CA2251745A1

CA2251745A1 - Non-a-non-e hepatitis virus having a translatable core region, reagents and methods for their use

Info

Publication number: CA2251745A1
Application number: CA002251745A
Authority: CA
Inventors: Daniele Primi; Sonia Mattioli; Gianfranco Fiordalisi
Original assignee: Individual
Current assignee: Diasorin International Inc
Priority date: 1996-04-17
Filing date: 1996-12-09
Publication date: 1997-10-23
Also published as: EP0914445A1; AU1109297A; WO1997039129A1; ITRM960245A1; IT1284630B1; ITRM960245A0

Abstract

It is described a non-A-non-E hepatitis virus (GBV hepatitis virus) having a tra nslatable functional core region, polynucleotides, peptides, antibodies, diagnostics and reagents thereof and methods for their use .

Description

NON-A-NON- E HEPATITIS VIRUS HAVING A TRANSLATABLE CORE REGION, REAGENTS AND METHODS FOR THEIR USE
The invention concerns nonA-nonE hepatitis virus (GBV hepatitis virus) having a translatable 5' core region, polynucleotides, peptides, antibodies, diagnostics and reagents thereof and methods for their use.
Simons J.N. et al. (Nature Medicine, 1, 564, l995) revealed a viral genome, Flavivirus-like, named GBV-C, in a small percentage of nonA-nonE hepatitis affected patient sera. The genome has some homology with genomes from two virus, GBV-A a GBV-B, identified in tamarins (Simons J.N. et al., Proc. Natl. Acad. Sci. USA, 92, 340l, I995). Said virus represent a phylogenetic separate group, not comprising hepatitis C virus (HCV).
PCT patent application N. W095/21922, in the name of Abbott Lab., shows a partial nucleotide sequence of a GHV-C isolate. A method to reveal antibodies against GBV
is claimed (claim 44, p. 6l8); however no enabling disclosure for such method able to detect with a statistic significative percentage positive samples from nonA-nonE patients is provided. PCT patent application N.
W095/21922 does not disclose aminoacid sequences to be effectively used to design diagnostic peptides. As a matter of fact, Figure 39 shows a panel of proteins, having an around 30d aminoacid minimal lenght. Only 2 out 512 sera from drug-addicted subjects result to be positive for these proteins in an ELISA assay. Moreover at p. 168, table 23, ELISA assays on nonA-nonE patients result to be positive in very few samples, thus rendering the assay not workable: 0/89 Japan subjects; 0/67 Greece subjects; 6/72 USA (set M) subjects; 1/64 USA (set T) subjects; 3/62 USA (set 1/3) subjects; 18/32 Egypt subjects. At p. 169, table 24, only 21 out 303 nonA-nonE
patients are positive with an ELISA assay using peptides sequence. It is evident that said assay may not be used neither for a screening of healthy subjects, nor for diagnosis.
PCT patent application N. W095/32291, in the name of Genelabs, identifies by ~epitope mapping different antigenic regions of the GBV polyprotein. However no peptides or a combination thereof for a diagnostic assay are disclosed.
EP patent application N. 73660l, in the name of Abbott Lab., discloses 88 nucleotides upstream of the sequence shown in the PCT patent application N.
W095/21922. However no evidence is provided for an open reading frame in this region able to code for a core-like protein.
The nucleocapsid/El junction of the viral isolate was identified by Leary, T. P., et al. 1996. J. Med.
Virol. 48:60-67. It is predicted to occur approximately 46 aminoacids downstream of the first methionine residue of the ORF, while Linnen, J. et al. 1996. Science 27 1:505-508 have also identified an isolate in which the putative capsid protein contains 83 aminoacid residues.
The apparent main difference among these two isolates is one base deletion causing different initiation sites.
Thus, in comparison with other Flaviviridae, in which the lenght of the capsid protein varies from 114 aminoacids in dengue fever virus to 175 residues in HCV, the GBV-C
nucleocapsid appears too short to be functional.
The presence of core-like proteins in GBV-C has also been challenged by recent translation studies demonstrating that the site of translation initiation of the viral polyprotein is immediately upstream of the putative signal sequence for the El glycoprotein (Simons, J. N., et al. 1996. J. Virol. 70:6126-6135).
Although it was originally suggested that the apparent lack of a functional core protein could be ascribed to artefacts of the sequencing process, due to compressions caused by the high GC contents (teary et al.
supra), it is now clear that these sequences represent the true status of GBV-C in the numerous isolates that have been characterized (Simons et al. 1996, supra).
It has been proposed, therefore, that the NH,-terminal end of the GBV-C polyprotein is a truncated derivative of an ancestral capsid protein and that GBV-C
requires a helper virus to replicate (Heringlake, S . , H.
L. Tillmann, and M. P. Manns. 1996. J. Hepatol. 25:239-247). Alternatively, since a limited number of virus clones from each isolate has been examined so far, it is possible that, after infection, a large population of single base deletion mutants arise and these mutants would have the highest probability of being examined.
The authors report the characterization of a GBV-C
isolate, obtained from a HIV and HCV positive patient, in which translation can initiate at an AUG codon located 273 nucleotides upstream of the previously identified initiator site (Simons et al. 1996, supra), leading to a potentially functional nucleocapsid core protein of 107 aminoacids. This phenotype remained unvaried over the one year monitoring of the patient, suggesting therefore that stable replication competent GBV-C isolates may exist in nature.
The authors of the instant invention have also identified NS3 peptides of nonAnonE associated virus and has set up an assay to detect antibodies able to recognize epitopes comprised in said peptides. Said antibodies are detected in chronic and acute hepatitis affected subjects, classified as nonA-nonE for the lacking ef known virus related markers, in a high percentage of studied subjects (around 300). Surprisingly the assay is able to detect said antibodies also in subjects which are affected by other pathologies, as hepatitis A, hepatitis B, hepatitis C, hepatitis Delta, ~.I~J~, ~j~~i" v~~,,-dStr:..~.L~e:-~ S.", ~"'~~..~'y dU.~CI:TL~,'_:i.~
i.'_~S~dSeS
(LES, s'~NA+) r Show~rig 3 ~''iatwCrn~:'z1p ~r=G:_ v:E''.' ;;~~
The assay is per formed preferentia? 1;: t~~ react' ng a treated bzolcgzca~. samp~.e, as serum, ~i.t=~. Je~.ected peptides to ge4 an eiFectve and d'agnos=is method.
Antigen~anti.bodyes complexes are revealed in an effective ar.d reprcducir' a way by means of one of assays ~.-~cwr to the expert .in the ficid, as ELIcA, RIB:, EIA, i:nmunoblottin, atc.
1G y.~_~_ the foliow;;ny "~L~I vir~:s" means v' wa associated t4 non.~~or.E ~:epa=i t::s; "izu~zunoloc~.cail~r r~omolcgcus epi tope" means an ept =ope ::,at% irlg an a:~r.inca:.~.d sequence w~ir_h d'_~fers 'nom th~ reFe=r'inCj v'~?ltd~L~2 Cv~ ~:Tln or m:J~~?
y am~.ncac~.ds, but whic:~ i.s able to recc~nrze anc ~oi:.c. ~o antibodies for the ~e''errng epitone, with 5u:os;.ar~~-ial' y the sa~:.e binui::g a'fi :i ty.
It is a n object of the ~.;~~:er_ticr_ a ~oeptic'e ab'e ~to s: eCif::cally bir:C tc no:~..~r_onE hepat_tis visas a~~~~~oa,zas, having a =equer_ce cf a= least ~C,.

