjuvant after 4 weeks. A control group received PBS in Freund's adjuvant. 17 Plates were coated with an immunoreactive phage clone and unrelated clone at a 10 concentration of 0.75x10 9 phage particles/ml in 50mM carbonate buffer. Unbound protein 11 was washed away by three washes. Following blocking, plates were washed as described 12 above. mAbs 3e5-1, 5E5/H7 and unrelated mAb were diluted 1/200 and added in duplicate 13 to immunoreactive and unrelated phage clones-coated wells and incubated for 3 hours. 14 Horseradish peroxidase-conjugated goat anti-mouse IgG was added to all wells. Following 15 washing as above, the substrate OPD was added to all wells and the colour was detected as 16 described above. 17 18

Peptide And E2 Protein Binding Assay 19

Plates were coated with 10µg/ml of mimotope K1, chimeric peptide or unrelated peptide 20 (LCM). For detection of binding to E2 protein, plates were coated with either 20µg/ml of 21 partially purified E2 protein, 0.5mg/ml of E2 cell lysate, mock cell lysate or coating buffer 22 only. The assays were carried out as above except that mouse sera from each group were 23 tested individually and added in duplicate to coated wells at 2 fold serial dilution starting 24 from 1/50. Positive control mAb 12D11 (1/800), 5E5/H7 (1/400) and 3e5-1 (1/400) were 25 For E2 protein assay, OD values obtained with mock cell lysate or coating buffer were 5 subtracted from values obtained with E2 cell lysate or partially purified E2 protein, 6 respectively. The cut off value was calculated as the mean of the corrected OD value of 7 the lowest dilution of the pre-bleed sera + SD. 8 9

Human Sera Assays 10

All conditions of ELISA using human sera were similar to those described for mouse sera, 11 with the following exceptions: assays were performed in a category three laboratory. All 12 sera were heat inactivated at 56°C for 30 minutes before use. Primary antibody incubation 13 was at 4°C overnight. Secondary antibody was Horseradish peroxidase-conjugated rabbit 14 anti-human IgG (Dako) diluted 1/8000. Blocking and diluent buffers were PBS, pH 7.4 + 15 0.5% Casein (sigma) + 0.2% Triton X-100. Washing buffer contained PBS, pH7.4 + and 16 1% Tween-20. 17 a) Anti-E2 antibody responses. Plates were coated with 0.5mg/ml of E2-lysate, β-18 galactosidase cell lysate or mock cell-lysate. Human sera were diluted two-fold starting 19 from 1/20 in ELISA diluent and added in duplicates to all three antigen coated plates. In 20 this assay 0.5% of mock antigen was incubated in the diluent to minimise the background. 21

The plates were then incubated at 4°C, overnight. Secondary antibody was added and the 22 assay was carried out as for the mouse sera. The cut-off was calculated as the mean of the 23 value of the OD obtained with mock cell lysate + 3 SD. The OD obtained for each serum 24 at 2-fold dilution starting from 100µg/ml) was incubated overnight at 4°C with fixed 4 dilution of human sera (1/20). The mixture was then added in duplicate to plates pre-5 coated with 0.5mg/ml of E2 cell lysate and mock cell-lysate at 4°C, overnight. Each 6 human serum (1/20) without competitor was also added in duplicate to pre-coated plates. 7

The assay was carried out as described above. The OD values obtained with mock-cell 8 lysate were subtracted from OD values obtained with E2-cell lysate. The percentage of 9 inhibition was calculated as the ratio between the net OD value in presence of inhibitors 10 and direct binding of human sera to E2 protein in absence of inhibitor x 100. 11 c) Competition ELISA. The assay was performed as in the inhibition assay except that a 12 decreasing concentration of partially purified E2 protein (2 fold dilution, starting from 13 20µg/ml) was added in duplicate simultaneously with the synthetic peptide competitor and 14 human sera to plates coated with the same concentration of protein. No mock cell lysates 15 were included in the diluent. The reactants were incubated for three hours. Sera were also [START_REF] Kato | Characterization of hypervariable regions in the putative envelope protein of 18 hepatitis C virus[END_REF] added in duplicate to the antigen-coated plates without competitors. 17 This assay was performed as described by [START_REF] Flint | Characterization of hepatitis C virus E2 glycoprotein 45 interaction with a putative cellular receptor, CD81[END_REF] [START_REF] Flint | Characterization of hepatitis C virus E2 glycoprotein 45 interaction with a putative cellular receptor, CD81[END_REF]. Briefly, 3 GST-CD81 (EC2) encoding the human (hCD81) and murine (mCD81) second 4 extracellular (EC2) loops were coated onto Immulon II ELISA plates at 5µg/ml in PBS. 5

Plates were blocked with 4% milk powder/TBS. A truncated E2 661 protein (H-Chiron) at 6 500ng/ml, the concentration resulting in 50% of maximal binding to CD81, was incubated 7

with human serum at final dilutions of 1/200 and 1/1000 at room temperature for 1 hour. 8

The E2-antibody mix was added to the CD81 coated plates in triplicate and allowed to 9 bind at room temperature for 2 hours. After washing with Tris phosphate buffer saline 10 (10mM Tris-HCl pH8.0, 0.15M NaCl), bound E2 was detected with rat mAbs 7/59, 6/1a 11 and 11/20a and anti-rat IgG horseradish peroxidase. Human mAb CB H7 and CB H9 12 positive and negative controls respectively were included in the assay to assess the validity 13 of the assay. 14 15

