The subject invention relates to a novel hepatitis B surface antigen mutant and methods of detecting this mutant, and/or antibodies thereto, in patient samples. In particular, the mutant contains a substitution of amino acid threonine for the amino acid alanine at position 123 in the amino acid sequence of the hepatitis B surface antigen (HBsAg) protein.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention relates to a novel mutant of hepatitis B virus (HBV) which has a modified "a" determinant as a result of an amino acid substitution (i.e., Thr to Ala) at amino acid position 123 of the S-HBsAg sequence. This amino acid substitution corresponds to a nucleotide substitution in the threonine codon of adenine to guanine at position 521 in the HBV genome.

In particular, the present invention includes the isolated nucleotide sequence of SEQ ID NO:1 which encodes the full envelope gene sequence of the mutant virus. Additionally, the present invention includes an isolated nucleotide sequence which corresponds to the "a" determinant sequence of the virus (SEQ ID NO:4), as well as the isolated nucleotide sequence of the full mutant virus. The invention also includes nucleotide sequences having at least 70% identity, preferably at least 80% identity, and more preferably at least 90% identity to the nucleotide sequences of the present invention, as well as complements thereof.

"Identity" is defined as the degree of sameness, correspondence or equivalence between the same strands (either sense or antisense) of two DNA segments. More specifically, sequence identity or percent identity is the number of exact matches between two aligned sequences divided by the length of the shorter sequence and multiplied by 100. The greater the percent identity, the higher the correspondence, sameness of equivalence between the two strands. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482 489 (1981). This algorithm may be extended to use with peptide or protein sequences (in terms of identity or similarity) using the scoring matrix created by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353 358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov Nucl. Acids Res. 14(6) :6745 66763 (1986). An implementation of this algorithm for nucleic acid and peptide sequences is provided by the Genetics Computer Group (Madison, Wis.) in the BestFit utility application. The default parameters for this method are described in the Wisconsin Sequence Analysis Package Program Manual, Version 8 (1995) (available from Genetics Computer Group, Madison, Wis.). Other equally suitable programs for calculating the percent identity or similarity between sequences are generally known in the art.

"Complementarity" is defined as the degree of relatedness between two DNA segments. It is determined by measuring the ability of the sense strand of one DNA segment to hybridize with the antisense strand of the other DNA segment, under appropriate conditions, to form a double helix. In the double helix, wherever adenine appears in one strand, thymine appears in the other strand. Similarly, wherever guanine is found in one strand, cytosine is found in the other. The greater the relatedness between the nucleotide sequences of two DNA segments, the greater the ability to form hybrid duplexes between the strands of two DNA segments.

The invention also includes the polypeptides encoded by the nucleotide sequences described above. In particular, the invention encompasses the polypeptide encoded by the isolated nucleotide sequence of the envelope gene comprising the nucleotide sequence of the "a" determinant of HBsAg of the mutant virus, polypeptides having at least 70% similarity to these amino acid sequences, preferably at least 80% similarity thereto, and more preferably at least 90% similarity thereto. Additionally, the invention includes the polypeptide sequence encoded by the nucleotide sequence of the mutant "a" determinant and the full mutant virus. The present invention also includes fragments of these sequences.

"Similarity" between two amino acid sequences is defined as the presence of a series of identical as well as conserved amino acid residues in both sequences. The higher the degree of similarity between two amino acid sequences, the higher the correspondence, sameness or equivalence of the two sequences. ("Identity" between two amino acid sequences is defined as the presence of a series of exactly alike or invariant amino acid residues of both sequences.) Percent similarity is calculated between the compared polypeptide sequences using programs known in the art (see above).

For purposes of the present invention, a "fragment" of a nucleotide sequence is defined as a contiguous sequence of approximately at least about 6, preferably at least about 8, more preferably at least about 10 12 nucleotides, and even more preferably at least about 15 18 nucleotides corresponding to a region of the specified nucleotide sequence.