a~ inoaci s, said non.Anc.~.~ hepat. ti s virus 'oei ng characterized by hcs7~.Il~~ a trar.slatabie =~nc~i~.na_ core-;ikA ~rorswn 'onger then ~~ arninr_acid res~d~~;es a:~d by CC="lpriSl::~ ~.-~ d~.'Ll._"lG3Cid Sac~u,eIlCE'.~.~ ~~aS~

hczrc'_cgeus to the secrlence of SEQ I~ ;do.

4:

25 '~SLrIPa.R'!PFULLTPT'IGFP,~PS, or an i.;unu:-:olcgically ho:noloc~ous variant cr the peptidA. Prefer aiy the peptide ~.as a 5equer~ce ccr~rrised in SEQ Iu Nc.4.

It is a Further object of tha i::vention a ccnposi~zor_ ccmpris~:~lQ at _east one o the peptides of the i nTrention. Prezerably said cor_p,csi ticn is adhered Jn a SO1 ~ a ptla$e .

A.ccarding tc a preferred. embcdi.-~,A-:t of the in~.Antion said c:ornpcsition comprises a'~ least tt~rc pepy~.~~ the irler.tion.
~.-'1"s ~ a.

AMEN~rp SH~.ET
r~ ~n" ~r,~. ,.: ~~ 7r, i ~Ln~ .~.; . ,nm _fty.. .,.~r_.'lrJ'', t7 ~,..~
_ . _. ~. , , _ . -.Z; r~. _ _ ~~ prr~-r nr~.
_ _..~.., ...,_.............,..~.~.._~.__ ... _...._....,a - ' ~ ~ : . _ ,.~
:j.. . I~: _ . ~ \ . ) ; ~