RESULTS

17

Selection Of HCV E2 Protein Mimotopes 18

A peptide mimic of the epitope recognised by mAbs 3e5-1 and 5E5/H7 was identified. 19 The percentage yield after the third round was significantly increased >200 for mAb 3e5-1 20 and ~500 times for mAb 5E5/H7, than the background library (Table I) giving rise to [START_REF] Pestka | 17 Rapid induction of virus-neutralizing antibodies and viral clearance in a single-18 source outbreak of hepatitis C[END_REF] The specificity of the mimotope enriched phage clones for binding to these anti-HCV E2 3 mAbs was confirmed by ELISA. mAbs 3e5-1 and 5E5/H7 gave OD 492nm values of 2.17 4 and 0.849, respectively (Figure 1). In contrast, the same antibodies gave OD 492nm values 5 of 0.228 and 0.593 when tested against an unrelated phage clone, respectively. These 6 results indicated that the immuno-reactive phage clones were specific to antibody 3e5-1 7 than to 5E5/H7. 5E5/H7 antibody had a substantial cross reactivity with the unrelated 8 phage clone. 9 10 Delineation Of HCV Epitope Mimicked By The Mimotope K1 11 The K1 peptide appears to be a mimic of the peptide 523 GAPTYSWG 530 of E2 protein 12 (alternatively we can say: the K1 peptide could substitute the authentic peptide 13 523 GAPTYSWG 530 of E2 protein). This peptide had the greatest binding among nine 7-mer 14 peptides recognised by mAb 5E5/H7 in PepScan analysis [El-Attar et al. in preparation]. [START_REF] Pestka | 17 Rapid induction of virus-neutralizing antibodies and viral clearance in a single-18 source outbreak of hepatitis C[END_REF] Alignment of this peptide, the mimotope K1, HCV prototype H strain and other HCV 16 types 1a, 1b, 2a, 2b, 3a, 4, 5a and 6 (Figure 2) revealed the presence of several identical 17 and conserved residues within this region of E2. The sequence/motif S 1 S 2 WG has the core 18 motif WG identical to all HCV types, whereas S 1 and S 2 was replaced by either identical 19 (S) or conserved residues (S, N, T and Y) with the exception of R in HCV-BK isolate. S, 20 N, T and Y residues are uncharged, polar amino acids. Interestingly the core motif WG 21 has been reported to be critical for binding E2 to CD81 [START_REF] Owsianka | Identification of conserved 5 residues in the E2 envelope glycoprotein of the hepatitis C virus that are critical for 6 CD81 binding[END_REF]

Immunogenicity And Antigenicity Of Mimotope K1 1

Mice immunised with chimeric peptide/in FCA induced antibody responses mainly to the 2 mimotope K1. The geometric mean titre to the chimeric and the mimotope K1 peptides 3 were log 10 3.3 and log 10 2.9, respectively. 4 5The murine anti-mimotope antibodies were found to recognise the native E2 protein of 6 HCV. Binding of the anti-mimotope antibodies to E2 protein was confirmed by repeating 7 the experiment 5 times. Antibody titres when using the COS-7 cell lysate infected with 8 RAd-79 as solid phase antigen were significantly higher in sera of immunised mice (Table 9 II) as compared to those from control mice (Table II) (p value was 0.0003 by student t 10 test). Similarly, in three independent experiments, the anti-mimotope antibody titre to 11 partially purified E2 protein was significantly higher (p values of 0.0292, 0.04920 and 12 0.0089, respectively by student t test) as compared to that of the negative group (Figure 3). 13 To avoid any false evaluation, a Bonferroni correction was calculated and p value was 14 equal to 0.0000. [START_REF] Pestka | 17 Rapid induction of virus-neutralizing antibodies and viral clearance in a single-18 source outbreak of hepatitis C[END_REF] 16

Prevalence Of Anti-E2 Antibody Response In HCV-patient Sera 17 18

All 15 (100%) human sera positive (on going infection) or weak positive (self-limited 19 infection) for HCV-RNA had anti-E2 antibodies compared to 7 out of 10 (70%) sera 20 negative to HCV RNA (resolved infection). However, among the 8 sera negative for anti-21 HCV antibodies, 3 sera (37%) showed the presence of anti-E2 antibodies. This may be due 22 to the low sensitivity of the commercial ELISA tests for screening anti-HCV antibodies 23 resulting from the lack of envelope protein in these assays. It may also indicate the 24 appearance of anti-E2 antibody earlier in the infection, and this could be missed out by a 25 commercial ELISA. These sera were excluded from this study. Antibody titres were 26 The binding of 5 out of 7 (71%) sera with a confirmed history of hepatitis C infection to 6 the E2-lysate was inhibited by the K1 mimotope at the concentration of 0.5mg/ml. The 7 inhibition value ranged between 24% -59% (Figure 4). Four (80%) of these sera were 8 negative or weakly positive for HCV RNA. These results highlight a possible correlation 9 between recovery from HCV infection and the presence of anti-E2 antibodies. [START_REF] Partidos | The effects of a flanking sequence on the immune 11 response to a B and a T cell epitope from the fusion protein of measles virus[END_REF] One sample, which had shown no reactivity, was initially anti-E2 antibody negative. This 11 particular serum was chosen to ensure the sensitivity of the assay. No reaction was 12 detected with the negative sera in presence or absence of the mimotope K1. One serum 13 with antibodies to the E2 lysate was inhibited by 18% with the unrelated peptide. 14 15 To emphasise the value of the mimotope in terms of resolving infection for use with 16 human sera, a further competition assay was performed with nine HCV sera from 17 individuals with self limited or resolved hepatitis C infection. The mimotope K1 was able 18 to compete with the recombinant E2 protein in four out of 6 cases where E2 had a degree 19 of inhibition. In three cases the inhibition induced by the mimotope was 18.3 %, 38.12 % 20 and 24.7% compared to 2.75 %, 8.04 % and 28.62% with recombinant E2 protein 21 respectively (Figure 5). In the fourth case the inhibition exerted by the recombinant E2 22 protein was much greater than seen using the mimotope where the percentage of 23 inhibition was 31.3% and 13.9% respectively. The titre of anti-E2 protein antibody ranged 24 To assess the value of the mimotope in terms of recognition by human sera that are 6 capable of inhibiting E2-CD81interaction, the nine sera used for competition assay were 7 tested for their ability to block E2 binding to CD81. Five sera had inhibited E2 binding to 8 CD81 at a 1/1000 dilution. Four of these sera were found to be reactive with the 9 K1mimotope (Table III), suggesting that the mimotope could be a mimic of an epitope on 10 E2 protein that is involved in E2-CD81 interaction. The human mAb CBH-7 showed 11 inhibition activity as expected and negative control CBH-9 mAb had no serum inhibition 12 activity. 13 14 DISCUSSION 15 16 There is accumulating evidence that CD81 is playing a central role in HCV entry to 17 hepatocytes through its interaction with E2 protein. Identification of an inhibitor to block 18 this interaction would assist future therapeutic vaccine to control HCV infection. 19 In this study a mimotope was identified that mimics a conformational B-cell epitope 20 involved in binding of E2 to CD81 using 15-mer random phage displayed peptide library. 21