Additionally, the present invention includes the isolated nucleotide sequence encoding the complete surface antigen or protein of the HBV mutant virus, the complement thereof, as well as fragments of the sequence and its complement. The present invention also encompasses isolated nucleotide sequences having 70% identity, preferably 80% identity, and more preferably at least 90% identity to the sequence of the mutant virus.

The invention also encompasses the purified polypeptide encoded by the isolated nucleotide sequence of the complete surface antigen gene of the mutant virus, as well as fragments of this sequence. Additionally, the present invention encompasses purified polypeptides having at least 70% similarity, preferably at least 80% similarity, and more preferably at least 90% similarity to the purified polypeptides encoded by the isolated nucleotide sequences, respectively.

Also, the present invention includes an isolated nucleotide sequence which is hybridizable, under moderately stringent conditions, to a nucleotide sequence corresponding to or complementary to the nucleotide sequence of the mutant genome, the nucleotide sequence of the envelope gene, the nucleotide sequence of the HBsAg or the nucleotide sequence encoding the "a" determinant of the mutant virus. A nucleic acid molecule is "hybridizable" to another nucleic acid molecule when a single-stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and ionic strength (see Sambrook et al., "Molecular Cloning: A Laboratory Manual", Second Edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The conditions of temperature and ionic strength determine the "stringency" of the hybridization. "Hybridization" requires that two nucleic acid sequences contain complementary sequences. However, depending on the stringency of the hybridization, mismatches between bases may occur. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acid sequences and the degree of complementarity. Such variables are well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm (i.e., melting temperature) for hybrids of nucleic acids having these sequences. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra). For hybridization with shorter nucleic acids, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra).

Once the nucleotide sequence encoding the amino acid sequence containing the variation (i.e., Thr to Ala at position 123 of the HBsAg) has been isolated, it may then be introduced into either a prokaryotic (e.g., E. coli) or eukaryotic host cell (e.g., mammalian cell (such as a HeLa cell or a Chinese hamster ovary cell) or a yeast cell (such as S. cerevisiae or S. carlsbergensis)) through the use of a vector or construct. The vector or construct of the present invention (e.g., a plasmid, a cosmid, a bacteriophage, etc.) may comprise the nucleotide sequence encoding the mutant protein sequence as well as any promoter which is functional in the host cell and is able to elicit expression of the protein encoded by the nucleotide sequence. The promoter is in operable association with or "operably linked" to the promoter. (A promoter is said to be "operably linked" with a coding sequence if the promoter affects transcription or expression of the coding sequence.) Suitable promoters include, for example, T7, TP1, lactase, and metallothionein and are well-known in the art. The vector may be introduced into the host cell of choice by methods known to those of ordinary skill in the art including, for example, transfections, transformation and electroporation (see Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., Vol. 1 3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press (1989)). The host cell is then cultured under suitable conditions permitting expression of the protein which is then recovered an purified.

It should be noted that expression in a host cell can be accomplished in a transient or stable fashion. Transient expression can occur from introduced constructs which contain expression signals functional in the host cell, but which constructs do not replicate and rarely integrate in the host cell, or where the host cell is not proliferating. Transient expression also can be accomplished by inducing the activity of a regulatable promoter operably linked to the gene of interest, although such inducible systems frequently exhibit a low basal level of expression. Stable expression can be achieved by introduction of a construct than can integrate into the host genome or that autonomously replicates in the host cell. Stable expression of the gene product of interest can be selected for through the use of a selectable marker located on, or transfected with, the expression construct, followed by selection for cells expressing the marker. When stable expression results from integration, the site of the construct's integration may occur randomly within the host genome or can be targeted through the use of constructs containing regions of homology with the host genome sufficient to target recombination with the host locus. Where constructs are targeted to an endogenous locus, all or some of the transcriptional and translational regulatory regions can be provided by the endogenous locus.

