CA2251904A1 - An antigenic epitope of the a determinant of hepatitis b surface antigen and uses thereof - Google Patents
An antigenic epitope of the a determinant of hepatitis b surface antigen and uses thereof Download PDFInfo
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- CA2251904A1 CA2251904A1 CA002251904A CA2251904A CA2251904A1 CA 2251904 A1 CA2251904 A1 CA 2251904A1 CA 002251904 A CA002251904 A CA 002251904A CA 2251904 A CA2251904 A CA 2251904A CA 2251904 A1 CA2251904 A1 CA 2251904A1
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- hbsag
- peptide
- amino acid
- motif
- polypeptide
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Classifications
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
- C07K16/082—Hepadnaviridae, e.g. hepatitis B virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5761—Hepatitis B
- G01N33/5764—Hepatitis B surface antigen
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- C12N2730/00—Reverse transcribing DNA viruses
- C12N2730/00011—Details
- C12N2730/10011—Hepadnaviridae
- C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
- C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The subject invention relates to a peptide sequence corresponding to amino acid residues (117 to 128) of hepatitis B surface antigen and uses thereof. In particular, the peptide is an antigenic epitope and may therefore be used, for example, as a diagnostic reagent or in the production of a vaccine. Furthermore, the present invention also relates to a C(K/R)TC motif present within the peptide as well as to other peptides containing this motif.
Description
CA 022~1904 1998-10-16 W097/39029 PCT~S97/06732 AN ANTIGENIC EPITOPE OF THE A DETERMINANT OF HEPATITIS
B SURFACE ANTIGEN AND USES THEREOF
BACKGROUND OF THE INVENTION
Technical ~ield The present invention relates to an isolated peptide corresponding to amino acid residues 117 to 128 of hepatitis B surface antigen (HBsAg) and uses thereof. The peptide is an antigenic epitope contributing to the a-determinant and may be used, for example, as a diagnostic reagent for the detection of hepatitis B virus (HBV) and in the production of vaccines against HBV. Furthermore, the invention more specifically relates to peptides containing amino acid residues 121-124 of HBsAg, and thus having a C(K/R)TC motif, as well as to uses of such peptides.
Backa~ound Information Hepatitis B Virus (HBV) is a serious and widespread human pathogen. Acute hepatitis causes significant morbidity and mortality, and chronic infection with the virus is associated with chronic hepatitis, cirrhosis and hepatocellular carcinoma. Currently, approximately 1 million chronically infected people live in the United States, and there are an estimated 300 million carriers worldwide. HBV is a blood born pathogen which is spread by contaminated serum and maternal-neonatal transmission.
Health care workers and others exposed to blood or blood products are at an increased risk of acquiring HBV
infection. Transmission from acutely infected individuals and from persistently infected carriers is well known. In endemic regions, where HBV infection may occur in 75 to 95%
of the population and where the carrier rate exceeds 5%.
The maternal-neonatal transmission and horizontal infections early in life are most critical because such early acquisition of infection, which is usually subclinical and unrecognized, is the ma~or risk factor in . . .
CA 022~1904 1998-10-16 W097/39029 PCT~S97/06732 chronic HBV infection. Worldwide research on the prevention of HBV infection has led to the detection of HBV carriers and vaccine development.
The specific diagnostic marker for acute and chronic HBV infection is the detection of the presence of hepatitis B surface antigen (HBsAg). HBsAg is the major envelope protein found in HBV. During the infectious cycle, a large amount of HBsAg is produced in serum. Only a very small portion of the total HBsAg exists as complete virions or Dane particles [Dane, et al., Lancet 1:695-698 ~1970)]. The majority of HBsAg assembles as small subviral particles, without viral cores, that are in high excess over HBV
virions. The presence of HBsAg in serum is detected by conventional sandwich immunoassays [Overby, et al., Vox San~uinis 24: 102-113 (1973); Wands, et al., Proc. Natl.
Acad. Sci. USA 78: 1214-1218 ~1981); Usuda, et al., J.
Immunol. Methods 87: 1300. (1986)i Zuckerman eds., Viral he~atitis and liver disease, Alan R. Liss Inc, New York, ~1988)]. These immunoassays are extensively used worldwide for diagnosis of HBV infection and for screening blood donors and pregnant women.
The HBsAg protein in the viral and subviral particles displays the major B-cell antigenic determinants which can induce a protective immune response. This has led to the use of native or recombinant HBsAg particles as vaccines for prevention of HBV infection [Szmuness, et al., N. Enal.
J. ~ç~. 303:833 (lg80); Zuckerman eds., Viral he~atitis and liver disease, Alan R. Liss Inc, New York, (1988)].
Serologically, HBsAg contains a common epitope, referred to as the a-determinant, and two sets of subtype determinants d or y and w or r that are mutually exclusive [Le Bouvier, J. Infect, Dis. 123: 671 (1971); Bancroft, et al., J.
Immunol. 109: 842-848 (1972)]. The combination of the common and subtype determinants results in four major subtypes: adw, ayw, adr and ayr. In general, the anti-HBsAg immune response in humans mainly targets the a-CA 022~1904 1998-10-16 W097/3~29 PCT~S97/06732 determinant associated with all subtypes of HBV [Iwarson, et al., J. Med. Virol. 16: 89-95 (1985); Zuckerman eds., Viral he~atitis and liver disease, Alan R. Liss Inc, New York, (1988)]. Immunization with one HBsAg subtype confers protection against HBV infection with all subtypes [Szmuness, et al., N. Encl. J. Med. 303:833 (1980)], indicating that the a-determinant is of the greatest clinical interest.
On a molecular level, HBsAg is a 226 amino acid membrane protein. Primary sequence analysis suggests that HBsAg contains four transmembrane domains and two hydrophilic loops with one loop in the extracellular space and one loop buried inside the HBV particle [Stirk, et al., Interviroloqv 33:148-158 (1992)]. The a-determinant is located in the extracellular loop and spans amino acid residues 101-159. This hydrophilic region (aa 101-159) is extremely rich in cysteine, containing eight cysteine residues. The formation of disulfide bonds among these cysteines is crucial in defining the structure of the a-determinant. The significance of the disulfide bonds was clearly demonstrated in early studies that showed HBsAg immunogenicity was greatly reduced by disulfide bond reduction followed by alkylation of the reduced sulfhydryl groups [Vyas, et al., Science 178:1300 (1972); Dreesman, et al., J. Gen. Virol. 19:129 (1973)].
Studies also suggest that the a-determinant contains several non-overlapping epitopes, indicating that it is not a single determinant, but it is most likely composed of several epitopes located on different regions of HBsAg [Gerin, et al., Proc. Natl. Acad. Sci. USA 80:2365-2369 (1983); Peterson, et al., J. Immunol. 132:920-927 (1984)].
More significantly, the a-determinant can be mimicked by synthetic peptides derived from the a-determinant region (aa 101-159) [Lerner, et al., Proc. Natl. Acad. Sci. USA
78:3403-3407 (1981); Bhatnagar, et al., Proc. Natl. Acad.