The mimotope was able to elicit HCV E2 specific antibody responses in vivo and bound to 22 human sera with the capacity to inhibit E2-CD81 interaction. Two murine mAbs, 3e5-1 23 and 5E5/H7, raised against the E2 protein were selected for screening. mAb 3e5-1 that 24 initially used to purify E1-E2 heterodimer complexes used in the chimpanzee protection 3 study [START_REF] Choo | Vaccination of chimpanzees against infection by the 30 hepatitis C virus[END_REF][START_REF] Rosa | A 24 quantitative test to estimate neutralizing antibodies to the hepatitis C virus: 25 cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells[END_REF], highlighting that an epitope recognised by 4 mAb 5E5/H7 would be a mimic of the authentic protein on the virus particle. mAb 5 5E5/H7 gave significantly higher yields of reactive clones after three rounds of 6 biopanning. The reactivity of each mAb for the selected clones was confirmed by 7 immuno-blot and ELISA, with mAb 3e5-1 giving the best discrimination between selected 8 phage clones and unrelated phage clones. This could be explained by the linear nature of 9 the epitope recognised by mAb 3e5-1 and the complex nature of the epitope recognised by 10 mAb 5E5/H7. This is supported by previous characterisation of these two monoclonal 11 antibodies (Michael Houghton Invention EP0980434B1 12 (http://www.freepatentsonline.com/EP0980434.html)) where evaluation of epitope 13 exposure by both antibodies showed mAb 3e5-1 giving approximately 3.9 higher signal to 14 intracelluarly E2 compared to conformational mAb 5E5/H7. 15 16 The conformational nature of the epitope recognised by mAb was initially assessed by 17

PepScan analysis (El-Attar et al, in preparation). PepScan revealed 9 reactive but distantly 18 located E2-overlapping peptides with the peptide 523 GAPTYSWG 530 (W) in the SWG motif is in agreement with the importance of aromatic amino acids in the 6 antigenic determinants of peptides [START_REF] Appel | Elucidation of discontinuous linear 12 determinants in peptides[END_REF]. 7 8 Interestingly, the predicted contribution to the epitope mimicked by K1 is in agreement 9 with the recent identification of critical conserved amino acid residues involved in binding 10 E2 to CD81 [START_REF] Johansson | 14 Human combinatorial libraries yield rare antibodies that broadly neutralize 15 hepatitis C virus[END_REF][START_REF] Owsianka | Identification of conserved 5 residues in the E2 envelope glycoprotein of the hepatitis C virus that are critical for 6 CD81 binding[END_REF] and overlaps with CD 81 11 binding region 2 (a.a 522-551) proposed by [START_REF] Yagnik | A model for the hepatitis C virus envelope glycoprotein E2[END_REF]. 12 Similar regions of E2 of conformational nature (a.a. 480-493 and 544-551) involved in 13 the CD81 interaction was also reported [START_REF] Flint | Characterization of hepatitis C virus E2 glycoprotein 45 interaction with a putative cellular receptor, CD81[END_REF]. Other investigators have 14 reported the existence of discontinuous B cell epitope that overlaps with the domain 15 mimicked by K1 [START_REF] Lechner | 37 Antibodies directed to envelope proteins of hepatitis C virus outside of 38 hypervariable region 1[END_REF][START_REF] Mink | 46 Characterization and mapping of a B-cell immunogenic domain in hepatitis C virus 47 E2 glycoprotein using a yeast peptide library[END_REF]. In one report, rabbit antibodies 16 recognised a domain spanning residues 511-530 of the E2 protein was found to block viral 17 attachment [START_REF] Lechner | 37 Antibodies directed to envelope proteins of hepatitis C virus outside of 38 hypervariable region 1[END_REF]]. Studies with human mAbs have revealed the presence 18 of conserved conformational-dependent epitopes [START_REF] Allander | Recombinant human monoclonal antibodies against 9 different conformational epitopes of the E2 envelope glycoprotein of hepatitis C 10 virus that inhibit its interaction with CD81[END_REF][START_REF] Bugli | Mapping B-cell epitopes 26 of hepatitis C virus E2 glycoprotein using human monoclonal antibodies from 27 phage display libraries[END_REF]19 Hadlock et al., 2000;[START_REF] Johansson | 14 Human combinatorial libraries yield rare antibodies that broadly neutralize 15 hepatitis C virus[END_REF]. Antibodies to these epitopes inhibited the 20 binding of E2 to CD81. 21 22 To verify that the mimotope K1 is a mimic of authentic HCV E2, anti-mimotope 23 antibodies were raised in mice and tested for their binding to the native E2 protein. [START_REF] Bugli | Mapping B-cell epitopes 26 of hepatitis C virus E2 glycoprotein using human monoclonal antibodies from 27 phage display libraries[END_REF] order to overcome immune unresponsiveness due to the low molecular weight of the 25 colinearly coupled to the N terminal of the mimotope K1 peptide. This T-helper epitope is 2 known to have a wide range of class II major histocompatibility complex molecules both 3 in mice and human [START_REF] Partidos | Prediction and identification of a T cell epitope in the 8 fusion protein of measles virus immunodominant in mice and humans[END_REF]]. 4

The antibody response induced following immunisation with the chimeric peptide was 5 mainly against the mimotope K1. The level of anti-mimotope antibody titre to the E2 was 6 significant in four out of five experiments (p values 0.0003-0.0492). These data 7 demonstrated that the mimotope K1 could substitute for the authentic HCV E2 epitopes in 8 provoking a specific immune response to the E2 protein. The low level of anti-E2 antibody 9 responses obtained in these experiments can be explained by the requirement of 10 considerable molar excess (10 3 -10 4 ) of peptide over native protein in order to reveal any 11 binding between peptide and antibody. Additionally, the affinity constant of the antibody 12 for the peptide is mostly several orders of magnitude lower than for the protein [ Van 13 Regenmortel, 1987]. 14 15 The fact that the mimotope K1 mimics the CD81 binding region 2 suggests that anti-16 mimotope antibodies would have the ability to block binding E2 protein to CD81. 17 agreement with previous reports. The mimotope inhibited the binding of all sera that were 1 either negative or weak for HCV RNA compared to 1 of 2 patients with circulating HCV 2 RNA. The specificity of the assay was confirmed as negative human sera as well as HCV 3 patients with no anti-E2 antibodies gave negative results, with no difference in the binding 4 of antibodies to the native protein in the presence or absence of the mimotope. These 5 results add to the value of the mimotope as representing a conformational epitope, which 6 may play a role in viral clearance. In this context, other studies [Keck et al., 2004; Lechner 7 et al., 1998] suggested that a substantial portion of anti-E2 antibodies in HCV infected 8 patients are directed to non-linear B cell epitopes of the HCV envelope protein. It was 9 also demonstrated in the previous study that the K1 mimotope mimics an epitope 10 recognised by convalescent human sera. These sera were examined in this study for their 11 capacity to inhibit E2-CD81 interaction. Five sera inhibited E2-CD81 interaction at 1: 12 1000 dilutions. Four of these sera were reactive with the mimotope by the competition 13 assay. Binding of HCV to cellular ligand e.g. CD81 may be mediated via a domain on E2 14 that overlaps with that defined by the synthetic mimotope K1. Residues 522-551 of E2 15 protein were also reported to be involved in binding to CD81 [START_REF] Flint | Characterization of hepatitis C virus E2 glycoprotein 45 interaction with a putative cellular receptor, CD81[END_REF]Zhang et 16 al., 2004]. Within the latter region of E2, five residues (W420, Y527, W529, G530 and 17 D535) were critical for CD81 binding [START_REF] Owsianka | Identification of conserved 5 residues in the E2 envelope glycoprotein of the hepatitis C virus that are critical for 6 CD81 binding[END_REF], three of these residues are 18 present in the peptide reported in this study giving the highest signal with mAb 5E5/H7 19 and mimicked by the mimotope K1. 20 Antibodies to E2 are found in over 90% of persistently infected individuals but resolution 21 may require antibody against selected epitopes. This antibody specificity may be absent in 22 chronic hepatitis C disease. Such a gap in the antibody repertoire has been suggested as 23 occurring in carriers of HBV [START_REF] Brown | Affinity of antibody 22 responses in man to hepatitis B vaccine determined with synthetic peptides[END_REF]. Notwithstanding this, the development 24 of peptide mimics of conformational E2 determinants could prove valuable for the fine 25 The mimotope K1 mimics an epitope that lies in an area that displays a high degree of 3 homology within HCV subtypes (92-97% identity) indicating that antibodies directed to 4 this mimotope would cross-react with different HCV isolates. The reactivity of two 5 broadly neutralising human monoclonal antibodies was reported to be dependent on 6 residues in this same region of E2 (amino acids 523-535) [START_REF] Johansson | 14 Human combinatorial libraries yield rare antibodies that broadly neutralize 15 hepatitis C virus[END_REF]. 7 8 In summary, the mimotope K1 was able to induce HCV E2 specific immune response in 9 vivo in mice and binds to human sera from patients infected with HCV which showed the 10 capacity to inhibit E2-CD81 interaction. This response was directed at a conformational B 11 cell epitope that displays a high degree of homology within HCV subtypes (92-97% 12 identity), thus would broadly cross-react with different HCV isolates. More work is 13 needed to enhance the anti-mimotope antibody response and to assess the ability of 14 mimotope K1 for the induction of neutralising antibody responses. In conclusion, the 15 results of this study offer an approach for the development of therapeutic vaccines against 16 hepatitis C. 17