In view of the above, the present invention includes the isolated nucleotide sequence encoding the purified polypeptide of the virus (i.e., the modified "a" determinant having a threonine residue rather than the wildtype alanine residue at position 123 of the amino acid sequence of the HBsAg), the isolated nucleotide sequence of the envelope gene of the mutant virus, the isolated nucleotide sequence encoding the HBsAg of the virus, the isolated nucleotide sequence of the mutant virus, the protein encoded by each sequence, the vector comprising either nucleotide sequence as well as the host cell into which the vector is introduced. It should also be noted that the proteins may be produced either recombinantly, as described above, or synthetically. Further, the proteins may be purified from the virus itself.

The polypeptides or proteins may also be used to develop monoclonal and/or polyclonal antibodies which bind to the immunological epitope(s) of interest of mutant HBV. Methods of producing such antibodies are well known to those of ordinary skill in the art (see, e.g., Kohler and Milstein, Nature 256:494 (1975), Mimms et al., Virology 176:604 619 (1990), Hammerling et al., Protein Purification, Principles and Practice, 2.sup.nd ed., Springer-Verlag, N.Y. (1984)). Also, one may simply immunize a mammal with the polypeptide or protein of the present invention in order to cause antibody production. Such antibodies may then be recovered. The polypeptide or protein used contains an epitope of the mutant HBV.

As noted above, the present invention also includes methods of detecting antibody to the mutant HBV using the proteins or polypeptides (i.e., antigens), in particular, the HBsAg containing the "a" determinant, described above (see, e.g., U.S. Pat. No. 5,595,739). More specifically, there are two basic types of assays, competitive and non-competitive (e.g., immunometric and sandwich). In both assays, antibody or antigen reagents are covalently or non-covalently attached to the solid phase. Linking agents for covalent attachment are known and may be part of the solid phase or derivatized to it prior to coating. Examples of solid phases used in immunoassays are porous and non-porous materials, latex particles, magnetic particles, microparticles (see published EPO application No. EP 0 425 633), beads, membranes, microtiter wells and plastic tubes. The choice of solid phase material and method of labeling the antigen or antibody reagent are determined based upon desired assay format performance characteristics. For some immunoassays, no label is required. For example, if the antigen is on a detectable particle such as a red blood cell, reactivity can be established based upon agglutination. Alternatively, an antigen-antibody reaction may result in a visible change (e.g., radial immunodiffusion). In most cases, one of the antibody or antigen reagents used in an immunoassay is attached to a signal generating compound or "label". This signal generating compound or "label" is in itself detectable or may be reacted with one or more additional compounds to generate a detectable product. Examples of such signal generating compounds include chromogens, radioisotopes (e.g., 125I, 131I, 32P, 3H, 35S, and 14C), fluorescent compounds (e.g., fluorescein, rhodamine), chemiluminescent compounds, particles (visible or fluorescent), nucleic acids, complexing agents, or catalysts such as enzymes (e.g., alkaline phosphatase, acid phosphatase, horseradish peroxidase, beta-galactosidase, and ribonuclease). In the case of enzyme use, addition of chromo-, fluoro-, or lumo-genic substrate results in generation of a detectable signal. Other detection systems such as time-resolved fluorescence, internal-reflection fluorescence, amplification (e.g., polymerase chain reaction) and Raman spectroscopy are also useful.

There are two general formats commonly used to monitor specific antibody titer and type in humans: (1) antigen is presented on a solid phase, as described above, the human biological fluid containing the specific antibodies is allowed to react with the antigen, and then antibody bound to antigen is detected with an anti-human antibody coupled to a signal generating compound and (2) an anti-human antibody is bound to the solid phase, the human biological fluid containing specific antibodies is allowed to react with the bound antibody, and then antigen attached to a signal generating compound is added to detect specific antibody present in the fluid sample. In both formats, the anti-human antibody reagent may recognize all antibody classes, or alternatively, be specific for a particular class or subclass of antibody, depending upon the intended purpose of the assay. These assays formats as well as other known formats are intended to be within the scope of the present invention and are well known to those of ordinary skill in the art.