_ 5 _ F~azther object cf the invention is a diagnostic ki: for nonRror.~, hepatitis carnprising as specific reagent at least one peptide o~ Lha invention.
Preferably said kit comprises at least twe oei t~.des of t'.:e invention.
Furtrer object o~ ~.he i nventicr is a rr.ethod to detect ::cn.~, ron~ hepa=iris virus antibodies in a sa.~.-~ple c..,rnprisi:~g the following steps:
- inc~,:,ating in conditions allow':.nc t_:e for~!a~ion cf a.. artigen-aztibcdy co~:p~.e:c said sample with at least or.e peptide ;:f the inve:aion;
reveali-g saia cemp~.ex.
Tie izvsnti=n is new desl~r:.bed i n a non- i imi c'_ng .way by reference tc the following figures wherein:
r - riguro 1 renYeser_ts ~he ~r~.i:.eacic sect:enea o_ GDv N53 region dod of used peptides, with their dEnomination:
A~nrnlnr~l ~:~rrT
IJILI'1'Jv. ' ~~. ~ I _ ~mr ~~n~ "~I, ~:on~~ ,,.,;~_.~.,.: o .-m;i n , i '.. . .. . , ~ ' ~ i f i , ,n _ . ~ ~ . a I r"1 ~1 ~ . .
- . . I , . , '. I y. , , ~ ' I , , i . v a 4 W~ 97/39l29 PCT/IT96/00248 - figure 2 represents a bar graphic of p3.1-p3.9 peptide reactivity in ELISA assay with sera from subjects affected by different pathologies;
- figure 3a a 3b represent aligned aminoacid sequences of GBV core region; first three from top: from literature, others: isolated from the authors; "-" means an identity with the U36380 sequence; "_" means an identity with the U45966 sequence;
- figure 4a represents aligned aminoacid sequences of a segment of GBV core region, from literature: the first from the top from U45966 of Fig. 3a, others are isolated from the authors; at the bottom the consensus sequence is shown (SEQ ID No. 3);
- figure 4b represents a bar graphic of consensus peptide of Fig. 4a reactivity in ELISA assay with sera from subjects affected by different pathologies;
- figure 5 represents the nucleotide sequence alignment of 10 GBV-C isolates obtained from 10 distinct GBV-C positive patients, compared with the sequences of the published isolates deposited in GenBank [GBV-C
isolate (teary et al., supra), PNF2161 isolate (Linnen et al. supra) named GBV1 in the figure, and R10291 isolate named GBV2 in the figure]. The sequences were analyzed with the CLUSTAL alignment program. The alignment includes the nucleotide sequence between AC1S and AS-CWP
primers (shadowed in grey), and comprises a partial 5'UTR, the putative core region and a partial E1 region (corresponding to nt I12-733 of GBV-C, nt I29-752 of GHV1 and nt 56-680 of GBV2). The nucleotides are numbered only for isolate 35 (identified as the isolate with the longer ORF) and nucleotide +1 identifies~the nucleotide A of its first putative start codon in frame with the long ORF.
Note that in the other isolates nucleotide insertions or deletions are present. Gaps, introduced in the sequences to preserve alignment, are represented by dashes. The sequences between nucleotides 316 and 446, that are conserved, are not shown for reason of space. The nucleotides conserved among a11 the sequences are pointed out by an asterisk. The ATG codons in frame with the long ORF of each isolate are boxed;
- figure 6 represents the aminoacid sequence alignment of the isolates described in Fig. 5 and deduced from their nucleotide sequences. The aminoacid sequence of isolate 35 is identified as 35/1, while the sequences deduced from the two additional isolates derived from distinct serum samples collected during the follow-up of this patient are pointed out as 35/2 and 3S/3. The sequences were analyzed with the CLUSTAL alignment program. The sequence of the longer published ORF (GBV2) is considered as the consensus sequence. Dashes denote identity with the consensus sequence, whereas individual variations are shown by the single aminoacid code. The methionines in-frame with the long ORF are boxed and pointed out with an arrow for each isolate. The areas shadowed in grey mean the regions of high variability detected in our isolates;
- figure 7 represents a SDS-gel pattern of [3~S]cysteine-labelled in vitro translation products of the constructs, containing the AC1S/AS-CWP amplified region from the pointed out isolates. Positions of molecular weight standards (in kilodaltons) are pointed out. The last lane contains the negative control (no RNA). The IVTT of pCR35/AC1S construct was performed with and without capped mRNA;
- figure 8 represents the construction of the deletion mutants of pCR35/AC1S, pCRl9/AC1S, pCR3064/AC1S
and pCR2095/AC1S vectors. In the figure, the strategy for the construction of the pCR35/AC1S derivatives is described. The location of the T7 promoter (in black), the Bam H1 cleavage site and the position of the primers (AC1S and AS-CWP) used for the construction of the parent plasmid are shown. The numeration of the nucleotides is relative to the first putative start codon of the sequence of isolate 35. The partial 5' non coding region is white, the partial ORF is shadowed in grey and the position of AUG codons are pointed out with the notation Met. The positions of the sense primers (primers 1, 2, 3, 4 and 5) used with the antisense AS-CWP primer for the construction of the deletion mutants are also shown. The same strategy was followed for obtaining the pCRl9/AC1S, pCR3064/AC1S and pCR2095/AC1S derivatives. The length of the partial 5'UTR, of the partial ORF and the position of the AUG codons of these isolates are shown in Fig. 5 and 6.
- figure 9 represents a SDS-gel pattern of ~35S~cysteine-labelled in vitro translation products gLenerated by the O2, 43, 44 and 05 deletion mutants of pCR35/AC1 (A), pCRl9/ACIS (B) and pCR2095/AC1S (C) plasmids, obtained as described in Fig. 8. Positions of molecular weight markers (in kilodaltons) are pointed out. In panel (A)-the products of capped and uncapped mRNAs derived from the pCR35/AC1S undeleted plasmid are shown. A11 the other mRNAs used in these experiments were capped transcripts. In the same panel the first lane represents the product of translation of an unrelated mRNA (UNR). In panel (B) the first lane represents the product of translation of the pCR35/~, mutant, that was used as control for the size of the product of isolate 19. In panel (C) the first lane contains the negative control (no RNA) .

Analysis of GBV protein sequences Protein sequences have been computer analyzed by means of ANTIGEN program 6.85 (PCGene). ANTIGEN utilizes Hopp and Wood algorithm to predict protein potentially hydrophilic regions.
Peptide synthesis Peptides have been synthesized with an automated synthesizer MilliGen 9050 (Septrim). A11 of aminoacids were ~noc a-amino protected and pre-activated at the carboxy terminus pentafluorophenyl esters; only serine and threonine were activated~as benzotriazine esters.
A11 of coupling reactions were performed with HOBT
0.33M/DMF; washings were made with DMF and the a-amino group was released with 20o piperidine in DMF. Peptides were released with from resin by treatment with a mixture of TFA and scavengers which differ according to the composition.
Cases sera from 52 healthy donors controlled to be HBV-, HCV-and HIV-;

sera from 27 acute nonAnonE hepatitis patients;

sera from 16 chronic nonAnonE hepatitis patients;

sera from 61 acute hepatitis B patients;

sera from 14 chronic hepatitis B patients;

sera from 15 acute hepatitis C patients;

sera from 15 chronic hepatitis C patients;

sera from 29 drug addicted HIV-, HCV+ patients;

sera from 31 HIV+ patients;

sera from 33 HIV+ piastrinopenic patients;

sera from 30 AIDS patients;

sera from 39 LES patients.

ELISA assay The solid phase was prepared onto polivinylchloride dishes (N unc), by using a peptide or a mixture thereof.