Sci. USA 79:4400-4404 (1982); Dreesman, et al., Nature CA 022~1904 1998-10-16 W O 97/39029 PCT~US97106732 295:158-160 (1982); Prince, et al., Proc. Natl. Acad. Sci.
USA 79:579-582 (1982); Gerin, et al., Proc. Natl. Acad.
Sci. USA, 80:2365-2369 (1983); Ohnuma, et al., J. Immunol.
145:2265-2271 (1990); Manivel, et al., J. Immunol.
5 149:2082-2088 (1992)~.
The finding that synthetic peptides can mimic the a-determinant and elicit protective immunoresponses presents a new approach to HBV vaccine development. Although native or recombinant HBsAg based vaccines against HBV are wildly available, there is still an urgent need for the development of an appropriate vaccine for economical mass immunization. Chemically synthesized peptides, therefore, may have advantages in terms of cost and safety of HBV
vaccination programs. Indeed, such a strategy is being employed for the development of potential vaccines against HBV.
Several research groups [Bhatnagar, et al., Proc.
Natl. Acad. Sci. USA 79:4400-4404 (1982); Prince, et al., Proc. Natl. Acad. Sci. USA 79:579-582 (1982); Tam, ed., Synthetic ~e~tide: a~roaches to biolo~ical problems, Alan R. Liss Inc, New York, (1989)] demonstrated that a nonapeptide sequence (aa 139-147) of HBsAg represents an essential part of the a-determinant, eliciting antibodies crossreactive with both ad and ay subtype of HBsAg. It was further discovered that the cyclic version of the peptide (aa 139-147), in which there is a disulfide bond between Cysl39 and Cysl47 as shown in Figure 6, more closely resembles the native conformation of the a-determinant [Tam, ed., Synthetic ~eDtide: approaches to bioloaical problems, Alan R. Liss Inc, New York, (1989)]. The well defined loop structure and immunogenicity of the cyclic peptide (aa 139-147) make it an ideal candidate for a potential synthetic vaccine. However, recent research have established that a vaccine-induced escape mutant of HBV contains an amino acid substitution located in the loop region, with Glyl45 being substituted by Argl45 [Carman, et CA 022~1904 1998-10-16 W O 97/39029 PCT~US97/06732 al., Lancet 345:1406-1407 ~1990); Carman and Thomas, Gastroentroloov 102:711-719 (1992)]. As a result of the Gly-Arg 145 mutation, the HBV mutant escapes from the vaccine-induced immunity and is undetectable by monoclonal based immunoassay [Carman, et al., Lancet 345:1406-1407 (1990); Carman and Thomas, Gastroentrolo~y 102:711-719 (1992)]. Thus, the cyclic peptide (aa 139-147) has limited utility as a synthetic vaccine and diagnostic reagent due to the emergence of new HBV mutants.
Dreesman, et al. [Dreesman, et al. Nature 295:158-160 (1982)] identified another cyclic synthetic peptide (aa 122-137), in which there is a disulfide bond between Cysl24 and Cysl37, as shown in Figure 6, contributing to the a-determinant. However, the cyclic peptide showed much lower(i.e., 3 order lower) affinity than the native HBsAg [Ionescu-Matiu, et al., J. Immunol. 130:1947-lg52 (1983)].
In addition, as shown by the sequence alignment in Figure 5, there is an extensive sequence variation in the region covered by aa 124 to 137 among subtypes and mutants of HBV, indicating that the whole loop structure (aa 124-137) is less likely to be a common epitope shared by all subtypes of HBsAg.
The same peptide sequence (aa 124-147), but presented as an oligomerized form, was also identified as a conformational epitope contributing to the a-determinant [Manivel, et al., J. Immunol. 149:2082-2088 (1992);
European Patent Application WO 94/05698 (1994)]. This oligomerized peptide elicits an immune response that predominantly targets the a-determinant. Compared to the corresponding monomeric peptide, the oligomerized peptide is more immunogenic [Manivel, et al., J. lmmunol. 149:2082-2088 (1992)]. However, the exact structural feature of the oligomerized peptide is undefined. The undefined structure of the oligomerized peptide and the extensive variation of the peptide sequence limit its utility as a synthetic vaccine and a diagnostic reagent.
CA 022~l904 l998-l0-l6 Gerin et al. ~Gerin, et al., Proc. Natl. Acad. Sci.
USA 80:2365-2369 (1983)i Milich and Chisari, U.S. Patent No. 4,599, 230] demonstrated that a synthetic peptide (i.e., 5 aa 110-137) can elicit a subtype specific antibody response against HBsAg. However, because of the peptide subtype specificity, only the HBsAg's subtype from which a peptide is derived can be immune recognized. For instance, an antibody elicited by a peptide sequence derived from the ayw subtype of HBsAg, will not be able to recognize the three other subtypes: adr, awy, awr of HBV [Gerin, et al., Proc. Natl. Acad. Sci. USA 80:2365-2369 (1983)]. This is due to the fact that the peptide (aa 110-137) contains several subtype specific amino acids (see Figure 5) which mimic the subtype determinant more effectively than the a-determinant. The subtype specific immune response of the peptide sequence (aa 110-137) prevents its use as a general vaccine against all subtypes of HBV infections.
More recently, using two shorter peptides ~i.e., aa 115-129 and aa 123-136) of HBsAg, Ohnuma, et al. found that the peptide (aa 115-129) bears an epitope contributing to the a-determinant while the peptide (aa 123-136) represents mainly a subtype specific epitope [Ohnuma, et al. J.
25 Immunol. 145:2265-2271 (1990)]. Their studies showed that 30% of human serum samples from HBsAg-immunized individuals recognized the peptide (aa 115-129), indicating that the peptide (aa 115-129) is an immunodominant epitope.
However, similar to the longer peptide sequence (aa 110-30 137), the shorter peptide sequence (aa 115-129) contains the same subtype specific amino acids at position 117, 120, 122, 125, 126, 127 and 128. Therefore, on a structural basis, it is unknown why the shorter peptide (aa 115-129) mimics the a-determinant better than the longer peptide (aa 35 110-137).
In summary, several non-overlapping peptide sequences derived from the region 110-160 of HBsAg have been CA 022~1904 1998-10-16 W097/39029 PCT~S97/06732 identified as antigenic and immunogenic epitopes contributing to the a-determinant. However, due to subtype sequence variations of HBsAg and the emergence of HBV
mutants, there still existed a need to identify peptide epitopes contributing to the a-determinant with consensus - sequences shared by all subtypes of HBsAg including most HBV mutants. Such peptide epitopes will enhance the antigenicity and immunogenicity to the a-determinant, and the induced immune response will target all subtypes of HBsAg including most HBV mutants.
All U.S. patents and publications referred to herein are hereby incorporated in their entirety by reference.
SUMMARY OF THE INVENTION
The main object of the present invention is to provide a common peptide epitope contributing to the a-determinant of HBsAg that is shared by all subtypes of HBsAg including most HBV mutants.