In view of the above, therefore, the present invention includes a method of detecting antibodies to mutant HBV in a test sample comprising the steps of: (a) contacting the test sample suspected of containing the antibodies with an antigen or protein comprising the modified "a" determinant and thus the Thr to Ala substitution (for example, the HBsAg, the "a" determinant or the full virus); (b) detecting the presence of the complex and thus antibodies present in the test sample. More specifically, the present invention includes a method of detecting antibodies to mutant HBV in a test sample comprising the steps of: (a) contacting the test sample suspected of containing the antibodies with the antigen for a time and under conditions sufficient to allow the formation of antibody/antigen complexes; (b) adding a conjugate to the resulting antibody/antigen complexes for a time and under conditions sufficient to allow the conjugate to bind to the bound antibody, the conjugate comprising an antibody (directed against the protein) attached to a signal generating compound capable of generating a detectable signal; (c) detecting the presence of the antibody which may be present in the test sample by detecting the signal generated by the signal generating compound. A control or calibrator may also be used which binds to the antigen.

Additionally, the present invention includes another method for detecting the presence of antibody which may be present in a test sample. This method comprises the steps of: (a) contacting the test sample suspected of containing antibodies with anti-antibody specific for the antibody in the sample (i.e., anti-mutant HBV antibody or fragment thereof), for a time and under conditions sufficient to allow for formation of anti-antibody/antibody complexes and (b) detecting the presence of antibody which may be present in the test sample. (Such anti-antibodies are commercially available and may be created, for example, by immunizing a mammal with purified mu-chain of the anti-mutant HBV antibody raised again the protein of the present invention or immunogen.)

More specifically, this method may comprise the steps of: (a) contacting the test sample suspected of containing the antibodies (i.e., anti-mutant HBV antibodies) with anti-antibody specific for the antibodies, under time and conditions sufficient to allow the formation of anti-antibody/antibody complexes; (b) adding a conjugate to the resulting anti-antibody/antibody complexes for a time and under conditions sufficient to allow the conjugate to bind to the bound antibody, the conjugate comprising the protein (i.e., antigen comprising the modified "a" determinant) being attached to a signal generating compound capable of generating a detectable signal; and (c) detecting the presence of the antibodies which may be present in the test sample by detecting the signal generated by the signal generating compound. A control or calibrator may be used which comprises antibody to the anti-antibody.

The present invention also encompasses a third method for detecting the presence of antibody to mutant HBV in a test sample. This method comprises the steps of: (a) contacting the test sample suspected of containing the anti-mutant HBV antibodies with anti-antibody specific for the antibody, under time and conditions sufficient to allow the formation of anti-antibody/antibody complexes; (b) adding protein (i.e., an antigen or polypeptide comprising the modified "a" determinant, for example, the surface antigen) to the resulting anti-antibody/antibody complexes for a time and under conditions sufficient to allow the antigen to bind to the antibody; and (c) adding a conjugate to the resulting anti-antibody/antibody/antigen complexes, the conjugate comprising a composition comprising monoclonal or polyclonal antibody attached to a signal generating compound capable of detecting a detectable signal, the monoclonal or polyclonal antibody being directed against the antigen; and (d) detecting the presence of the antibodies which may be present in the test sample by detecting the signal generated by the signal-generating compound. Again, a control or calibrator may be used which comprises antibody to the anti-antibody.

It should also be noted the one or more of the monoclonal antibodies of the present invention may be used as a competitive probe for the detection of antibodies to the mutant HBV protein of the present invention. For example, a mutant HBV protein of the present invention can be coated on a solid phase. A test sample suspected of containing antibody to the mutant antigen may then be incubated with an indicator reagent comprising a signal-generating compound and at least one monoclonal antibody of the present invention for a time and under conditions sufficient for the formation of antigen/antibody complexes of the test sample and indicator reagent to the solid phase or the indicator reagent to the solid phase. The reduction in binding of the monoclonal antibody to the solid phase can be measured. A measured reduction in the signal as compared to the signal generated from a confirmed negative HBV test sample indicates the presence of anti-HBV antibody in the test sample.