Peptides were dissolved separately in a mixture of 500 CH3CN/H20 and diluted to a single final concentration of 10 ug/ml (total peptide concentration of 20 ug/ml) in carbonate buffer 50 mM, pH 9.6. 200 ul were added to each well and incubated for 2 hrs at 37C. When a single peptide was used, the final concentration was 20 ~g/ml.

Following to peptide adsorption, solid phases were saturated for 1 hr at 37~C with 300 ul/well of a BSA 2' solution in Tris-HC1 0.1M pH 7.5.
200 ul/well of 1:21 diluted sera in a solution of 5 PBS containing 0.3 g/1 EDTA,. 2 g/1 BSA, 2 ml/1 Triton x100 and 2 ml/1 Tween 20 were incubated 2 hrs at 37~C.
Following to 4 washes with PBS/Tween 20 0.1;, dishes were treated with 100 ul/well of horseradish peroxidase conjugated anti human Ig (Amersham). 1:30.000 diluted in 10 a PBS solution containing 0.04o Tween 20 and 20o non immune goat serum.
Following to 4 washes with PBS/Tween 20 O.lo, l00 ul/well of chromogen/substrate were added (Sorin Biomedica Diagnostics, DEIA KIT). The colorimetric reaction was stopped after 30 min. by adding 200 ul/well of sulphuric acid 1N. The optical density was read at 450 nm by referring to 630 nm.
CORE REGION
Reverse transcription (RT)-PCR
Analysis was performed with serum samples from 10 distinct subjects resulted positive for the presence of GBV-C RNA with the RT-PCR and hybridization assay as previously described (Fiordalisi, G., et al. J. Infect.
Dis. 174:l81-183). Four of them (patients identified with the codes 35, 19, 47 and MG) were HIV+/HCV+ patients with chronic liver disease; DM and 1159 were non A-E acute hepatitis patients; BZ was a patient with hepatocellular carcinoma; 3Q64 was a patient with fulminant non A-E
hepatitis; patient 2095 was an HCV+ subject with chronic hepatitis; and finally DS39 was a healthy blood donor.
Total RNA was extracted from 100 ul of serum samples of these subjects with TriPure reagent (Boehringer Mannheim Germany) and subjected to RT with random hexanucleotides using Superscript II (Life Tecnologies, Gaithersbul-g, MD). The obtained cDNA was used as template for PCR
reaction with degenerated primers, designed in our laboratory and derived from the sequences of GBVC and GBV
isolates deposited in Genbank database (Leary et al., Linnen et al. supra). The sequences of these primers, located in the 5'-UTR and putative structural regions, were:
AC1S sense primer:
5'AGG GTT SGW WGG TSG TAA ATC C 3' (SEQ ID No. S) AS-CWP antisense primer:
5'CGC CTG RTA NAR NGG CCA RCA 3' (SEQ ID No.6) wherein S = C or G; W = A or T; R = A or G; N = A or C or G or T.
Each of the 45 cycles of PCR included 1 min at 94~C, 1 min at 52~C and 1 min at 72~C, with an elongation time of 10 min in the last cycle. The sequence of the PCR
product obtained with these primers corresponds to nt 129 to 752 of GenBank accession no. U44402, the GBV-C isolate with the longest core-like protein reported to date (Linnen et al. supra).
Cloning and sequencing The amplification products obtained with AC1S and ASCWP primers were inserted into pCR2.1 vector (Invitrogen, San Diego, CA) and the sequences of the cloned inserts were obtained using the A.L.F. DNA
sequencer (Pharmacia Biotech, Uppsala, Sweden). The sequenced plasmids were named pCR X/AC1S, where X
corresponded to the isolate derived from the 10 patients considered in this study. Nucleotide and aminoacid sequences were analyzed with the CLUSTAL alignment program included in PC/GENE software package (Intelligenetics Inc., Mountain Vlow, CA).
Construction of deletion mutants' Mutants with deletions of different portions of the 5' region of pCR35/AC1S, pCRl9/AC1S, pCR3064/AC1S and pCR2095/AC1S constructs were derived from these plasmids by PCR amplification using synthetic primers. The antisense primer was the AS-CWP primer described above, while the sense primers, designed in our laboratory and derived from the sequences of our GBV-C isolates, mapped just upstream of the first 5 putative start codons on the basis of the sequence of isolate 35 (Fig. 5 and 6), as 5 described in Fig. 8.
Primer 1: nt -43/-27 5'ACGTAAGCTT A GGT GTW GGY CCT ACC G 3' (SEQ ID. No.7) Primer 2: nt +48/+64:
5'ACGTAAGCTT G TAC GGY CCA CGT CGC C 3' (SEQ ID. No.8);
Primer 3: nt +99/+117:
5'ACGTAAGCTT G CGA GTT GRC AAG GAC CAG 3'(SEQ ID. No.9);
Primer 4: nt +167/+Z83:
5'ACGTAAGCTT GR RVC GGG AAA TGC ATG 3'(SEQ ID. No.lO);
Primer 5: nt +239/+255:
15 5'ACGTAACCTT TG AGG GCG GGT GGC ATT 3'(SEQ ID. No.l1), wherein underlined nucleotides correspond to specific sequences of GBV-C, W = A or T; Y = C or T; R = A or G; V
- C or A or G. The position of the primers corresponded to the nucleotide numbers obtained considering as +1 the 20 nucleotide A of the first ATG in the ORF of isolate 35 (see Fig. 5) . The products of PCR with 1, 2, 3, 4 and 5 sense primers and AS-CWP antisense primer were cloned in pCR2.1 and named respectively pCRX/01, pCRX/02, pCRX/~3, pCRX/~4 and pCRX/05, where X corresponded to the 25 identification code of the isolate (35, 19, 3064 and 2095y .
In vitro transcription and translation (IVTT) Plasmids were linearized by digestion with BamHl and transcribed in vitro with T7 RNA polymerase (mCAP mRNA
30 Capping kit, Stratagene, La Jolla, CA) according to the manufacturer's instructions. In most cases, a m'GpppG cap structure was incorporated at the 5' end of the RNA
transcripts. In vitro translation reactions were performed with a rabbit reticulocyte lysate (RRL) system 35 (Amersham, Buckinghamshire, England), according to the manufacturer's instructions. Reactions (25 ul) contained 20 U of rRNasin (Promega, Madison,WI), 20 uCi of ['SS]
cysteine (l.000 Ci/mmol, Amersham, England) and 1.5 ~g of in vitro transcribed RNA. After incubation at 30~C for 1 hr, 2 ul aliquots were denatured for 5 min at 100~C in an equal volume of 2 X sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and analyzed on 18o SDS-polyacrylamide gels.
Protein gels were dried and exposed to X-ray film.
IO RESULTS