More specifically, the present invention encompasses an isolated or purified linear or cyclic peptide showing cross reactivity with anti-hepatitis B surface antigen (HBsAg) antiserum which comprises the "a" epitope of HBsAg.
The peptide is a 12 mer of the amino acid sequence corresponding to amino acid residues 117 to 128 of HBsAg.
This peptide may contain a disulfide bond between amino acid residues 121 and 124. This bond yields approximately an eight to ten fold increase in affinity as compared to the linear peptide.
The peptide may contain the C(K/R)TC motif and have the amino acid sequence Xlx2x3x4c~K/R)Tcxsx6x7xs wherein:
CA 022~1904 1998-10-16 x1 is selected from the group consisting of serine, glycine, alanine, valine and an aliphatic amino acid of from two to six carbon atoms;
X2 is selected from the group consisting of threonine, serine, alanine and glycine;
X3 is glycine or alanine;
X4 is selected from the group consisting of proline, serine and threonine;
Xs is selected from the group consisting of threonine, methionine, alanine, serine and glycine;
X6 is selected from the group consisting of threonine, serine, alanine, isoleucine and an aliphatic amino acid of from two to six carbon atoms;
X7 is selected from the group consisting of proline, leucine, threonine, serine, alanine and an aliphatic amino acid of from two to six carbon atoms; and X8 is selected from the group consisting of alanine, glycine, and valine.
Preferably, the peptide has the amino acid sequence STGPC(K/R)TCTT PA or AAGPC( K/R) TCATPA.
Furthermore, the present invention also includes a vaccine against hepatitis B comprising a pharmacologically effective dose of a cyclic peptide showing cross-reactivity with HBsAg antiserum which comprises the "a" epitope of HBsAg, wherein said peptide comprises amino acid residues 121-124 of HBsAg having a C(K/R~TC motif, and wherein said peptide is prepared by synthetic means and a pharmaceutically acceptable carrier. Preferably, the pharmaceutically acceptable carrier is alum. The peptide of the vaccine may further comprise a myristic acid residue added to the amino terminus.
Additionally, the present invention encompasses an antibody directed against the peptides of the invention as well as any fragments thereof. More specifically, it CA 022~1904 1998-10-16 includes those antibodies produced in response to peptides comprising residues 121-124 and having a C(K/R)TC motif.
The antibody may be either monoclonal or polyclonal.
Moreover, the present invention also includes a kit for detecting the presence of hepatitis B surface antigen or antibody in a test sample comprising a container containing a polypeptide comprising a cyclic peptide showing cross-reactivity with HBsAg antiserum which comprises the "a" epitope of HBsAg. The peptide comprises amino acid residues 121-124 of HBsAg having a C (K/R)TC
motif. The polypeptide may be prepared by synthetic means and may be attached to a solid phase.
Additionally, the present invention includes a method for detecting hepatitis B virus surface antigen (HssAg) in a test sample suspected of containing HBsAg comprising the steps of: a. contacting the test sample with an antibody or fragment thereof which specifically binds to a cyclic peptide showing cross-reactivity with HBsAg antiserum which comprises the "a" epitope of HBsAg, wherein the peptide comprises amino acid residues 121-124 of HBsAg having a C(K/R)TC motif, for a time and under conditions sufficient to allow the formation of antibody/antigen complexes; and b. detecting the complexes containing the antibody, wherein said antibody is producing by utilizing a polypeptide prepared by synthetic means. The antibody may be attached to a solid phase and may be monoclonal or polyclonal.
The invention also includes a method for detecting hepatitis B antibodies in a test sample suspected on containing these antibodies comprising the steps of:
a. contacting the test sample with a probe polypeptide wherein the polypeptide comprises a cyclic peptide showing cross-reactivity with HBsAg antiserum which comprises the - "a" epitope of HBsAg, wherein the peptide comprises amino acid residues 121-124 of HBsAg having a C (K/R)TC motif, for a time and under conditions sufficient to allow the CA 022~1904 1998-10-16 formation of antigen/antibody complexes; and b. detecting the complexes which contain the probe polypeptide said antibody. The probe polypeptide may be attached to a solid phase. This solid phase may be selected from the group 5 consisting of beads, microtiter wells, wall of test tube, nitrocellulose strips, magnetic beads and non-magnetic beads.
Furthermore, the present invention also includes a 10 method for producing antibodies to HBsAg comprising administering to an individual an isolated, immunogenic polypeptide or fragment thereof comprising a cyclic peptide showing cross-reactivity with HBsAg antiserum which comprises the "a" epitope of HBsAg, wherein the peptide comprises amino acid residues 121-124 of HBsAg having a C (K/R)TC motif, in an amount sufficient to produce an immune response. The polypeptide may be prepared by synthetic means.
The invention also includes a diagnostic reagent comprising a polypeptide or fragment thereof derived from hepatitis B surface antigen, wherein the polypeptide or fragment thereof comprises a cyclic peptide showing cross-reactivity with HBsAg antiserum which comprises the "a~
25 epitope of HBsAg, wherein the peptide comprises amino acid residues 121-124 of HBsAg having a C(K/R)TC motif. The polypeptide may be produced by synthetic means.
Further, the invention includes all of the above 30 entities and uses wherein the peptide may be, for example, amino acids 117-128 of HBsAg or any other length of an amino acid sequence or fragment of HBSAg provided the peptide contains amino acid residues 121-124 and thus the C (K/R)TC motif.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 ~A) represents binding of mAb H166 to rHBsAg subtype ac~ and ay with equal affinity in ELISA. (B) CA 022~1904 1998-10-16 W097/39029 PCT~S97/06732 represents inhibition of mAb H166 binding to rHBsAg (ay) by free rHBsAg (ay and ad). Degree of inhibition was determined using competitive ELISA as described in the examples.
Figure 2 represents inhibition of mAb H166 binding to rHBsAg (ay) by cyclic peptides derived from HBsAg sequence.
Degree of inhibition was determined using competitive ELISA
as described in the examples.
Figure 3 represents inhibition of mAb H166 binding to rHBsAg (ay) by the linear and the cyclic peptide I
(STGPCKTCTTPA). Degree of inhibition was determined using competitive ELISA as described in the Examples.
Figure 4 represents a plot of ~GmUt-wt at the various alanine substitution sites in peptide I derived from HBsAg.
Figure 5 represents a sequence alignment of the region from residues 101 to 160 of subtypes of HBsAg and HBsAg mutants.
The alignment was performed as described in the Examples.
All sequences are denoted by their Genbank accession codes (source). The original sequences and their authors can easily be retrieved from the NCBI (National Center for Biotechnology Information, Bethesda, MD) or from GCG
(Genetic Computer Group, Madison, WI) using the Genbank accession codes as keywords. The listed subtypes (SUBT) and genotypes (GTYPE) are from the original references. HBsAg mutants are denoted as 'mut' and several undefined subtypes of HBsAg sequences are labeled as 'nd'. The C(K/R)TC motif is shadowed.