Figure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 5 .

 12345 Figure 1. Analysis by ELISA of phage clone selected by biopanning. The mAb 3e5-1 showed stronger binding to its reactive phage clone with high degree of discrimination to unrelated clones compared to mAb 5E5/H7. No binding was observed using an unrelated antibody to either of the clones. 254x190mm (96 x 96 DPI)

5 [

 5 have been identified as putative receptors for HCV. The CD81 1 tetraspanin, scavenger receptor class B type I (SR-BI) and the recently reported co-2 receptor; Claudin-1 are the most promising candidates [Evans et al., 2007; Pileri et al., 3 1998; Scarselli et al., 2002]. Studies using assays of retroviral pseudoparticles (HCVpp) 4 and infectious clones support a central role for CD81 in mediating viral infection Bartosch et al., 2003b; Cormier et al., 2004; Lavillette et al., 2005; Lindenbach et al., 6 2005]. Furthermore HCV E2 has been shown to interact with the large extra-cellular loop 7

8 9Sera 10 A 21 Screening of a 15 -mer Phage Library 22 A

 810211522 interaction capacity were sourced by Prof. Jane McKeating and used as a 1 positive and negative control, respectively in E2-CD81 binding assays. CBH-7 has 2 neutralisation activity to multiple HCV genotypes recognises conformational epitope on 3 the HCV E2 domain C proposed by Keck et al. [Hadlock et al., 2000; Keck et al., 2005; 4 Keck et al., 2004; Owsianka et al., 2008]. CBH-9 was negative in Nt Ab assay (patent 5 application titled: prevention and treatment of HCV infection employing antibodies //www.faqs.org/patents/app/20090202482 ). Human panel of 25 sera was generously supplied by J. Craske, at the Public Health 11 Laboratories, Manchester, UK. The samples were obtained from a longitudinal study of 12 patients infected previously with HCV through the use of contaminated blood clotting 13 factors. All sera were negative for HBV and HIV. Evidence of HCV infection was shown 14 by the presence of anti-HCV antibodies (Ortho Diagnostics) and confirmed by RIBA-II. 15Only sera containing anti-E2 antibodies were used in this study. Sera were divided into 16 those positive or weak positive for HCV RNA by PCR, (15 patients) and those that did not 17 have demonstrable RNA in their circulation but were positive for anti-HCV antibodies by 18 commercial serological assays (10 patients). Sera from individuals with no history of HCV 19 infection were used as HCV-negative sera (8 individuals). 20 random 15-mer peptide library displayed on filamentous phage and E.coli K91kan was 23 kindly provided by Dr. G. Smith, University of Missouri, USA. Polystyrene beads (1/4˝ 24 diameter) (Pierce, Rockford, IL) were coated with 1/1000 dilution of mAb3e5-1 or 25 in 0.1M carbonate buffer pH9.5 at 4 o C, overnight. All other steps were as 1 described before [D'

3 E2 Protein 4

 34 ; Partidos and 19 Steward, 1992] were synthesised using the Millipore 9050 synthesiser and Fmoc 20 chemistry as described by D'Mello et al [1997]. Peptides were characterised by high-21 performance liquid chromatography (HPLC), mass spectrometry and amino acid 22 sequencing. Mimotope K1 Peptide and the chimeric peptide had a single major peak with 23 a retention time of 5.7 and 20.79 minutes and molecular mass of the expected values of 24 1763 and 3309, respectively. The composition of the peptides was also verified by amino 25 . A synthetic peptide representing an epitope from the lymphocytic 1 choriomeningitis virus (LCM) was used as an unrelated negative control peptide. 2 Recombinant adenovirus RAdCMV-E2 (RAD-79) expressing E2 protein of HCV-BK type 5 1b (aa 368-693) [Takamizawa et al., 1991] and Adenovirus expressing β-galactosidase 6 (RAd-35) were generously provided by Dr. Gavin Wilkinson (University of Wales, 7 College of Medicine, Cardif). Detailed RAd-79 propagation, E2 purification method and 8 results are described elsewhere (El-Attar et al., in preparation). Briefly, RAd-79 was 9 propagated and titrated in 293 cells. 200pfu/cell of RAd-79 was used to infect COS-7 10 cells. RAd-35 was treated in same way as RAd-79 and used as negative control antigen to 11 allow discrimination between anti-E2 antibody and anti-adenovirus antibody which might 12 exist in human sera. 293 and COS-7 cells were grown in Glasgow's essential medium and 13