The analysed peptides are comprised in the NS3 region, from aa. 1346 to aa. 1550 (SEQ TD No.l), and are shown in Figure 1.
All of peptides were adsorbed to the solid phase at different concentrations and confronted with a panel of sera from nonAnonE affected patients and healthy donors.
ELISA assays were performed with different concentrations and adsorption conditions. Results show that peptides of Table 1 react with sera with different binding affinities. The reactivity may be increased by previous adsorption to the solid phase of a mixture of different peptides.
The G20T peptide has a decreased activity given to a cysteine dimer at position 1467. The cysteine to serine (G20T(C->S)) substitution solves this stability problem.
In order to get good reactivities solid phases should be prepared either with a mixture of l0ug/ml S16V
and 10 ug/ml G20T(C->S) or with E21A peptide at 20 ug/ml.
To determine the cut-off reactivities of 200 sera from healthy donors were tested and the following formula was applied:
OD cut-off = average OD healthy donors + 8 s.d. (standard deviation) The value was for both of solid phases of 0.450 ODqsonm. A11 of samples with an OD>0.450 were considered to be positive. Results from sera screening were reported in Table 1.
Table 1 S16V+G20T(C-S) E21A

n. patientsa patients patients+ + + +

DONORS

HEP. acute 27 8 32 7 28 nAnE chronic 16 8 32 7 28 B acute 61 30 46, 17 26, 6 HEP. B 14 1 3,1 0 0 chronic C acute 15 B 19,5 3 7,3 C 15 9 22,5 5 12,5 chronic TD HIV-,HCV+ 29 17 32,7 19 36,5 HIV+ 31 HIV+ 33 piastrinopenic I,ES 3 9 Results obtained with p3.1-p3.9 peptides of Figure 1 are shown in Figure 2.
Data show trhat some of analyzed peptides are able to specifically detect antibodies against epitopes of NS3 region of GBV virus.
CORE REGION
Identification of specific epitopes of "core" region Aminoacid sequences (as in figure 3a a 3b) show methionines which may be aligned with either the first or the second methionine already described. The 35HIV clone sequencing revealed a new aminoacid sequence, at the N
terminus region, starting with a methionine residue.
By clone sequencing aminoacid sequences of GBV virus N-terminus were revealed. The "consensus" sequence is shown in Figure 4a.