Figure 6 illustrates the proposed structures of the HBsAg a determinant. (A) Amino acids 124-147 of H3sAg form two putative loops via the disulfide bridges between cysteines at 124-137 and 139-147 [Bhatnagar, et al., Proc. Natl.
Acad. Sci. USA 79: 4400-4404, (1982); Dreesman, et al., Nature 295:158-160, ~1982); Brown, et al., J. Immunol CA 022~1904 1998-10-16 W O 97/39029 PCTrUS97/06732 Methods, 72: 41-48, (1984)]. (B) The C(K/R)TC motif forms a loop structure via the disulfide bridging between Cysl21 and Cysl24.
DETAILED DESCRIPTION OF THE INVENTION
The subject invention relates to an isolated peptide corresponding to amino acid residues 117 to 128 of hepatitis B surface antigen (HBsAg) and uses thereof. The peptide is an antigenic epitope contributing to the a determinant and may be used as a diagnostic reagent for the detection of hepatitis B virus (HBV) and in the production of vaccines against HBV. Further, the invention relates to the C(K/R)TC motif (aa 121-124) within the epitope which is the main binding site for recognition by anti-a monoclonal antibody. This motif was discovered to be a common epitope shared by all subtypes of HBsAg including most HBV mutants.
The present invention also encompasses peptides and kits containing this motif.
Initially, the peptides of the invention were identified by using an anti-a monoclonal antibody (H166).
The peptide sequence corresponding to amino acid residues 117 to 128 of HBsAg specifically binds to this anti-a monoclonal antibody. In particular, the peptide specifically inhibits the native protein HBsAg binding to the anti-a monoclonal antibody with a IC50 (50% inhibition concentration) in the range of 10-6 to 10-7 M, and the maximum inhibition by the peptide is about 80% (Figure 2 and Figure 3). Therefore, the peptide (aa 117-128) contains an antigenic epitope contributing to the a-determinant.
In addition to the peptides noted above, a previously unidentified disulfide bond between residues Cysl21 and Cysl24 of HBsAg, as shown by Figure 6B, is disclosed by the present invention. As stated in the background, disulfide bond formation among the cysteine residues in the a-CA 022~1904 1998-10-16 W097/39029 PCT~S97/06732 determinant is crucial in defining the structure of the a-determinant. Formation of a correct disulfide bond will enhance the ability of the peptide to mimick the a-determinant, resulting in increased binding affinity of the peptide for anti-a monoclonal antibody. Indeed, compared to the native protein HBsAg, the cyclic form of the peptide (aa 117-128) is only 20-fold less effective, whereas the linear form of the peptide is 160-fold less effective in the inhibition of the anti-a monoclonal antibody binding to HBsAg. Therefore, the loop structure constrained by the disulfide bond between Cysl21 and Cysl24 represents more closely the native conformation of the a-determinant.
It is well known in the art that appropriate restriction of the conformational freedom of synthetic peptides lead to their enhanced performances.
Conformational restriction is usually achieved by crosslinking amino acids within the synthetic peptide. The simplest way to achieve this is to cyclize the peptide via disulfide bridges. There are numbers of examples in which the procedure has improved the antigenic and/or immunogenic properties of synthetic peptides (Kennedy, et al., J.
Virol. 46:653-655 (1983), Ferguson, et al., Virolo~v 143:505-515 (1985)). Similarly, formation of the disulfide bond between Cysl21 and Cysl24 imparts an eight fold increase in affinity to the linear peptide (aa 117-128) for immune recognition by the anti-a monoclonal antibody (F igure 3).
Similar to the previously identified peptide sequences (aa 110-137 and aa 115-129), both linear and cyclic peptide sequences (aa 117-128) of the present invention contain the same subtype specific amino acids at position 117, 120, 122, 125, 126, 127 and 128. The effect of these subtype specific amino acids on the peptides mimicking the a-determinant is unknown. In order to identify the most critical residues in the peptide (aa 117-128) for mimicking the a-determinant, a set of alanine-substituted analogs of CA 022~l904 l998-l0-l6 W O 97/3gO29 PCT~US97/06732 the peptide (aa 117-128), which represent single amino acid substitutions by alanine, were used. Using the alanine-substituted analogs of the peptide (aa 117-128), three residues Cysl21, Thrl23 and Cysl24 were found to be the most critical residues for the anti-a monoclonal antibody recognition (see Example VII). Substitution of alanine for any one of the three residues completely eliminated the peptide (aa 117-128) binding to the anti-a monoclonal antibody (see Table I and Figure 4). Minor effects (i.e., a less than 10 fold decrease or increase in binding affinity) were observed for the remaining residues in the sequence (aa 117-128) (see Table I and Figure 4). These results indicate that the CXTC motif, where position 122 can be any amino acid as represented by the x residue (see Summary section), is the most important structural feature for antibody recognition and for mimicking the a-determinant. In other words, it is the CXTC motif that contributes to the a-determinant, not the remaining residues in the peptide sequence.
Furthermore, sequence analysis indicated that the CXTC
motif is highly conserved among 100 subtypes and mutants of HBsAg isolates. As shown by the sequence alignment (see Figure 5), although there are many sequence variations in the region of residues 101 to 160, the CXTC motif is fully conserved in all of the sequences shown in Figure 5. In addition, the residue at position 122 is relatively conserved; it is either Lys or Arg in all of the sequences shown in Figure 5. Also, the alanine-substituted alalogs showed that keeping Lys or Arg at position 122 imparted approximately a ten-fold increase in affinity (see Table I). Therefore, using both alanine-substituted alalogs and computational sequence analysis, the CXTC, or more accutately the C(K/R)TC motif was identified as the key element or the essential core epitope within the peptides ~aa 117-128 or aa 115-129) for mimiking the a-determinant.
Sequence analysis also indicated that the C(K/R)TC motif is a common epitope shared by all subtypes of HBsAg including CA 022~1904 1998-10-16 W097/39029 PCT~S97/06732 most HBV mutants. Thus, the conservation of the C(K/R)TC
motif explained on a structural basis why immunization with one subtype of HBsAg can confer protection against HBV
infection with all subtypes. Since all subtypes contain the C(K/R)TC motif, immunization with one subtype will induced neutralizing antibodies targeting the C(K/R)TC
motif. Thus, the antibodies will target all HBsAg subtypes as long as they contain the C(K/R)TC motif.
The highly conserved C(K/R)TC motif and its antigenic nature permit the use of the peptide (aa 117-128) or synthetic peptides containing the C(K/R)TC motif as synthetic vaccines. Since the loop structure of the C(K/R)TC motif is well constrained by the disulfide bond between Cysl21 and Cys 124, synthetic peptides containing this motif will mimic the native structure of the a-determinant on HBsAg and elicit neutralizing antibodies that target the C(K/R)TC motif on all subtypes and most mutants of HBsAg. Furthermore, the finding that the high binding affinity can be maintained by the C(K/R)TC motif establishes the feasibility of using the C(K/R)TC motif in tracer epitopes for the detection of HBsAg in a homogeneous immunoassay format. These potential utilities are described below.