In connection with probes, one may use the nucleic acid sequences of the present invention to synthesize DNA oligomers of about 8 10 nucleotides, or larger, which are useful as hybridization probes in detect the presence of the viral genome in, for example, the sera of subjects suspected of harboring the virus or for screening donated blood for the presence of the virus. The nucleic acid sequences of the present invention also allow for the design and production of mutant HBV specific polypeptides which may be used as diagnostic reagents for the presence of antibodies raised during infection with the virus.

Primers may also be developed using the nucleic acid sequences of the present invention.

It should also be noted that the antibodies of the present invention, or fragments thereof, may be utilized in various diagnostic assays in order to determine the presence of mutant HBV proteins (or nucleic acid sequences corresponding thereto) in a test sample. For example, an antibody directed to one or more of the proteins (i.e., antigens or polypeptides comprising the modified "a" determinant) of the present invention may be added to the test sample for time and under conditions sufficient for the formation of antibody/antigen complexes. If such complexes are detected, then the antigen (i.e., protein) is present in the test sample.

In yet another method, a polyclonal or monoclonal anti-mutant HBV antibody or fragment thereof, or a combination of these antibodies, which has been coated on a solid phase is contacted with a test sample suspected of containing mutant HBV proteins, in order to form a first mixture. This mixture is then incubated for a time and under conditions sufficient to form antigen (i.e., protein)/antibody complexes. An indicator reagent comprising a monoclonal or polyclonal antibody, or fragment thereof, which specifically binds to a mutant HBV region (e.g., the "a" determinant of the virus described herein), or a combination of these antibodies, to which a signal-generating compound has been attached, is then contacted with the antigen/antibody complexes in order to form a second mixture. This second mixture is then incubated for a time and under conditions sufficient for the formation of antibody/antigen/antibody complexes. The presence of mutant HBV protein in the sample and captured on the solid phase is determined by detecting the presence of a measurable signal generated by the signal generating compound. The amount of mutant protein or antigen in the sample is proportional to the signal generated.

Additionally, one may use a different method in order to detect the presence of mutant HBV protein in a test sample. More specifically, a polyclonal or monoclonal anti-mutant HBV antibody (as described above), or a combination thereof, bound to a solid support, the test sample, and an indicator reagent comprising a monoclonal antibody or polyclonal antibody (or fragments thereof) which specifically binds to the mutant HBV antigen (e.g., surface antigen comprising the "a" determinant or the "a" determinant alone), or a combination of these antibodies to which a signal generating compound is attached, are contacted to form a mixture. This mixture is incubated for a time and under conditions sufficient to form antibody/antigen/antibody complexes. Mutant HBV proteins of the present invention and captured on the solid phase are determined by detecting the measurable signal generated by the signal-generating compound. The amount of mutant HBV protein in the test sample is proportional to the signal generated.

It should be noted that one may also detect the presence of antibody and/or antigen to the mutant HBV in a simultaneous assay. More specifically, a test sample is simultaneously contacted with a capture reagent of a first analyte, which comprises a first binding member specific for the first analyte, attached to a solid phase, and a capture reagent of a second analyte, which comprises a first binding member for a second analyte. (A binding member of a pair is defined as a molecule which, through chemical or physical means, specifically binds to the second molecule of the pair.) A mixture is thus formed. This mixture is then incubated for a time and under conditions sufficient to form capture reagent/first analyte and capture reagent/second analyte complexes. These complexes are then contacted with an indicator reagent comprising a member of a binding pair specific for the first analyte labeled with a signal-generating compound and an indicator reagent comprising a member of a binding pair specific for the second analyte labeled with a signal-generating compound. A second mixture is formed. This second mixture is then incubated for a time and under conditions sufficient to form capture reagent/first analyte/indicator reagent complexes and capture reagent/second analyte/indicator reagent complexes. The presence of one or more analytes is determined by detecting a signal generated in connection with the complexes formed on either or both solid phases as an indication of the presence of one of more analytes in the test sample.