Figure 4b shows the reactivity obtained by assaying different sera with the peptide having the consensus sequence of Fig. 4a.
Peptide synthesis 5 Peptides were synthesized with an automated synthesizer MilliGen 9050 (Septrim) as shown for peptide for NS3 region.
Cases sera from 20 healthy donors;
10 sera from 23 acute nonAnonE hepatitis patients;
sera from 10 chronic nonAnonE hepatitis patients;
sera from 22 acute hepatitis B patients;
sera from 33 drug addicted HIV-, HCV+ patients;
sera from 7 HIV+ patients;
15 sera from 17 HIV+ piastrinopenic patients;
sera from 47 Community Aquired HCV+ patients;
sera from 11 AIDS patients.
ELISA assay Solid phase was prepared on polivinylchloride dishes (Nunc) . l00 ul of a 20 ug/ml solution of G11C, D23C-VA, M37C-LVA, D13G-TG and M27C-CTG peptides, in carbonate buffer 50 mM, pH 9.6 were added to each well and incubated for 2 hrs at 37~C.
Further to peptide adsorbtion solid phases were saturated for 1 hr at 37~C with 300 ul/well of a BSA 20 solution in Tris-HC1 0.1M pH 7.5.
100 ul of 1:21 diluted serum in PBS containing 0.3 g/1 EDTA, 2 g/1 BSA, 2 m1/1 Triton x100 and 2 m1/1 Tween 20 were incubated into wells for 2 hrs at 37~C.
Following to 4 washes with PBS/Tween 20 0.1%, 100 ul/well of horseradish peroxidas'e conjugated goat anti human Ig (Sorin Biomedica), diluted l:100 in a solution of PBS containing 0.04% Tween 20 and 200 of preimmune goat serum. were added to each well.
Following to 4 washes of PBS/Tween 20 0 . 1 o BSA, 100 ul of chromogen and substrate were added to each well (Sorin DEIA KIT). The colorimetric reaction was blocked after 30 min with 200 ul/well of sulphuric acid 1N. The OD reading was performed at 450 nm by referrng to OD at 630 nm.
Results show the presence of antibodies against peptides of the "core" region of GBV virus.
Sequence varialbility of the 5' terminal region of different GBV-C isolates The GBV-C isolates so far analyzed have revealed putative truncated core proteins of about 83 and 46 amino acids in length (Linnen et al. supra, Simons et al. 1995 supra). These differences are due to nucleotide deletions or insertions that introduce in the sequences a frameshift causing the generation of distinct in-frame starting codons. It is conceivable, therefore, that the intrinsic genomic variability of this virus may introduce, in some particular isolates, additional mutations, insertions or deletions that could generate potentially functional core proteins.
In order to evaluate this possibility, we analyzed the nucleotide sequences, and their deduced translation products. On the basis of this analysis, we found 4 different types of sequences. The sequences of isolates 19, DM, DS39, BZ, 1l59 and 47 encoded for a putative core protein of approximately 83 aminoacids in length, corresponding to the published sequence of R10291 isolate (GenBank accession no. U45966) (Linnen et al. supra) (GBV2 in Fig. 5 and 6). The most 5' and unique inframe AUG codon of isolates 3064 and MG, on the other hand, could initiate the translation of a putative core protein of 46 aminoacids in length similar to the one potentially encoded by the published sequences of GBV-C isolate (GenBank accession no. U36380) (teary et aI. supra) and PNF2161 isolate (Linnen et al. supra) (GenBank accession no. U44402) (GBV-C and GBV1 in the Fig. 5 and 6). The sequence of isolate 2095 was more peculiar since, due to one base deletion (Fig. 5), it contained only one potential in-frame initiator AUG codon, downstream of the putative start codon of GBV-C and HGVI and located 9 nucleotides only upstream of the sequence that specifies the putative E1 signal sequence (Leary ET al. supra). The mostly unexpected finding, however, was that the sequence derived from the isolate 35 (35/1 in Fig. 5 and 6) could potentially encode for a putative core protein of approximately 106 aminoacids in length. This particular feature was due to a point mutation that introduces an additional in-frame AUG codon 69 nucleotides upstream of the putative initiation codon of the HGV 2 reference sequence.
In order to exclude the possibility of errors (intrinsic to the polymerase chain reactions) or of sequence misinterpretations (possibly due to the frequent sequence compressions), we confirmed our results by amplifying and sequencing several clones of each isolate (data not shown). Moreover, in the case of patient 35, we cloned and sequenced RT-PCR products derived from 2 additional sequential serum samples collected during the one year follow-up of this patient (sequences 35/2 and 35/3 in Fig. 6). While this analysis confirmed the presence of the upstream AUG codon in a11 the samples of patient 35, it also established that this particular isolate was stable over the time and that it was not dominantly replaced by a population of mutants., Thus, our data provide the first evidence for the existence of additional GBV-C sequences, potentially leading to a core protein longer and not described in the previously cDNA clones derived from the numerous viremic patients analyzed so far (Leary et al., Simons et al.
1995, 1996, supra) .
Finally, our results establish that the putative core proteins of our isolates are characterized by discrete regions of high variability, that are separated by highly conserved motifs (Fig. 6).
Translation of isolates with distinct putative initiator codons 5 The deduced translation products derived from the nucleotide sequences of our isolates demonstrate that these GBV-C sequences are very heterogeneous with respect to the potential translation initiation site. Thus, in order to understand the potential replicative competence 10 of these viral isolates, it was important to assess whether initiation at any of these sites can indeed occurr. This issue is particularly relevant since, recently, Simons et al. (1996 supra) have provided evidence suggesting that, in their GBV-C isolates, 15 initiation of translation occurred only immediately upstream of the putative El signal sequence and that the upstream in frame AUG seemed to be unable to direct the synthesis of GBV-C polyprotein.
To determine the effect of the upstream in frame AUG
20 codons on translation of our viral isolates, mRNAs with capped methylated (m'GpppG) 5' end, synthesized from linearized pCRX/AC1S constructs, were examined for mRNA
activity in a cell-free translation system prepared from rabbit reticulocytes. For this purpose we considered 25 isolates with distinct putative initiator codons.
Isolate 35 had 5 in-frame AUG codons in the putative core region (Fig. 5 and 6) and therefore the pCR35/AC1S
construct (covering the partial 5'UTR, the putative core and a portion of the E1 protein) can potentially mediate 30 the synthesis of different transcription products with expected sizes of about 17, 14,11, 10 or 7 kDa. Figure 7 shows that efficient translation occurred and a polypeptide with the apparent size of 17 kDa, compatible with the activity of the first in-frame AUG, was observed 35 in a SDS-PAGE analysis. It is important to note that efficient translation was also obtained with IVTT

reactions programmed with uncapped RNA derived from pCR35/AC1S. Thus, these data strongly suggest that, at least in this construct, the first available in-frame AUG
from the 5' end of the RNA can efficiently act as the initiator codon. Furthermore, since the cap stmcture was not determinant for efficient translation, the data also suggest that, as recently proposed by Simons et al.
(Simons et al. 1996, supra), GBV-C RNA may not utilize the classical ribosome scanning mechanism.
IO IVTT reaction mixtures were also programmed with pCRDS39/AC1S, pCRDM/AC1S, pCRl9/AC1S, pCR3064/AC1S, pCRMG/AC1S and pCR2095/AC1S constructs.
Isolates DS39, DM and 19 had the first in-frame AUG
corresponding to the second in-frame AUG of isolate 35 (Fig. 5 and 6). Moreover, isolate DS39 had also two additional putative initiatior codons so that the pCRDS39/AC1S construct could potentially direct the synthesis of three possible proteins of 14, 10 and 7 kDa.
The pCRDM/ACIS construct, on the other hand, can potentially initiate translation at 4 different AUG
codons, producing polypeptides of I4, II, 10 and 7 kDa.
Two of these plasmids, pCRDS39/AC1S and pCRl9/AC1S, directed the translation of products of about 14 kDa as detected by SDS-PAGE (Fig. 7). The size of these proteins is compatible with the activity of the AUG sequence just downstream of the start codon of the pCR35/AC1S
construct, suggesting that, also in these cases, translation was initiated at the first available in-frame AUG of the ORF. Since pCRl9/AC1S contained only one AUG
inframe with the long ORF, and since on the basis of the nucleotide sequence we could also exclude the possibility that the expressed proteins represented unrelated products of translation directed by AUG codon located in the other two frames, we conclude that the observed band is the correct product of translation.