Vaccine Pre~aration The peptides of the invention can serve as synthetic vaccines by conjugating the peptides to immunogenic carriers. Suitable carriers include proteins, polysaccharides such as latex functionalized sepharose, agarose, cellulose, cellulose beads, and polymeric amino acids such as polyglutamic acid and polylysine. Examples of protein substrates or carriers include serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, and yet other proteins known to those skilled in the art.
CA 022~l904 l998-l0-l6 WO 97~9029 PCTrUS97/06732 Conjugation methods are known in the art and include but are not limited to using N-succinimidyl-3-(2-pyridylthio)propionate (SPDP) and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC). Either the amino or the carboxyl terminal of the peptides disclosed here can be modified by adding a cysteine residue. These reagents create a disulfide linkage between themselves and peptide cysteine residues on one protein and an amide linkage through the epsilon-amino on a lysine, or other free amino group in the other. A variety of such disulfide/amide-forming agents are known. Other bifunctional coupling agents form a thioester rather than a disulfide linkage. Many of these thio-ether-forming agents are commercially available and are known to those of ordinary skill in the art. The carboxyl group of the peptides also can be activated by combining them with succinimide or 1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt, and the conjugation of the peptides to carriers can be achieved by the formation of an amide bond.
Typically, such synthetic vaccines are prepared as injectables, either as liquid solutions or suspensions;
solid forms suitable for solution in or suspension in liquid prior to injection also may be prepared. The preparation may be emulsified or the protein may be encapsulated in liposomes. The active immunogenic ingredients often are mixed with pharmacologically acceptable excipients which are compatible with the active ingredient. Suitable excipients include but are not limited to water, saline, dextrose, glycerol, ethanol and the like; combinations of these excipients in various amounts also may be used. The vaccine also may contain small amounts of auxiliary substances such as wetting or emulsifying reagents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine. For example, such adjuvants can include aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-CA 022~1904 1998-10-16 W097t3~29 PCT~S97/06732 acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred to as nor-MDP), N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'2'-dipalmitoyl-sn-glycero-3-hydroxphosphoryloxy)-ethylamine (CGP 19835A, also referred to as MTP-PE), and RIBI (MPL + TDM+ CWS) in a 2%
squalene/Tween-80~ emulsion.
The vaccines usually are administered by intravenous or intramuscular injection. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include but are not limited to polyalkylene glycols or triglycerides. Such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10%, preferably, about 1% to about 2%.
oral formulation include such normally employed excipients as, for example pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions may take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 10% to about 95% of active ingredient, preferably about 25% to about 70%.
Vaccines are administered in a way compatible with the dosage formulation, and in such amounts as will be prophylactically and/or therapeutically effective. The quantity to be administered generally is in the range of about 5 micrograms to about 250 micrograms of antigen per dose, and depends upon the subject to be dosed, the capacity of the subject~s immune system to synthesize antibodies, and the degree of protection sought. Precise amounts of active ingredient re~uired to be administered also may depend upon the ~udgment of the practitioner and may be unique to each subject. The vaccine may be given in a single or multiple dose schedule. A multiple dose is one in which a primary course of vaccination may be with one to CA 022~l904 lsss-l0-l6 W 097/39029 PCTrUS97/06732 ten separate doses, followed by other doses given at subsequent time intervals required to maintain and/or to reinforce the immune response, for example, at one to four months for a second dose, and if required by the individual, a subsequent dose or doses after several months. The dosage regimen also will be determined, at least in part, by the need of the individual, and be dependent upon the practitioner~s judgment.
Pre~aration of antibodies a~ainst the C(K/R)TC motif The peptides prepared as described herein are used to produce antibodies against the C(K/R)TC motif, either polyclonal or monoclonal. The peptide could be the sequence aa 117-128 of HBsAg, it also could be any peptide as long as it contains C(K/R)TC motif (aa 121-124). When preparing polyclonal antibodies, a selected mammal (for example, a mouse, rabbit, goat, horse or the like) is immunized with the peptide-carriers disclosed herein.
Serum from the immunized animal is collected after an appropriate incubation period and treated according to known procedures. If serum containing polyclonal antibodies to the C(K/R)TC motif contains antibodies to other antigens, the polyclonal antibodies can be purified by, for example, immunoaffinity chromatography. Techniques for producing and processing polyclonal antibodies are known in the art and are described in, among others, Mayer and Walker, eds., Immunochemical Methods In Cell and Molecular BioloaY, Academic Press, London (1987).
Antibodies specifically against the C(K/R)TC motif also may be obtained from a mammal previously immunized with HBsAg.
An example of a method for purifying antibodies specific to the C(K/R)TC motif from serum of an individual immunized with HBsAg using affinity chromatography is provided herein.
Monoclonal antibodies directed against the C(K/R)TC
motif also can be produced hy one skilled in the art. The CA 022~1904 1998-10-16 general methodology for producing such antibodies is well-known and has been described in, for example, Kohler and Milstein, Nature 256:494 (lg75) and reviewed in J.G.R.
Hurrel, ed., Monoclonal Hvbridoma Antibodies: Techniaues and A~lications, CRC Press Inc., soca Raton, FL (1982), as well as that taught by L. T. Mimms et al., Viroloov 176:604-619 (1990). Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus.
See also, M. Schreier et al., Hybridoma Techniques, Scopes (1980) Protein Purification, Principles and Practice, 2nd Edition, Springer-Verlag, New York (1984); Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas (1981);
Kennet et al., Monoclonal Antibodies (1980). Examples of uses and techniques of monoclonal antibodies are well known to those of skill in the art.
Monoclonal and polyclonal antibodies thus developed, directed against the C(K/R)TC motif, are useful in diagnostic and prognostic applications as well as in passive immunotherapy. Monoclonal antibodies especially can be used to produce anti-idiotype antibodies. These anti-idiotype antibodies are immunoglobulins which carry an ~linternal image~ of the antigen of the infectious agent against which protection is desired. See, for example, A.
Nisonoff et al., Clin. Immunol. Immuno~ath. 21:397-406 (1981), and Dreesman et al., J. Infect. Dis. 151:761 (1985). Techniques for raising such idiotype antibodies are known in the art and exemplified, for example, in Grych et al., Nature 316:74 (1985); MacNamara et al., Science 226:1325 (1984); and Uytdehaag et al., J. Immunol. 134:1225 (1985). These anti-idiotypic antibodies also may be useful for treatment of HBV infection.
CA 022~1904 1998-10-16 W O 9713gO29 rcTrusg7/o6732 Immunoassa~ and Diaanostic Kits Both the peptides of the present invention and the antibodies raised against the C(K/T)TC motif are useful in immunoassays to detect the presence of HBsAg or HBV in biological test samples. The design of these immunoassays is subject to variation, and a variety of these are known in the arti a variety of these have been described herein.
Examples of assays which utilize labels as the signal generating compound and those labels are described herein.
Signals also may be amplified by using biotin and avidin, enzyme labels or biotin anti-biotin systems.
