WO1991004341A1 - Hdv-specific peptides - Google Patents

Abstract

The human Delta virus is a defective viroid-like agent causing exacerbated liver disease in Hepatitis B virus-infected individuals. To date, the only marker of Delta virus superinfection demonstrated is a p24/p27 protein, originating from the antigenomic strand in a region designated ORF5. While the in vivo genesis and metabolism of the protein encoded by this reading frame is still not completely understood, it is clear that antibodies of high titer do develop to this protein in all infected individuals, and that antigen can be shown to be circulating in affected sera early in infection. In order to generate a specific, well-defined virus antigen reagent for immunodiagnostic studies, we have localized the pertinent Delta antigen epitope to a 55 amino acid region near the amino terminus of ORF5. We have synthesized this region and successfully expressed this epitope as a specific high-yield fusion protein in E. Coli. This novel recombinant not only localizes a critical epitope, but serves as an efficient mimic of native virus-specific protein. Together, the Delta antigen and specific antibody provide a reagent panel to diagnose and monitor the course of Hepatitis Delta virus infection in humans.

Description

HDV-SPECIFIC PEPTIDES

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to polypeptides presenting an epitope of the human delta virus ORF5 antigen. Such polypeptides are useful in diagnosis and treatment of HDV infections.

Information Disclosure Statement

The human Delta virus was first described by Rizzetto et al. (1977) as a serological marker, superimposed upon a Hepatitis B infection. Individuals expressing this new antigenic determinant often develop severe chronic active hepatitis and cirrhosis. While originally characterized as a heterotrophic agent endemic to the Mediterranean basiπ (Rizzetto, 1983), there is strong evidence of an anti-Delta response in patients from both U.S. coasts, reaching an incidence of almost 12% among asymptomatic S antigen carriers (Nath et al., 1984) .

The Delta virus is a defective RNA virus that requires the replication and/or products of Hepatitis B for its persistence. The infectious particle is 35-37 nm in diameter, and consists of a lipoprotein envelope containing Hepatitis B S antigen enclosing a 1,679 nucleotide single-stranded circular RNA genome (Bonino et al., 1986). The envelope liberates a Delta virus-specific protein antigen afte. disruption with the detergent NP-40 (Rizzetto et al, 1980) , indicating a probable association with the Hepatitis Delta virus RNA genome itself.

Two polypeptides of 24Kd and 27 d have been detected in partially purified Hepatitis Delta particles using Western blot analysis when reacted with sera from patients with chronic Delta agent infection (Bergman & Gerin, 1986; Bonino et al., 1986) . To date, these are the only identified antigens, in relative abundance, consistent with the original Delta antigen described by Rizzetto et al. (1977) . The antigenomic strand of Hepatitis Delta virus is capable of encoding Delta antigen polypeptides in bacteria, as recently shown by Weiner et al. (1988) . Using bacterial expression vectors employing hSOD leader sequences and cDNA clones of the viral genome, the ORF 5 region has been expressed to produce immunoreactive p24kd and p27kd (Wang et al., 1986).

As a result of a nucleotide sequence ambiquity, the amino acid sequence of the delta antigen ORF5 has been given as either 195 or 211 amino acids. This sequence ambiguity may be the result of an allelic variation, or it may be attributable to a sequencing error.

Delta virus infection is diagnosed by the detection of Delta antigen in the serum, by immunofluorescent staining of liver biopsy material, or by the appearance of Delta agent- specific antibodies later on in the infection (Rizzetto and Verme, 1985) .

Delta antigen reagent used in ELISA and other immunodiagnostic formats have been isolated from infected liver, either human (Crivelli et al. , 1981) , chimpanzee (Puig et al., 1988) or woodchuck (Choi et al., 1988). In general, such procedures are cumbersome, have only modest yields, and involve the handling of highly infectious materials.

SUMMARY OF THE INVENTION

Jameson and Wolf, "The Antigenic Index: A Novel Algorithm for Predicting Antigenic Determinants," CABIOS, 4: 181-186 (1988) describe an algorithm which integrates the predicted influences of hydrophilicity and flexibility to obtain an indication of the likelihood that a particular residue will belong to an antigenic determinant. Application of this algorithm to the known delta antigen sequence allows one to identify several maxima and minima.

Surprisingly, a fusion protein carrying the delta sequence from 20-74, which includes a stretch (residues 54-57) having a negative antigenic index (-.450), is immunoreactive in ELISA and Western Blot assays with commercially available anti- HDV polyclonal antibodies and with sera of patients exhibiting clinical signs of HDV infection.