In contrast, no bands were detected in the reactions programmed with the pCRDM/AC1S plasmid. Since this construct contained the same potential in-frame initiator AUG codon than pCR39/AC1S and pCRl9/AC1S, the results 5 rise the possibility that Sequences upstream of the potential initiator codon may influence the translation either positively or negatively. This possibility is also reinforced by the fact that the plasmids derived from isolates 3064 and MG (pCR3064/AC1S and pCRMG/AC1S), that 10 could potentially direct the synthesis of a band of 10 kDa, did not produce detectable quantities of specific proteins, in accordance to the data of Simons et al.
(Simons et al. 1996 supra). Negative results were also obtained with the pCR2095/AC1S construct, derived from 15 isolate 2095, that could potentially produce a protein of about 7 kDa.
Analysis of deletion mutants To examine in details the activity of the different potential in-frame initiator AUG codons, to verify the 20 possibility of a regulatory role of the upstream sequences and to unequivocally establish the size of the obtained translation products, cell-free protein synthesis experiments were performed with capped mRNA
transcribed by truncated forms of the 35, 19, 3064 and 2095 constructs described in Fig. 8. Figure 9A shows the results obtained when IVTT reaction mixtures were programmed with the 35 deletion mutant plasmids. In these experiments, translation of the mRNA derived from pCR35/AC1S yielded the major precursor band of 17 kDa as well as 3 additional faint bands of 14, 11 and 7 kDa, probably corresponding to translation products that utilize the second, third and the fifth in-frame AUG as initiator codons. A very similar pattern was obtained with the pCR35/~1 construct. In vitro translation of pCR35/02, pCR35/03, pCR35/~4 and pCR35/05 transcripts yielded bands of progressively lower molecular weights of about 14, 11 and 7 kDa, implying therefore that, in this isolate, a11 the in-frame AUG codons, with the exception of the fourth AUG, might serve as potential site of translation initiation. In the lane containing the translation products of pCR35/02 mutant we found two bands of 14 and 11 kDa corresponding to translation products that utilized both the second and the third available AUG initiation codons.
As expected, the analysis of the deletion mutants of pCRl9/AC1S, that contains only one in-frame AUG, gave different results (Fig. 9B). The 14 kDa bands obtained with reactions programmed with pCRl9/AC1S, pCRl9/~1 and pCRl9/~2 demonstrate that the first and unique in-frame AUG is the site of translation initiation and that., at least for this mutant, deletion of 5' UTR does not seem to significantly influence the level of translation.
A quite unexpected finding of these experiments was that the reactions programmed with some of the deletion mutants of pCRl9/AC1S construct, that lacked the AUG
codon immediately upstream of the putative E1 signal sequence, yielded a band with the same apparent molecular mass of the 7 kDa protein detected in the reactions programmed with pCR35/44 and pCR35/~5. Although we can not exclude that this band may have resulted from partially degraded RNA, it is interesting that in this template the AUG triplet correspcnding to the fifth in-frame start codon of isolate 35 is replaced by an ACG
codon, that has been found to act as initiation codon in other mRNAs (Boeck, R., and D. Kolakofsky. l994. EMBO J.
13:3608-3617), including a transcript derived from a GBV-C mutated sequence (Simons et al. 1996 supra).
Reactions programmed with the deletion mutants derived from pCR3064/AC1S construct provided no detectable bands (data not shown). Since in this constructs the only available initiation codon is the AUG
corrisponding to the fourth start codon of the isolate 35, these results confirmed the data obtained with the mutants of isolate 35 and with the pCR3064/AC1S and pCRMG/AC1S plasmids, suggesting that this particular codon, as already reported (Simons et al. 1996 supra), is silent, probably due to the composition of the flanking sequences.
Finally, evidence for a regulatory role of the partial 5'UTR were obtained with the deleted 2095 plasmids (Fig. 9C). In this isolate the only available site of transcription initiation corresponds to the fifth AUG of isolate 35, that is however silent in construct pCR2095/AC1S. The expected 7 kDa product was surprisingly detected as a faint band with pCR2095/01, pCR2095/02 and pCR2095/~3 and the intensity of this band increased progressively by programming the reactions with pCR2095/04 and pCR2095/05. These data clearly mean that, in this particular template, the translation of the polypeptide is strongly influenced by the partial 5'UTR
present in the undeleted construct that, in this case, seems to inhibit the translation process.
Finally, we must stress that we have verified that none of the bands obtained with the deletion mutants can be accounted for by unrelated products of translation encoded by the other two possible reading frames.

o . ~ CA 02251745 1999-04-27 - 2 3 -~ ;
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: WABCO BV

(ii) TITLE OF INVENTION: Non-A non-E Hepatitis virus having a translatable core region, reagents and methods for their use (iii) NUMBER OF SEQUENCES: 11 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Dennison Associates (B) STREET: 133 Richmond Street West, Suite 301 (C) CITY: Toronto (D) PROVINCE: Ontario (E) COUNTRY: Canada (F) POSTAL CODE: M5H 2L7 (v) COMPUTER READABLE FORM:

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(A) APPLICATION NUMBER: 2,251,745 (B) FILING DATE: 09-DEC-1996 (C) CLASSIFICATION:

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(A) TELEPHONE: 416-368-8313 (B) TELEFAX: 416-368-1645 (2) INFORMATION
FOR
SEQ
ID
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(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 205 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 1:

Arg Val Arg Asp Val Ala Arg Gly Cys Gly Val Gln Leu Leu Tyr Val Ala Thr Ala Thr Pro Pro Gly Ser Pro Met Thr Gln His Ser Ile Pro Ile Glu Thr Lys Leu Asp Val Gly Glu I1e Pro Phe Tyr His Gly Gly Ile Pro Leu Glu Arg Met Arg Thr Gly Arg His Leu Val Phe Cys His Ser Lys Ala Glu Cys Glu Arg Leu Ala Gly Gln Phe Ser Ala Arg Gly Val Asn Ala Ile Ala Tyr Tyr Arg Gly Lys Asp Ser Ser Ile Ile Lys Asp Gly Asp Leu Val AlaThrAsp AlaLeu SerThrGly Tyr Val Cys Thr Gly Asn Phe Ser ThrAspCys GlyLeu ValValGlu Glu Asp Val Val Val Glu Val Leu ProThrIle ThrIle SerLeuArg Thr Thr Asp Val Pro Ala Ser Glu SerMetGln ArgArg GlyArgThr Gly Ala Leu Arg Gly Arg Ser Arg TyrTyrAla GlyVal GlyLysAla Pro Gly Tyr A1a Gly Val Val Ser ProValTrp SerAla ValGluAla Gly Arg Gly Val Thr Trp Tyr Met ProAspLeu ThrAla Asn Gly Glu (2) INFO RMATION FOR ID NO: :

3 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 aminoacids (B) TYPE: o acid amin (C) STRANDEDNESS: le sing (D) TOPOLOGY:linear (ii) MOLECULE TYPE:peptide (xi)SEQUENCE DESCRIPTION: 2:
SEQ ID NO:

Met Ser Leu Asn Arg Ala TyrPro ProGlyLeu ThrThr ProThr Arg Tyr Gly Pro Arg Arg Pro MetSer LeuLeuThr AsnArg PheAsn Ser Arg Arg Val Asp Lys Asp TrpGly ProGlyVal MetGly LysAsp Gln Pro Lys Pro Cys Pro Ser ArgThr GlyLysCys MetGly ProPro Arg Ser Ser Ala Ala Ala Cys ArgGly SerProArg IleLeu ArgVal Ser Arg Ala Gly Gly Ile Ser ProTyr ThrIleMet GluAla LeuLeu Leu Phe Leu Leu Gly Val Glu GlyAla IleLeu Ala (2) INFORMATION
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(A) LENGTH: 84 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (xi)SEQUENCE DESCRIPTION: 3:
SEQ ID NO:

Met Ser Leu Leu Thr Asn PheAsn ArgArgVaI AspLys AspGln Arg Trp Gly Pro Gly Val Met LysAsp ProLysPro CysPro SerArg Gly Arg Thr Gly Lys Cys Met ProPro SerSerA1a AlaAla CysSer Gly Arg Gly Ser Pro Arg Ile Leu Arg Val Arg Ala Gly Gly Ile Ser Leu Pro Tyr Thr Ile Met Glu Ala Leu Leu Phe Leu Leu Gly Val Glu Ala Gly A1a Ile Leu (2) INFORMATION FOR SEQ ID N0: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 amino acids 1 5 (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 4:
Met Ser Leu Asn Arg Ala Arg Tyr Pro Pro Gly Leu Thr Thr Pro Thr Tyr Gly Pro Arg Arg Pro Ser (2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
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(2) INFORMATION FOR SEQ ID NO:
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(A) LENGTH: 21 base pairs (B) TYPE; nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleicacid (xi) SEQUENCE DESCRIPTION: SEQ NO: 6:
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1 (2) INFORMATION FOR SEQ ID NO:
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(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single 3 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleicacid (xi) SEQUENCE DESCRIPTION: SEQ NO: 8:
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(2) INFORMATION FOR SEQ ID NO:
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(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ N0: 9:
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(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid 2 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid 2 (xi) SEQUENCE DESCRIPTION: SEQ NO: 10:

(2) INFORMATION FOR SEQ ID NO:
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(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single 3 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ NO: 11:
ID

Claims

29

1. Peptide able to specifically bind to nonAnone hepatitis virus antibodies having a sequence of at least 10 aminoacids, said nonAnone hepatitis virus being characterized by having a translatable functional core-like protein longer than 84 aminoacid residues and by comprising an aminoacid sequence at least 60% homologous to the sequence of SEQ ID NO. 4:
MSLNRARYPPGLTTPTYGPRRPS; or an immunologically homologous variant of the peptide.

2. Peptide according to claim 1 having a sequence comprised in SEQ ID No. 4.

3. Composition comprising at least one of the peptides according to claim 1 or 2.

4. Composition according to claim 3 which is adhered on a solid phase.

5. Composition according to claim 3 or 4 comprising at least two peptides according to any of claim 1 or 2.

6. Diagnostic kit for nonAnone hepatitis comprising as specific reagent at least one peptide according to any of claim 1 or 2.

7. Diagnostic kit for nonAnone hepatitis comprising as specific reagent at least two peptides according to any of claim 1 or 2.

8. Method to detect nonA nonE hepatitis virus antibodies in a sample comprising the following steps:
- incubating in conditions allowing the formation of an antigen-antibody complex said sample with at least one peptide according to any of claims 1 or 2;
revealing said complex.