One of the competitive assay formats using solid phase can be designed which utilize the signal labeled ( with radioactive isotope, such as 1125, or with an enzyme, or with biotin) synthetic peptides detailed herein and a monoclonal or polyclonal antibodies directed against the C(K/T)TC motif. In the assay format to detect the presence of HBsAg in a human test sample, a known amount of signal labeled peptides is first added to the human test sample, then the human test sample containing certain amount signal labeled peptides is contacted and incubated with a solid phase coated with a monoclonal or polyclonal antibodies directed against the C(K/T)TC motif. If HBsAg and HBV
particles are present in the test sample, they will compete with the labeled peptides binding to the monoclonal or polyclonal antibodies on the solid surface. After removal of unbound materials and peptides by washing the solid phase, the amount of peptides bound by the antibodies can be determined by determining the level of radioactivity or by adding an enzyme substrate or by adding the anti-biotin conjugate following the addition of substrate. ~he reduced signal is proportional to the amount of HBsAg and HBV
particles in the human test sample.
CA 022~l904 l998-l0-l6 In fluorescence polari~ation immunoassay (FPIA), which is a homogeneous competitive assay,; i.e. it does not reguire wash or separation steps, synthetic peptides, particularly the C(K/R)TC motif, of the present invention may be used as tracer epitopes for the detection of HBsAg.
Using synthetic peptides as tracer epitopes in FPIAs of high molecular weight proteins is known in the art and is described in, among others, Geysen, et al., J. Immunol.
Methods 102:259-274 (1987); Houghten, et al., Nature 354:84-86 (1991); Lam, et al., Nature 354:82-83 (1991);
U.S. Patent No. 4,833, 092. In the FPIA, peptides of the present invention covalently linked to labeling group (such as fluorescein) will be used as a tracer; and an antibody (monoclonal or polyclonal) against the C(K/R)TC motif will be used to form a complex with the peptide epitope tracer.
The system containing the complex of the epitope tracer and the antibody then can be used to detect and quantitate the presence of HBsAg in human test sample by monitoring the change of fluorescence polarization.
The present invention can be illustrated by the use of the following non-limiting examples: ' EXAMPLES
EXAMPLE I
PEPTIDE SYNTHESIS
All Fmoc protected amino acid and reagents used for peptide synthesis were purchased from Applied Biosystem (Foster City, CA) or Rainin Instrument Co. Inc.
(Emeryville, CA) Monoclonal antibody (mAb) H166 [Peterson, et al., J. Immunol. 132: 920927 (1984)] was obtained from the Abbott Monoclonal Antibody Development Group, Abbott Park, Illinois. Recombinant HBsAg [Mimms, et al., J Virol.
Methods 25:211-232, (1989)], subtype ad and ay, was obtained from Abbott Rare Reagent Development Group (Abbott Park, IL). Alkaline phosphates-conjugated goat anti-mouse CA 022~1904 1998-10-16 W O 97t39029 PCTrUS97/06732 IgG and IgM were purchased from Pierce Chemical Co., (Rockford,IL).
All peptides were synthesized by the stepwise solid-phase method of Merrifield ~Merrifield, J. Am. Chem. Soc.
85:2149-2154, (1963)] on an Applied siosystem 431A
Synthesizer (Foster City, CA) or a Rainin Symphony Synthesizer (Emeryville, CA) using standard Fmoc (9-fluorenylmethoxycarbonyl) chemistry. Peptides were cleaved and deprotected with a mixture of 82% trifluoroacetic acid (TFA), 5% phenol, 5% H2O, 5% thioanisole and 2.5%
ethanedithiol for 2-4 hours at 25~C and precipitated by addition of cold ether. Crude peptides were purified by reverse phase HPLC using a 5-50% acetonitrile gradient containing 0.1% TFA. The homogeneity and identity of the purified peptides were confirmed by electro-spray mass spectrometry. All peptides were determined to be at least 95% pure. Cyclic peptides formed by intramolecular disulfide bridging were synthesized using the air oxidation method [Tam, et al., Proc. Natl. Acad. Sci. USA 83: 8082, (1986)]. Briefly, purified linear peptides were dissolved in 0.1 mM Tris-HCl buffer (pH 8.4) at a concentration of 0.5 mg/ml and stirred at 25 C for varying periods of time exposed to air. Cyclization was monitored by HPLC. Cyclic peptides were purified as described above and formation of intramolecular disulfide bonds was confirmed by electro-spray mass spectra.
EXAMPLE II
DETERMINATION OF AFFINITY
Enzyme-linked immunoassays (ELISA~ were used to evaluate the affinity of monoclonal antibody H166 to the rHssAg, subtype (ad and ay, 5 ~g/ml) in 0.2 M carbonate-bicarbonate buffer (pH 9.0) and blocked with 1% ssA in Pss buffer (pH 7.4). Twofold dilutions of H166, starting concentration (1 ~g/ml), were subsequently added (100 ~l/well) and the ad and ay. Briefly, microtiter plates were coated with ~l/well of rHBsAg 100 plates were incubated for 1 hour at 25 C. The wells were washed with CA 022~1904 1998-10-16 W 097139029 PCTrUS97/06732 PBS buffer containing 0.5% Tween 20, and 100 ~Ll/well of alkaline phosphates-conjugated goat antimouse Ig at 5000 fold dilution was added and incubated for 1-2 hr at 25 C.
After washing, 100 ~l/well of p-nitrophenyl phosphate substrate was added, and the absorbance was measured at 405 nm using an automated microtiter plate reader (Molecular Devices Corp., Menlo Park, CA). ELISA results were used to fit an analog of the Michaelis-Menten equation [Langone and Van Vunakis eds, Methods in Enz~moloov, Academic Press, San Diego, (1986)]:
[Ag--A~] = [Ag--Ab]ma~. x [Ab] + K
Where [Ag-Ab] is the antigen-antibody complex concentration, [Ag-Ab] maX is the maximum complex concentration, [Ab] is the antibody concentration, and Kd is the dissociation constant. During the curve fitting, the [Ag - Ab] was substituted with the value of OD405mm at the given concentration of antibody. Both Kd and [OD405nm]max~ which corresponds to the [Ag - Ab]~aX, were treated as fitted parameters.
CA 022~1904 1998-10-16 W O 97/39029 PCTrUS97/06732 EXAMPLE III
COMPETITIVE ELISAs Competitive ELISAs were used to identify the potential antigenic determinants and to compare their affinity through the determined IC50s. Microtiter plates were coated with rHBsAg and blocked with ~SA as described above.
Aliquots of peptides or rHBsAg competitors (50 ~l/well) at varying dilutions were added together with 100 ~l of H166 at 0.4 ~l/ml, and incubated for 1 hour at 25'C. The wells were washed with phosphate buffered saline (PBS) containing 0.5% Tween 20, and alkaline phosphatase-conjugated goat anti-mouse Ig at 5000 fold dilution (100 ~l/well) was added. The plates were incubated for 1-2 hr at 25 C, washed, and 100 ~l/well of p-nitrophenyl phosphate substrate was added, and the absorbance was measured at 405 nm. The percent inhibition of mAb H166 binding to rHBsAg by peptides was calculated according to the 'following equation:
. . OD40swith peptide - bkgd Inhlbltlon% = 100 x 1- . .