7 8

 7 Mice were bled at day 0 and two weekly intervals thereafter.18 19Antibody Binding Assays 20All assays were performed in 96-well plates (Maxisorb, Nunc or Immunolon2, Dynatech) 21 with reaction volumes of 50µl/well except for the blocking and stopping reaction steps 22 where 100µl/well and 25µl was added, respectively. Plates were coated overnight at 4 o C 23 (for peptides and phage clones) and at 37 o C (for E2 protein). All primary antibody 24 incubations were at 37 o C. Secondary antibody was horseradish peroxidase-conjugated 25 mouse IgG (Jackson) or rabbit anti-mouse (Dako) diluted at 1/1000 and applied 1for 1 hour at 37 o C. Washing buffer was PBS, pH 7.4 +0.05%Tween-20. Blocking buffer 2 was PBS, pH7.4 +10% pig sera. ELISA diluent was blocking buffer supplemented with 3 0.05% Tween-20. The reaction development was detected by the substrate ortho-4 phenlenediamine (OPD) from Sigma for 20 minutes at room temperature in the dark and 5 stopped by 2M H 2 SO 4 . The developed colour was detected by measuring the absorbance at 6 492nm using Multiscan plus, version 3.0.Phage Clones Binding Assay 9

  protein binding assays to insure the validity of the assay. Antibody titres 1 were determined as log 10 values of antibody titres obtained 4 weeks after boosting. The 2 cut off value for peptide assays was the mean OD of the highest concentration of the 3 negative group sera + 3SD. 4

  as the solid phase antigen was corrected by subtracting the OD value 1 obtained with the cell lysate expressing β-galactosidase. 2 b) Inhibition assay. A decreasing concentration of the competitor mimotope K1 or LCM 3

  Competition was measured as the inhibition exerted by any of the competitors for the 18 binding of anti-E2 containing human sera to the E2-coated ELISA plates, and calculated 19 as a percentage of the direct binding to the E2-coated plates. The assay was done in 20 duplicate on each occasion. The results were calculated as the percentage of binding of the 21 human sera in the presence of the competitor to direct binding to the E2-coated

21 and 21 Page

 2121 clones respectively. All 36 clones showed reactivity when screened by immuno-dot 22 blot to their specific mAb (results are not shown). DNA sequencing of the insert of all 23 enriched phage clones for both mAbs revealed an identical 15 deduced amino acids, F G E 24 F S F Q Y D C S S W G C, named mimotope K1. Analysis using the FASTA computer 25 and Lipman, 1988] showed a lack of similarity between the mimotope 1 K1 with any known human protein. 2

24 25

 24 and the 22 peptide mimicked by K1 overlaps with the sequence defined as CD81 binding region 2 23[START_REF] Yagnik | A model for the hepatitis C virus envelope glycoprotein E2[END_REF].Page

3 The

 3 all anti-E2 antibody positive sera detected, ranging between log 10 1.3 1 -3.1 (results not shown) compared to <1.3 in the negative sera. 2

3 Recognition

 3 1.9 -log 10 2.2 in these four sera. Some inhibition was observed with the 1 control peptide in two patients (15.61% and 19.11%). 2 Of The Mimotope K1 By HCV Human Sera With The Capacity[START_REF] Hadlock | Human monoclonal antibodies that inhibit binding of hepatitis C 6 virus E2 protein to CD81 and recognize conserved conformational epitopes[END_REF] Block E2 Binding To CD81. 5

  epitope and mAb 5E5/H7 that recognises a conformational epitope 1[Houghton M, personal communication]. mAb 5E5/H7 was of more interest as it was 2

Page

  giving the highest19 ELISA signal. The pattern of reactivity of mAb 5E5/H7 with distantly located peptides 20 suggested that these peptides are likely closely juxtaposed on the surface of the native E2 21 protein confirming the conformational dependant nature of this mAb 5E5/H7. Visual 22 inspection of the mimotope K1 showed possible contact residues within the domain 23 represented by the peptide 523 GAPTYSWG 530 namely SWG. The motif is common among 24 several HCV-1 isolates with the core motif WG identical in all HCV types including HCV 25 , the most divergent isolates and flanked by conserved residues, highlighting that 1 the mimotope K1 mimics a conserved domain among different HCV strains. Additionally, 2 the epitope mimicked by the peptide K1 overlaps with a region of E2 protein spanning a.a. 3 522-535, that is rich in turns and with an antigenicity index inclusive of a substantial 4 amount of β and coil regions (El-Attar et al, in preparation). The presence of tryptophan 5

  T-helper epitope (288-302) of the fusion protein of measles virus was 1

  However it was not feasible in the course of this study to measure the inhibition capacity 18 of E2-CD81 interaction and Nt activity in anti-mimotope mice sera due to the low anti-E2 19 antibody titre and the limited volume of available mouse sera. 20 Human antibodies elicited in response to HCV infection are anticipated to react with 21 native conformation of the viral envelope structure. We evaluated the mimotope K1 in 22 terms of recognition by a panel of human sera with various HCV profiles and examined its 23 capacity to inhibit their binding to the native protein. These sera were first screened for the 24 presence of anti-E2 antibodies. The level of anti-E2 antibody was generally low in 25

  responses against this protein, possibly taking into account the role of 1 protein and adjacent E1 sequences in determining the correct folding of E2. 2

18 ACKNOWLEDGEMENT 19 20Page

 19 We gratefully acknowledge Prof. Houghton for the gift of the mAbs 5E5/H7 and 3e5-1 21 and Dr. Wilkinson for the gift of the Adenovirus recombinant expressing HCVE2 (RAD-22 79) and β-galacotosidase. Also our sincere acknowledge to Prof. McKeating for the 23 professional assistance with regards to E2-CD81 binding assay. This work was sponsored 24

  reduced peak viremia after hepatitis C virus infection in 1 the chimpanzee. Hepatology 42(6):1429-1436. 2 Zhang J, Randall G, Higginbottom A, Monk P, Rice CM, McKeating JA. 2004. CD81 is 3 required for hepatitis C virus glycoprotein-mediated viral infection.

Table I . Phage yields obtained from phage display peptide library after three rounds of biopanning using/with mAbs 3e5-1 and 5E5/H7.

 I

				3e5-1		5E5/H7
		Incubation	Input	Output	Input	Output
	Round			% yield			%yield
		time	(TU/ml)	(TU/ml)	(TU/ml)	(TU/ml)
	1	overnight	1 x10 9 2.2 x10 4 0.001 5.8x10 10 3.45 x10 4 0.00006
	2	4 hrs	8 x10 9 9.3 x10 6 0.116	2x10 10	1.86x10 5	0.0009
	3	10 min	1.2 x10 9 2.8 x10 6 0.230 F o r	2x10 10	5.8 x10 6	0.029
				P
				e
				e r
				R
				e
					v i e
					w

Table II . Assessment of binding anti-mimotope peptide antibodies to E2 protein.