This discovery was serendipitous, as a person of routine skill in the art would have been guided by the antigenic index to avoid including amino acid residues fro which a negative antigenic index had been calculated in a peptide designed for use as an antigen. Jameson and Wolf, studying myohemerythrin, stated that the "non-reactive peptides correspond to local minima on the antigenic index plot." (page 184) While this is not necessarily true for all proteins, it certainly teaches away from preparation of HDVAg 20-74.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Hydrophilicity, surface probability, flexibility, and antigenic index of ORF 5 region of Hepatitis Delta Virus. Black bars outline the expressed region corresponding to amino acids 20 to 74. Actual amino acid sequence is indicated below using single letter code.

Figure 2 Strategy of oligonucleotide synthesis. A total of eight oligonucleotides were synthesized as shown in lower half of diagram. In addition to the 55 amino acids of Delta, a polylinker was designed that contained EcoRV, Ball and PvuII sites, enzymes sites not found in either Delta or the intermediate assembly vector (KS/SK M13+) .

Figure 3 Cloning and expression scheme for a subregion of 0RF5 which codes for Delta antigen. Stepwise intermediate clones for Delta antigen assembly are shown.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This application reports the chemical synthesis and cloning of a nucleotide sequence within the ORF 5 reading frame of the Delta virus. The nucleotide sequence codes for amino acids corresponding to Delta antigen amino acids 20 to 74. It is expressed as a Trp E fusion protein that when partially purified displays an epitope that is immunologically reactive against anti-Delta sera as detected using Elisa and Western blot analysis.

The diagnostic reagents of the present invention may be a polypeptide essentially including amino acids 20-74 of the delta antigen. A polypeptide "essentially includes" this sequence if it will competitively inhibit the binding of our preferred polypeptide to delta-specific antisera, and if it contains a region substantially homologous in amino acid sequence with the 20-74 region of delta antigen. It may, however, differ from our preferred polypeptide by one or more substitutions, deletions or insertions either internally or at the termini. Preferably, no more than about 10% of the aforementioned 20-74 sequence is deleted. It may include up to 5-10 amino acids of additional delta sequence before 20 or after 74, and it may contain unrelated additional amino acid sequence at either end (as in the bacterial fusion protein) . Preferred substitutions are indicated by the table below of groups of normally equivalent amino acids (with single letter codes indicated in parenthesis) :

(a) Ala(A) Ser(S) Thr(T) Pro(P) Gly(G) ;

(b) Asn(N) Asp(D) Glu(E) Gln(Q) ;

(c) His(H) Arg(R) Lys(K);

(d) Met(M) Leu(L) Ile(I) Val(V);

(e) Phe(F) Tyr(Y) Trp(W) ; and

(f) Cys(C).

This delta-specific polypeptide or protein may be labeled with any label conventional in the immunochemical art, such as an enzyme, radioactive, or fluorescent label, or it may be immobilized on a carrier for use in the capture of a delta- specific antibody in a sample. In the latter form, it also has utility for immunopurification of delta-specific antibodies.

The fusion protein may also be useful as a vaccine, with the delta 20-74 region eliciting the desired delta- specific immune response, and the remainder of the molecule acting as an immunogenic carrier. The immunogen may be expressed directly as a fusion protein, or the HDV-specific polypeptide may be conjugated in vitro with a macromolecular carrier, which in that event need not be peptide in character, but may instead be, e.g., a polysaccharide.

The DNA sequence used for the expression of the delta 20-74 region may be the native sequence, see, e.g., Wang, et al.. Nature, 328:456 (1987), and GENBANK, locus HPDXX, accession no. X04451, both incorporated by reference herein, or it may be a modified sequence which encodes the desired amino acid sequence. The sequence may be modified to add or subtract restriction sites, to match the codon preference of a particular host organism, to eliminate undesirable secondary structure of the messenger RNA, or to encode a mutant amino acid sequence as taught above. (The aforementioned nucleotide sequence ambiquity does not affect amino acids 20-74 of HDV ORF5.) The preferred amino acid sequence, which is that found in nature in the delta antigen, is given in Figure 1.

While production of the HDV-specific polypeptide by recombinant DNA techniques is preferred, it may also be prepared by other means such as solid phase peptide synthesis.

Example 1

MATERIAL AND METHODS

Construction and synthesis of oligonucleotides: A study of available restriction sites were analyzed for the entire ORF 5 sequence of nucleotides. Sites were found that would facilitate chemical synthesis and subsequent cloning (Figure 2) . Oligonucleotides were generated on a Model 381A oligonucleotide synthesizer (Applied Biosystems) with beta- cyanoethyl phosphoramidites. Synthesized nucleotides were then purified using DNA cartridges (Applied Biosystems) following instructions as provided. To simplify final assembly of oligos encoding the amino acid region of interest, cloning was performed in units into polylinker vectors pKS M13+ and pSK M13+ (Stratagene) . A 450 base pair Pvu II fragment containing the assembled Delta sequence was isolated and cloned into the Sma I site of the TrpE expression vector pATH 2 (obtained from Professor A. Tzagaloff, Columbia University) .