OD40swlthout peptlde-bkgd where the bkgd is the absorbance from the well without coating of rHBsAg.
EXAMPLE IV
SEOUENCE ANALYSIS
Nucleotide sequences corresponding to ~BsAg coding sequences were obtained from Genbank (release 89.0, 6/95) and EMBL (release 42.0, 3/95). The nucleotide sequences were translated into protein sequences and analyzed using programs from the Wisconsin Genetic Computer Group sequence analysis package (GCG, Version 8.0). Multiple sequence alignment of protein sequence was performed using progressive pairwise alignment (PILEUP, GCG), and sequences were displayed using the program PRETTY ( GCG). Final alignment and editing were performed manually.
CA 022~1904 1998-10-16 W O 97/39029 PCTrUS97/06732 EXAMPLE V
IDENTIFICATION OF PEPTIDE EPITOPE CONTRIBUTING TO THE A-DETERMINANT
Monoclonal antibody H166 is specific for the a-determinant, recognizing nine different subtypes of HBsAg as observed by Peterson et al.[Peterson, et al., J.
Immunol. 132: 920927 (1984)]. In order to quantitatively compare the affinity of mAB H166 for different HBsAg subtypes, the apparent dissociation constants (Kd) derived from the fitting curves for both subtype ad and ay of rHBsAg were determined via ELISA. As shown in Figure lA, mAb H166 binds the ad and ay subtypes with equal affinity.
The Kd derived from the fitting curves for both subtypes ad and ay, is approximately 0.9 ~g/ml, corresponding to lx10-8M. Competitive ELISAs were also used to evaluate the crossreactivity of mAb H166 with subtype ad and ay .
Figure lB shows the inhibition curves using free ad and ay subtypes as competitors. It is clear that both subtypes inhibit mAb H166 binding to rHBsAg with similar affinities.
The IC50s for subtype ay and ad are 0.026 ~M and 0.030 ~M
respectively. Thus, H166 recognizes a common epitope shared by the ad and ay subtypes of HBsAg.
Since H166 is a monoclonal anti-a antibody, it can be used to screen for an epitope that contributes to the a -determinant of HBsAg. Identifying the epitope of H166 will help to define the a determinant, or part of the a-determinant, on HBsAg. To identify the H166 epitope, three cyclic peptides derived from the extracellular hydrophilic region of HBsAg were synthesized. The peptide sequences are shown in Figure 2. Although the cyclic peptides II (aa 124-137) and III (aa 139-147) are known to be a major part of the a-determinant [Bhatnagar, et al. Proc. Natl. Acad.
Sci. USA 79:4400-4404 (1982); Dreesman, et al. Nature, 295:158-160 (1982)] only cyclic peptide I (aa 117-128~
binds specifically to H166. As shown in Figure 2, the cyclic peptide I inhibits 70% of HBsAg binding to the H166 -CA 022~l904 l998-l0-l6 W 097/39029 PCTrUS97/06732 at a concentration of 2 mM. Therefore, it is clear that peptide I represents the epitope of H166. It is interesting to note that peptide II exhibits about 20%
inhibition at a concentration of 2 mM, probably because part of its se~uence (aa 124-128) overlaps with peptide I
(aa 117-128).
EXAMPLE VI
AFFINITY OF LINEAR VS CYCLIC PEPTIDE I (aa 117-128) To further characterize the identified epitope of mAb H166, the IC50 of the cyclic peptide I (aa 117-128) was compared to the IC50s of its linear sequence and the native protein, rHBsAg (subtype ad), using competitive ELISA. As shown in Figure 3, the IC50 of cyclic peptide I is 0.54 ~1, only 20 fold less than the IC50 of the native protein, rHBsAg (0. 026~1M). The high potency of the cyclic peptide indicates that it contains the essential part of the epitope on HBsAg recognized by the mAb. More importantly, the IC50 of the linear peptide (4.0 ~M) is 8 fold higher than the IC50 of the cyclic peptide, although both peptides exhibit similar inhibitions at higher concentration (>0.1 mM). These result indicate that the cyclic peptide I
contains a micro conformation that is closer to the native structure of the a determinant on HBsAg compared to the linear peptide I.
An experiment was performed to verify that the sulfhydryl groups of the linear peptide I (aa 117-128) remained in a reduced form under the experimental conditions. Linear peptide I was first incubated with H166 in PBS buffer for 1 hour, and the mixture was analyzed by electro-spray mass spectrometry. No trace of the oxidized molecular ion was observed in the mass spectra, confirming that no oxidation of the sulfhydryl groups occurred during the time course of the ELISA. These results indicate that the linear peptide can be recognized by the mAb H166, although the cyclic form is the favorable confirmation.
CA 022~1904 1998-10-16 W097/39029 PCT~S97/06732 EXAMPLE VII
RESIDUES IN PEPTIDE I (aa 117-128) CRITICAL FQR BINDING
AFFINITY
The cyclic peptide I (aa 117-128) contains a critical residue at position 122 that differentiates the d and y subtypes. For the d subtype, position 122 is Lys, and Arg for the y subtype [Okamoto, et al. J. Virol. 61: 3030-3034, (1987)]. The insensitivity of mAb H166 to the subtype-specific residue implies this residue is not critical for the binding affinity. To prove this hypothesis and to identify the critical residues for mAb H166 recognition, a set of alanine-substituted analogs of peptide I, which represent single amino acid substitutions by alanine, were synthesized. Inhibition of H166 binding to HBsAg by the alanine-substituted peptides were determined using competitive ELISA. Table 1 shows the alanine-substituted peptide sequences and their determined IC50s.
Substitutions of Cysl21 and Cysl24 with Ala almost completely eliminate specific binding to H166. IC50s could not be obtained for these two peptides (121C and 124C) because no concentration dependent inhibition curves were observed in the concentration range 2.9x10-9 to 2.9x10-4 M.
Both peptides (121C and 124C) exhibited only 20% inhibition at the highest peptide concentration (3 mM).
Under the assay conditions, the IC50 is approximately inversely proportional to the binding affinity constant Ka [Cheng, et al., Biochem. Pharmacol. 22: 3099. (1973);
Munson, et al., ~n~l. Biochem. 107:220-239, (1980)]. Thus, the differences in binding free energy between alanine-substituted peptide and the wild type peptide (~Gmut-wt) were derived based on the equation:
~Gm~t-WI = - RT lnlc5ow~
where R is the gas constant and T is the temperature.
Figure ~ shows the plot of ~GmUt-wt at the alanine-substitution site. It is very clear that Cysl21, Thrl23 CA 022~1904 1998-10-16 W097/39029 PCT~S97/06732 and Cysl24 are the most critical residues for H166 recognition, since alanine substitutions at these three residues cause substantial loss in binding energy (>4.5 kcal/mol). Minor effects (0.5-1.5kcal/mol) were observed for the remaining residues, including residue 122. These results indicate that the CXTC motif is the main binding site or the essential core epitope of H166 on HBsAg where position 122 can be any amino acid as represented by the X
residue. These results also explain why mAb H166 binding is insensitive to the ad and ay subtype-specific residue.