While the present invention discloses the use of solid phase diagnostic assays, it is contemplated that the proteins of the present invention may be utilized in non-solid phase diagnostic assays. These assays are well-known to those of ordinary skill in the art and are considered to be within the scope of the present invention.

Additionally, the present invention also includes a vaccine comprising the protein of the present invention and a pharmaceutically acceptable adjuvant (e.g., Freund's adjuvant or Phosphate Buffered Saline (PBS)). Such a vaccine may be administered if one desires to raise antibodies in a mammal. Similarly, the present invention includes a particle which is immunogenic against mutant HBV infection comprising a non-mutant HBV polypeptide having an amino acid sequence capable of forming a particle when the sequence is produced in a eukaryotic host, and an epitope (e.g., the "a" determinant) of the mutant HBV of the present invention.

Kits are also included within the scope of the present invention. More specifically, the present invention includes kits for determining the presence of antibodies. In particular, a kit for determining the presence of antibodies in a test sample comprises a) the protein (i.e., antigen); and b) a conjugate comprising an antibody (directed against the antibody in the test sample) attached to a signal generating compound capable of generating a detectable signal. The kit may also contain a control or calibrator.

The present invention also includes another type of kit for detecting antibodies in a test sample. The kit may comprise a) an anti-antibody specific for the antibody in the test sample (i.e., that produced in response to the mutant HBV), and b) the protein (i.e., the "a" determinant containing the Thr to Ala substitution or a polypeptide such as the surface antigen comprising the "a" determinant). A control or calibrator comprising a reagent which binds to the protein may also be included. More specifically, the kit may comprise a) an anti-antibody specific for the antibody in the sample, and b) a conjugate comprising the protein, the conjugate being attached to a signal-generating compound capable of generating a detectable signal. Again, the kit may also comprise a control of calibrator comprising a reagent which binds to the protein.

In addition, the isolated nucleotide sequences of the present invention, as well as the related sequences described above with respect to sequence identity, may be used in order to create primers and probes. The probes may be used to detect nucleic acids in test samples, and the primers may be used for amplification purposes.

The design of such probes, for optimization in assays, in well within the knowledge of one of ordinary skill in the art. Generally, nucleic acid probes are developed from non-conserved regions when maximum specificity is desired, and nucleic acid probes are developed from conserved regions when assaying for nucleotide regions that are closely related to, for example, different members of a multi-gene family or in related species.

The probes (nucleotide sequences) of the present invention may be used, for example, to discover other antisense oligonucleotides related to those of the present invention. Thus, the probes would hybridize to portions of the Chk1 nucleotide sequence which may then be utilized, for example, for therapeutic purposes.

Primers may also be developed, using the nucleic acid sequences of the present invention, for utilization in the polymerase chain reaction (PCR) (see U.S. Pat. No. 4,683,195 and U.S. Pat. No. 4,683,202). PCR is a technique for amplifying a desired nucleic acid sequence contained in a nucleic acid or mixture thereof. The primers are each extended by a polymerase using the target nucleic acid as a template. The extension products become target sequences, following dissociation from the original target strand. New primers are then hybridized and extended by a polymerase, and the cycle is repeated in order to increase the number of target sequence molecules.

The present invention also encompasses a tissue culture-grown cell infected with the mutant HBV as well as the isolated mutant hepatitis B virus itself. Additionally, the present invention includes an immunogenic composition comprising the virus wherein the virus is attenuated or inactivated.

 

Claim 1 of 3 Claims

1. An isolated polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 1.

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