Culture conditions and induction of TrpE fusion peptides: Bacterial cultures were grown overnight in M9+Amp containing 20μg/ml tryptophan at 37°C. Overnight cultures were then diluted 1:100 with fresh M9+Amp without tryptophan and grown until A600=0.2. The tryptophan operon was induced with the addition of one ml indole acrylic acid (IAA) (lOmg/ml in ethanol) and grown for an additional 6-8 hours at 37°C.

Preparation of bacterial lysates: Cells were collected by centrifugation and pellets resuspended in 0.1 ml cold TEN buffer (50mM Tris, pH 7.5, 0.5mM EDTA, 0.3M NaCl) . Ten μl of lysozyme (10 mg/ml) was added and incubated for 15 minutes on ice. Lysis was performed with the addition of 2 μl of 10% NP-40 and further incubated 10 minutes on ice. Cell lysates were diluted with 150μl of NaCl-Mg buffer (1.5M NaCl, 12mM MgCl₂) containing a 1:300 dilution of DNase I (1 mg/ml) and left on ice for one hour with agitation. After centrifugation, the insoluble pellet was washed three times with lOOμl of TEN. The final pellet was then resuspended in 50μl of cracking buffer (0.01M (NaP0₄ ) , pH 7.2, 1% beta- mercaptoethanol, 1% SDS, 6M urea) . Identical procedures were used to prepare cell pellets from bacteria containing plasmids with the Delta sequence, without the Delta sequence, and with the Delta sequence in the wrong orientation.

Protein Assay: Protein concentration was determined using a standard Biorad Assay Kit. Unknown protein concentrations were compared to a standard curve using bovine serum albumin. All Elisas and Western blots were then normalized for protein concentration.

ELISA: Cell samples were serially diluted as follows: 1:1, 1:10, 1:100 and 1:1000, and bound down on microtiter plates overnight at 4°C or for 2 hours at 37°C. Plates were then washed with PBS-tween and blocked with 10% normal goat serum for one hour at 37°C. Delta antigen was detected using the HRP-conjugated Abbott positive control antisera, or clinical patients' sera followed by HRP-conjugated goat anti-human IgG. Color was developed using ortho- phenyldiamine (OPD) and H₂0₂ and absorbance was measured at 492 nm. The same antibodies, but differently labeled, were also used in the Western blot analysis.

Western Blot Analysis: Cell samples were diluted 1:5 and 20 ul aliquots were electrophoresed on 12% Laemmli. gels. Proteins were transferred overnight at 0.2 amps at 4°C onto 0.2μ pore nitrocellulose sheets (Schleicher and Schuell) . Filters were washed and blocked with 8% bovine serum albumin (BSA) in phosphate buffer (120mM NaCl, 2.7mM KC1, 1.5mM (KH P0₄ ) , 8mM (Na HP0₄ ) 12H₂0, pH 7.4). In one procedure, the blot was incubated with alkaline phosphatase-labeled delta- specific antibody and developed with 5-bromo-4-chloro-3-indoxyl phosphate p-toluidine salt (BCIP) , nitro blue tetrazolium chloride (NBT) , and MgCl₂. In a different procedure, the blot was incubated with delta-specific antibody, overlaid with radioiodinated staphylococcal protein A, and autoradiographed.

RESULTS

To prepare a gene which encoded amino acids 20 to 74 (Figure 1) of the human delta virus antigen ORF5, a series of oligonucleotides were designed using restriction sites that would facilitate stepwise ligation. In addition, a polylinker - including EcoRV, Ball and PvuII sites - was placed on the 5* end, abutting amino acid 20 to aid in subsequent expression vector manipulation (Figure 2) .

Ligations were carried out in pKS M13 or pSK 13, which served as intermediate vectors containing specific Delta oligonucleotides cloned into them. A final reconstituted hybrid pKS M13 was obtained that held the assembled Delta sequence. In order to express the Delta antigen, a 450bp PvuII fragment containing the complete sequence and downstream sequence of pKS M13 was cloned into the Smal site of pATH 2. Clones were screened by restriction analysis and hybridization. Bacterial cultures containing plasmids with and without the Delta sequence, and the Delta sequence in the wrong orientation, were induced. Cell lysates were prepared as described and bound to icrotiter plates in serial dilution. Samples containing the Delta sequence in the right orientation resulted in lysates that were immunologically reactive with positive human Delta antisera (Table I) . A small amount of immunoreactivity was also seen in uninduced bacterial lysates containing the Delta sequence in the correct orientation.