 II

	Exp. No.	E2 source	Mock antigen source		Anti-mimotope antibody (log10) titre	Mean titre (SD)	NMS (log10) antibody titre	Mean titre (SD)	P value
	1	supernatant PBS		1.72			1.69
					1.87		1.87 (0.16)	1.39	1.46 (0.20) 0.0492
					2.03			1.3
	. 2	supernatant PBS F o r	1.675 1.915 2.075		1.88 (0.20)	1.57 1.3 1.39	1.42 (0.14) 0.0292
			P
	3	supernatant PBS	1.97 e			1.39
					2.0 2.45 e r 2.14 (0.27)	1.40 1.41	1.4 (0.01)	0.0089
	4	supernatant PBS		1.675 1.9 2.15	R e 1.91(0.24) v 1.6 1.495 1.495 i e	1.53 (0.06) 0.0557
	5	E2-cell lysate*	Uninfected cell-lysate		2.0 1.96		2.02 (0.08)	1.39 1.44 w	1.42 (0.03) 0.0003
					2.12			1.43

Table III . Measurement of inhibition of E2-CD81 interaction in convalescent human HCV sera.

 III

							Reactivity with
	Patient No.		CD81 in presence of		inhibition
		PCR			% inhibition1		mimotope K1
	(all anti-HCV +ve)		serum (OD 450nm )1		status 1	peptide
	1	+/-	0.02* 0.50**	97%	++	+
	2 3	-ve +/-F 0.88 0.02 o r 0.87 0.19	-97%	-++	++ -
	4	+ve	0.03	0.56 P	96%	++	+
	5 6	-ve +/-	0.78 0.11	0.82 0.75 e e -86% r	-++	-++
	7 8 9 CBH-7 £ CBH-9 £	+/--ve +/-NA NA	1.11 0.83 0.02 0.11 1.07	0.92 0.82 0.22	--97% 86% -R e v --++ ++ -i e w	--+ NA NA

John Wiley & SonsJournal of Medical Virology

John Wiley & Sons

Journal of Medical Virology

John Wiley & Sons

Journal of Medical Virology


Thomas Reiberger

Nikolaos Malisiovas

Fylaktou Asimina

Papaventsis Dimitrios

Daoudaki Maria

Moskophidis Mathaios

Malisiovas Nicolaos

PhD Maria Daoudaki
email: daoudaki@med.auth.gr

Molecular epidemiology of chronic Hepatitis B virus infection in Greece

Keywords: Hepatitis B virus, Epidemiology, Greece, Genotypes, Mutations Genotypes, Mutations, An antigenic determinant

INTRODUCTION

Chronic hepatitis B virus (HBV) infection is an important public health problem, despite the availability of a vaccine. Worldwide, according to the World Health Organization (WHO), 360 million people suffer from chronic HBV infection [START_REF] Shepard | Hepatitis B virus infection: epidemiology and vaccination[END_REF], with an estimation of 600,000 deaths yearly due to the acute or chronic consequences of HBV infection [START_REF] Lok | Chronic hepatitis B[END_REF].

In Greece, HBV infection is considered a low to moderate endemic disease and HBsAg carrier prevalence ranges from 0.29% to 2.6% [Raptopoulou et al., 2008].

HBV is a partially double-stranded DNA virus of the family Hepadnaviridae with a genome of 3.2 kb containing four partially overlapping open reading frames encoding the polymerase (P), core (C), HBsAg and X proteins. Based on the antigenic determinants of the HBsAg, HBV was classified into nine serological types, adw2, adw4, adr, adrq-, ayw1, ayw2, ayw3, ayw4, and ayr, [Couroucé-Pauty et al., 1978]. HBsAg contains the 'a' determinant, (amino acid residues 124 and 147) which is common to all the HBV genotypes [START_REF] Weber | The diagnostic and clinical impact of the genetic variability of the S (surface) gene of the hepatitis B virus[END_REF][START_REF] Lada | Coexistence of hepatitis B surface antigen (HbsAg) and anti-HBS antibodies in chronic hepatitis B virus carriers: influence of "a" determinant variants[END_REF].

The heterogenicity of HBV serotype (based on single amino acid substitutions of the HBsAg), might have a positive selective value which can act as an immune evasion strategy [Simmonds, 2001]. Based on complete genome nucleotide sequence divergence of more than 8%, eight naturally occuring HBV genotypes (A-H) have been described [START_REF] Norder | Genetic diversity of hepatitis B virus strains derived worldwide: genotypes, subgenotypes, and HbsAg subtypes[END_REF][START_REF] Kurbanov | Geographical and genetic diversity of the human hepatitis B virus[END_REF]. There is a correlation between genotypes and antigenic subtypes, with a genotype associated with two or more different subtypes. Furthermore, subgenotypes have been identified within specific genotypes, based on more than 4% sequence divergence over the entire genome [START_REF] Norder | Genetic diversity of hepatitis B virus strains derived worldwide: genotypes, subgenotypes, and HbsAg subtypes[END_REF][START_REF] Kurbanov | Geographical and genetic diversity of the human hepatitis B virus[END_REF].

The prevalence of genotypes varies geographically, with genotype D being more common in the Mediterranean region. Subgenotypes within each genotype show distinct geographic distribution, except for those within genotype D which are found throughout the world [START_REF] Norder | Genetic diversity of hepatitis B virus strains derived worldwide: genotypes, subgenotypes, and HbsAg subtypes[END_REF].

Information concerning the genotype distribution of HBV in Greece is very limited [Katsoulidou et al., 2009]. In order to provide data on current HBV molecular epidemiology, the distribution of HBV genotypes, HBsAg subtypes and the mutations in the "a" determinant region, a retrospective study was conducted (years 2000-2007) among patients with chronic HBV infection in Thessaloniki, the second largest city in Greece. HBV genotypes were studied by partial sequencing of the surface gene, containing the HBsAg "a" antigenic determinant.

F

Serological assays

Routine ALT and AST determinations were performed (normal range 10-37U/l for both enzymes). The HBsAg, anti-HBc (total), anti-HBc IgM, HBeAg, and anti-HBe were determined by a commercial enzyme immunoassay method (Abbott Laboratories, Dallas, TX, USA).

HBV DNA quantitative analysis

The PCR-based assay COBAS-AMPLICOR (Roche Molecular Diagnostics, Indianapolis, IN, USA), was used for serum HBV DNA quantitation according to the protocol of the manufacturer [START_REF] Didomenico | COBAS AMPLICOR: fully automated RNA and DNA amplification and detection system for routine diagnostic PCR[END_REF][START_REF] Noborg | Automated quantitative analysis of hepatitis B virus DNA by using the Cobas Amplicor HBV monitor test[END_REF]. The HBV-DNA quantitation ranges from 60 to 38,000 IU/ml (300 to 200,000 HBV DNA copies/ml). (5'-CAA GGT ATG TTG CCC GTT TGT -3') and b) ΗΒ2 (5'-AAA GCC CTG CGA ACC ACT GAA -3'). The amplified region corresponds to the amino acid (aa) residues 101-186 of the S antigen chain which contains the adeterminant region (a.a 124-147). PCR products were separated by agarose gel electrophoresis. The clean-up of PCR products was performed according to the QIAquick PCR Purification Kit Protocol (Qiagen GmbH, Hilden, Germany) using a microcentrifuge and at least 20 ng/µl of viral DNA was present in the purified product.

Direct Sequencing of PCR products

The purified HBV DNA was sequenced directly by Qiagen Sequencing Services using an ABI 3700 DNA automated sequencer (Applied Biosystems, Foster City, CA, USA.). All sequences were analyzed in both, forward and reverse directions. The nucleotide sequence data reported in this study has been submitted to the DDBJ/EMBL/GenBank under accession numbers FJ178438 to 178573.

Determination of HBsAg Subtypes

The nucleotide sequences were translated into amino acid sequences according to the ORF of the partial S gene and the HBsAg subtypes were predicted from the amino acids at positions 122 (K for d and R for y determinants), 127 (P for w1-2, T for w3 and L-I for w4) and 160 (K for w and R for r) [START_REF] Magnius | Subtypes, genotypes and molecular epidemiology of the hepatitis B virus as reflected by sequence variability of the S-gene[END_REF]. Discrimination between ayw1 and awy2 was based on positions 134 and 159 (F and A, for ayw1 and Y and G for ayw2, respectively) [START_REF] Norder | Molecular basis of hepatitis B virus serotype variations within the four major subtypes[END_REF].

HBV Genotypes determination and Phylogenetic Analysis

HBV genotype was determined by sequence analysis of the 195 bp fragment from the HBV "a" determinant, using the genotyping tool available at the National Library of Medicine's National Center for Biotechnology Information (NCBI) [START_REF] Rozanov | A web-based genotyping resource for viral sequences[END_REF]. In addition, HBV genotype of each isolate was deduced by comparison of the partial surface protein sequences with the sequences of the corresponding genomic regions of all HBV strains available in the GenBANK/EMBL/DDBJ database by using the BLAST program, version 2.2.8 [START_REF] Altschul | Basic local alignment search tool[END_REF][START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF]. A phylogenetic analysis was carried out by pairwise comparison of the partial S gene sequences of representative HBV strains for all genotypes from the GenBANK/EMBL/DDBJ database with the 135 sequences obtained from the sera of the patients with chronic HBV infection, by using ClustalW [START_REF] Thompson | CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[END_REF].

Construction of the phylogenetic tree was carried out with ClustalX (version 1.83) by using the alignment file, obtained by analysis with ClustalW. By using this program the distances between all pairs of sequences were calculated, and this was followed by application of the neighbor-joining method to the distance matrix. Confidence values for the groups in the tree (bootstrap values on a scale from 1 to 1000) were also calculated by using ClustalX.

RESULTS

HBV serology and DNA viral load

A total of 135 HBV carriers (93 males, 42 females) with ages ranging from 9 to 92 years (mean value 45.5 years), were enrolled in the study (Table 1). Of the 135 patients, one hundred twenty-four patients were ethnic Greek residents of Thessaloniki (Northern Greece) while the rest were immigrants from Albania (five patients), China (two patients), Turkey (two patients), and Georgia (two patients).

All patients with chronic HBV infection had elevated aminotransferase levels. Mean ALT and AST levels were 238 IU/L (SD= 576.84) and 152 IU/L (SD= 282.65), respectively. All patients were HBsAg positive, and the majority of them (62.5%; 84/135) were anti-HBe positive. Thirty-five per cent (47/135) of patients were HbeAg positive, while 3% (4/135) were both HbeAg positive and anti-HBe positive. Anti-HBc seropositivity was detected in 98% of the study population. In 4.4% (6/135) of patients, simultaneous presence of HBsAg and anti-HBs was detected. Serum HBV-DNA levels ranged from 1.02x10 5 to 2.2x10 7 IU/ml. Patients positive for both HBeAg and anti-HBe, had significantly higher DNA levels than HBeAg negative patients (2.29x10 6 vs. 1.3x10 6 ; p<0.01).

Determination of HBV Genotypes

The results obtained by analysis of the nucleotide sequences from the 135 serum samples tested, are summarised in tables 1-3 and the phylogenetic trees in Figures 1 and2

Determination of HBsAg Subtypes and association with HBV Genotypes

Partial sequencing of the S gene predicted the HBsAg subtypes in all but one case. Isolates from genotype D specified exclusively subtype ayw2 (96/132, 73%) and subtype ayw3 (36/132, 27%). Almost all these isolates grouped together in HBsAg subtypes in the phylogenetic tree (Fig. 2). The only strain belonging to genotype A specified subtype adw2, was isolated from a chronic HBV native Greek carrier. In addition, the one strain belonging to genotype C was detected in a Chinese immigrant and belonged to subtype adr. The second strain detected from another immigrant from China belonged to genotype B, but the HBsAg subtype was not specified, as the HBsAg aminoacid sequence was only readable between positions 121 and 154. The No unusual genotype/HBsAg subtype combinations were found. The association of HBsAg subtypes with genotypes and seroconvertion status is shown in tables 2 and 3, respectively. The ayw2 HBsAg subtype was predominant in both the HBeAg positive and the anti-HBe positive patients with 74% (32/43) and 71% (58/82), respectively. The ayw3 subtype was less common with 26% (11/43) in the HBeAg positive patients and 29% (24/82) in the anti-HBe positive patients.

HBV genetic diversity

Single or multiple point mutations were found in 26% (35/135) of the cases as shown in Tables 1 and3. Some of the most common mutations occurred at amino acid positions 129 and 134 (5 strains), 133 and 145 (4 strains, including the 'vaccine escape' mutant G145R), and 128, 144, 164, 174 (3 strains each). Most common mutations observed were M133T, Y134N, and S174N (3 isolates). In the anti-HBe positive group, the mutation rate was significantly higher in patients with the ayw3 subtype compared to the ayw2 group (42% vs. 15%; p<0.01). Such difference was not observed in the HBeAg positive group (27% vs 22%; p>0.01). Multiple point mutations were almost exclusively observed in the anti-HBe positive group and in two of the four patients belonging to the HBsAg positive /anti-HBs positive group (Fig. 3).