Having determined the presence of an immunoreactive peptide by ELISA, Western blot analysis was performed to characterize the molecular weight species expressed. Bacterial cultures with and without the Delta sequence, and the Delta sequence in the wrong orientation, were induced respectively. Cell lysates were prepared as described. Samples were then electrophoresed on 12% Laemmli gels, transblotted onto nitrocellulose, and incubated with hepatitis Delta positive antisera. As predicted, a band of 43,000 molecular weight was seen in induced samples where the Delta sequence was present in the right orientation. An additional band was also seen at approximately 37,000-39,000. These identical bands, at a lower level, were also seen in uninduced bacterial containing the Delta sequence in the correct orientation. No immunoreactive bands were seen in other sample controls tested.

Example 2

Competitive Inhibition Assay for Detection of Antibody to Hepatitis Delta Antigen: A one step competitive inhibition enzyme linked immunosorbant assay was used for the detection of antibody to hepatitis delta antigen in human serum and plasma. The assay utilized the recombinant fusion peptide of Example 1 as the antigen. The antigen was immobilized onto microtiter wells for antibody capture. The wells were incubated with 50μl of human sera or plasma and 50μl of human anti-delta antibodies conjugated with horseradish peroxidase. Incubation is for two hours at 37°C. Color is developed with hydrogen peroxide and tetramethylbenzidine at room temperature for thirty minutes. Of 154 clinical positive specimens tested, the specificity was 98.7%. Of 940 clinical negative specimen tested, the sensitivity was 99.8%. The assay may be complete in two hours.

The assay demonstrated dilutional sensitivity a least comparable to that obtained with the Abbott Anti-delt EIA Kit marketed by Abbott Laboratories. The Abbott Ki utilized Woodchuck delta antigen.

TABLE 2: Base Sequence encoding the 20-74 Peptide

20

Trp Val Ala Gly Arg Lys Lys Leu Glu Glu

TGG CTG GCC GGA AGA AAG AAG TTA GAG GAA

30

Leu Glu Arg Asp Leu Arg Lys Thr Lys Lys

CTC GAG AGA GAC CTC CGG AAG ACA AAG AAG

40

Lys Leu Lys Lys lie Glu Asp Glu Asn Pro

AAA CTC AAA AAG ATT GAG GAC GAA AAT CCC

50

Trp Leu Gly Asn lie Lys Gly lie Leu Gly

TGG CTG GGG AAC ATC AAA GGA ATT CTC GGA

60

Lys Lys Asp Lys Asp Gly Glu Gly Ala Pro

AAG AAG GAT AAG GAT GGA GAG GGG GCT CCC

70

Pro Ala Lys Arg Ala

CCG GCG AAG AGG GCC REFERENCES:

1. Rizetto, M. , Hepatology 2:729-737 (1983).

2. Govindarajan, S., Chin, K.P., Redeker, A.G. and Peters A.L. , Gastroenterology 8jS :1417-1420 (1984).

3. Nath, N., Fang, C, Berberian, H. , et al., The 1984 Symposium on Viral Hepatitis, 3A21 p893 (1984) .

4. Bonino, F., Hoyer, B. , Shih, J. , Rizetto, M. , Purcell, R. and Gerin, J. , Infect Immunol. 43:1000-1005

(1984) .

5. Rizetto, M. , Hoyer, B., Canese, M. , Shih, W. , Purcell, R. and Gerin, J. , Proc. Natl. Acad. Sci USA 77:6124- 6128 (1980) .

6. Rizetto, M. , Canese, M. , Arico, S., Crivelli, O., Bonino, F. , Trepo, C. and Verme, G. Gut 18:997-1003 (1977).

7. Wong, K., et al., Nature 321:508-514 (1986).

8. Wong, K. , et al., Nature 328:456 authors corrigendum (1987) .

9. Chong, M. , et al., J. Virol. 62:2403-2410 (1988) .

10. Rizetto, M. , et al., Lancet 2.:986-990 (1979).

11. Crivelli, 0., et al., J. Clin. Microbiol 2.:173- 177 (1981) .

12. Puig, J. , et al., Viral Hepatitis and Liver Disease pp 403-405 (1988) Alan R. Liss Inc.

13. Shattock, A. and Morgan, B. J. Med. Virol.13:73- 82 (1984) .

Claims

1. An antigenic reagent comprising a polypeptide essentially including the amino acid sequence WVAGRKKLEELERD)__RKTKKKI_KKIEDENPWLGNIKGILGKKDKDGEGAPPAKRA.

2. The antigenic reagent of claim 1, in labeled form,

3. The antigenic reagent of claim 1, in immobilized form.

4. The antigenic reagent of claim 1, said polypeptide being attached to an immunogenic carrier.

5. A method of diagnosing the presence of a human delta virus infection which comprises obtaining a sample of a body fluid from an individual suspected of harboring such an infection, contacting the sample with the reagent of claim 1, and determining whether said polypeptide immunologically reacts with a component of said sample.

6. An assay kit comprising an antigenic reagent according to claim 1 and a delta virus-specific antibody.