Most strains were specified as subtype ayw2. Interestingly, 5 and 4 point mutations were found in the two patients from the HBsAg positive / anti-HBs positive group (Fig. 3). In the first case, an HBeAg positive leukemic patient

DISCUSSION

In this 8-year retrospective study, important data were obtained on the HBV genotypes/subtypes and mutations in HBsAg in chronic HBV carriers in Thessaloniki, Greece. Two HBV genotypes A and D, were found among native Greeks, with genotype D being almost prevalent exclusively (D: 98% vs. A: 1%). Genotype D was also the only genotype found in Turkey [START_REF] Bozdayi | Complete genome sequence and phylogenetic analysis of hepatitis B virus isolated from Turkish patients with chronic HBV infection[END_REF]Ozasln et al 2007] and the predominant genotype in Serbia [START_REF] Lazarevic | Distridution of HBV genotypes, subgenotypes and HBsAg subtypes among chronically infected patients in Serbia[END_REF].

The remaining 1% derived from two HBV strains from Chinese immigrants, were specified as genotypes B and C. The one isolate belonging to genotype A in this study was grouped with a reference sequence for A2, suggesting a European origin. As expected for samples from the European population, this strain belong to subtype adw2.

The presence of both HBsAg and anti-HBs, was found in 4% of the study population. The selection of HBsAg variants is a possible mechanism underlying the presence of both HBsAg and anti-HBs. Residue changes in the "a" determinant of the S protein, the main target of anti-HBs antibodies, could lead to escape from recognition by the host immune system. However, HBV escape mutants may also occur naturally in chronic HBV carriers due to the exclusive pressure of the host immune system. The results of the present study are comparable to those reported by [START_REF] Yamamoto | Naturally occurring escape mutants of HBV with various mutations in the S gene in carriers seropositive for antibody to hepatitis B surface antigen[END_REF], [START_REF] Kohno | Mutations in the envelope gene of hepatitis B virus variants co-occurring with antibody to surface antigen in sera from patients with chronic hepatitis B[END_REF] and [START_REF] Lada | Coexistence of hepatitis B surface antigen (HbsAg) and anti-HBS antibodies in chronic hepatitis B virus carriers: influence of "a" determinant variants[END_REF].

The direct sequencing of a relatively short fragment of the HBV genome was shown to be efficient for the determination of both viral genotype and HBsAg subtype, as well as mutations with potential impact on the HBsAg

  This was an 8-year retrospective analysis of samples of 135 chronic HBV carriers admitted to Papageorgiou Regional General Hospital of Thessaloniki during the period from December 2000 until January 2007 for routine HBV DNA detection and quantitation. All patients, all their parents/guardians in the cases of children signed an informed consent for the protocol study, which was approved by the Local Ethics committee. Socio-demographic characteristics (gender, place of birth, place of residence and age) and medical history were recorded. Patients with HCV or HIV co-infection were excluded from the study.

  from 200 µl of each serum sample by using QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. The HBV DNA sequence of 259 nucleotide pairs (329-587) from the HBsAg region, was amplified by PCR with primers a) HB1

9

  Dendrograms showing the phylogenetic relationships among the HBV isolates of the present study and prototype strains was plotted in the PHYLIP format output by using the TreeView software (version 3.0), which was obtained from the website of the University of Glasgow.StatisticsStatistical analysis of data was performed using the Statistical Program for social sciences (SPSS version 16.0). Results were presented as mean values +/-standard deviation (SD). Comparisons between patients groups were performed using the students t-test or nonparametric Mann-Whitney U test. A p value <0.05 was considered significant.

  . The sequences grouped within clusters A, B, C, and D of HBV. None of them grouped within genotypes E, F, G or H. All genotype clusters were supported by significant bootstrap values. Agreement between the plylogenetic analysis and the NCBI genotyping tool results was observed in all cases and an HBV genotype was assigned to all but one samples. The distribution of HBV genotypes found among the 135 serum samples was 98% for genotype D, which was almost exclusively prevalent, 1% for genotype A, and 0.5% for each genotype B and C, which were exclusively found in Asian immigrants.Recombination of the HBV genes from different genotypes was not observed.The sequences of the partial S gene from the 135 patients participated in the study are available at the GenBank/EMBL/DDBJ Database with accession numbers FJ17843-FJ 178573.

  the sequence seemed to specify subtype ayw (Thr 126 ), while the second part seemed to specify subtype adw (Thr 143 ).

  immunosupression, the point mutations were S136F, P142L, S143L, D144E, and the 'vaccine escape' mutation G145R. In the latter case, an anti-HBe positive chronic HBV carrier, the point mutations were T123N, M133L, G145R, and V177A.

  studies on HBV S genes are important in understanding the failure of protection with current HBV vaccines and the conflicting results of HBsAg detection with different diagnostic kits. This can be due to the fact that the specificity of the antibodies used in diagnostic assays and vaccines are targeted to these regions, and especially the "a" determinant. The results from this study, confirmed the good performance of the tests used, even in the presence of multiple point mutations.In conclusion, data from this study contribute to the investigation of HBV genotype and HBsAg subtype distribution in Greece. Partial S gene sequence analysis determined that the HBV genotype D and ayw2 HBsAg subtype, were predominant in patients with chronic HBV infection from northern Greece. The clustering of all DNA sequences into genotype groups corresponded to their respective HBsAg subtype. Analysis of the point mutations revealed that amino acid substitutions at immunodominant epitopes involved in B or/and T cell recognition did not affect detection by commercial immunoassays. Additional data will be required in order to determine the frequency of HBV genotypes in Greece.

  Fig.1

Table 2 .

 2 HBsAg Subtypes association with HBV Genotypes

	Genotype

Table 3 : Correlation of the HBV serology profile with the different subtypes, mutations in the a determinant.

 3

	HBV serology profile	Total	ayw2	ayw3	Mutations	HBV-	ALT
		no (%)				DNA	IU/ml
						(IU/ml)
	HBsAg+/HBeAb+
		82	58 (71%)	24 (29%)	ayw2 (15%), ayw3 (42%)
		(61%)
				HBV829GR	A128L
				HBV1010GR	A128V
				HBV548GR	A128V
				HBV641GR	D144E
			HBV998GR		E164G, S174N, S167L
				HBV205GR	F158L
				HBV275GR	M133T
				HBV400GR	M133T
				HBV895GR	M133T
				HBV1059GR	P127A, Q129R
			HBV1387GR		P142T, D144V
			HBV868GR		Q129G
			HBV552GR		Q129H,G130R
			HBV1141GR		S174N
			HBV204GR		T126S
			HBV982GR		T131I
			HBV785GR		Y134H
			HBV863GR		Y134N
				HBV1586GR	Y134N

ACKNOWLEDGEMENTS

The authors would like to thank all medical doctors and technicians working in the Virology Laboratory, Papageorgiou Regional General Hospital, Thessaloniki, Greece, that assisted in the study. The authors would also like to acknowledge Abbott Hellas for financing the S gene partial sequencing of the HBV strains.

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