Since H166 recognition only requires the CXTC motif, it will recognize all subtypes of HBV as long as the CXTC
motif is conserved in their HBsAg sequences.
EXAMPL E VI I I
SEOUENCE ALIGNMENT OF HBsAa In order to evaluate the conservation of the CXTC
motif in all subtypes of HBsAg and HBsAg mutants, analysis of 100 HBsAg sequences derived from human HBV genomes retrieved from the Genbank and EMBL databases was performed. The retrieved nucleotide sequences were translated into protein sequences and aligned using PILEUP.
From the sequence analysis, 43 unique sequences in the extracellular hydrophilic region from residues 101 to 160 HBsAg were identified and the sequence alignment is shown in Figure 5. Although there are many sequence variations in the entire region, it is very clear that the CXTC motif is fully conserved in all retrieved sequences regardless of the genotypes or subtypes. The alignment also shows that residue 122 is relatively conserved; it is either Lys or Arg in all sequences shown in Figure 5. From the sequence analysis results, it can be concluded that the CXTC, or more accurately the C(K/R)TC motif, is a common epitope shared by all subtypes of HBsAg including HBsAg mutants.
B SURFACE ANTIGEN AND USES THEREOF
BACKGROUND OF THE INVENTION
Technical ~ield The present invention relates to an isolated peptide corresponding to amino acid residues 117 to 128 of hepatitis B surface antigen (HBsAg) and uses thereof. The peptide is an antigenic epitope contributing to the a-determinant and may be used, for example, as a diagnostic reagent for the detection of hepatitis B virus (HBV) and in the production of vaccines against HBV. Furthermore, the invention more specifically relates to peptides containing amino acid residues 121-124 of HBsAg, and thus having a C(K/R)TC motif, as well as to uses of such peptides.
Backa~ound Information Hepatitis B Virus (HBV) is a serious and widespread human pathogen. Acute hepatitis causes significant morbidity and mortality, and chronic infection with the virus is associated with chronic hepatitis, cirrhosis and hepatocellular carcinoma. Currently, approximately 1 million chronically infected people live in the United States, and there are an estimated 300 million carriers worldwide. HBV is a blood born pathogen which is spread by contaminated serum and maternal-neonatal transmission.
Health care workers and others exposed to blood or blood products are at an increased risk of acquiring HBV
infection. Transmission from acutely infected individuals and from persistently infected carriers is well known. In endemic regions, where HBV infection may occur in 75 to 95%
of the population and where the carrier rate exceeds 5%.
The maternal-neonatal transmission and horizontal infections early in life are most critical because such early acquisition of infection, which is usually subclinical and unrecognized, is the ma~or risk factor in . . .
CA 022~1904 1998-10-16 W097/39029 PCT~S97/06732 chronic HBV infection. Worldwide research on the prevention of HBV infection has led to the detection of HBV carriers and vaccine development.
The specific diagnostic marker for acute and chronic HBV infection is the detection of the presence of hepatitis B surface antigen (HBsAg). HBsAg is the major envelope protein found in HBV. During the infectious cycle, a large amount of HBsAg is produced in serum. Only a very small portion of the total HBsAg exists as complete virions or Dane particles [Dane, et al., Lancet 1:695-698 ~1970)]. The majority of HBsAg assembles as small subviral particles, without viral cores, that are in high excess over HBV
virions. The presence of HBsAg in serum is detected by conventional sandwich immunoassays [Overby, et al., Vox San~uinis 24: 102-113 (1973); Wands, et al., Proc. Natl.
Acad. Sci. USA 78: 1214-1218 ~1981); Usuda, et al., J.
Immunol. Methods 87: 1300. (1986)i Zuckerman eds., Viral he~atitis and liver disease, Alan R. Liss Inc, New York, ~1988)]. These immunoassays are extensively used worldwide for diagnosis of HBV infection and for screening blood donors and pregnant women.
The HBsAg protein in the viral and subviral particles displays the major B-cell antigenic determinants which can induce a protective immune response. This has led to the use of native or recombinant HBsAg particles as vaccines for prevention of HBV infection [Szmuness, et al., N. Enal.
J. ~ç~. 303:833 (lg80); Zuckerman eds., Viral he~atitis and liver disease, Alan R. Liss Inc, New York, (1988)].
Serologically, HBsAg contains a common epitope, referred to as the a-determinant, and two sets of subtype determinants d or y and w or r that are mutually exclusive [Le Bouvier, J. Infect, Dis. 123: 671 (1971); Bancroft, et al., J.
Immunol. 109: 842-848 (1972)]. The combination of the common and subtype determinants results in four major subtypes: adw, ayw, adr and ayr. In general, the anti-HBsAg immune response in humans mainly targets the a-CA 022~1904 1998-10-16 W097/3~29 PCT~S97/06732 determinant associated with all subtypes of HBV [Iwarson, et al., J. Med. Virol. 16: 89-95 (1985); Zuckerman eds., Viral he~atitis and liver disease, Alan R. Liss Inc, New York, (1988)]. Immunization with one HBsAg subtype confers protection against HBV infection with all subtypes [Szmuness, et al., N. Encl. J. Med. 303:833 (1980)], indicating that the a-determinant is of the greatest clinical interest.
On a molecular level, HBsAg is a 226 amino acid membrane protein. Primary sequence analysis suggests that HBsAg contains four transmembrane domains and two hydrophilic loops with one loop in the extracellular space and one loop buried inside the HBV particle [Stirk, et al., Interviroloqv 33:148-158 (1992)]. The a-determinant is located in the extracellular loop and spans amino acid residues 101-159. This hydrophilic region (aa 101-159) is extremely rich in cysteine, containing eight cysteine residues. The formation of disulfide bonds among these cysteines is crucial in defining the structure of the a-determinant. The significance of the disulfide bonds was clearly demonstrated in early studies that showed HBsAg immunogenicity was greatly reduced by disulfide bond reduction followed by alkylation of the reduced sulfhydryl groups [Vyas, et al., Science 178:1300 (1972); Dreesman, et al., J. Gen. Virol. 19:129 (1973)].
Studies also suggest that the a-determinant contains several non-overlapping epitopes, indicating that it is not a single determinant, but it is most likely composed of several epitopes located on different regions of HBsAg [Gerin, et al., Proc. Natl. Acad. Sci. USA 80:2365-2369 (1983); Peterson, et al., J. Immunol. 132:920-927 (1984)].
More significantly, the a-determinant can be mimicked by synthetic peptides derived from the a-determinant region (aa 101-159) [Lerner, et al., Proc. Natl. Acad. Sci. USA
78:3403-3407 (1981); Bhatnagar, et al., Proc. Natl. Acad.
Sci. USA 79:4400-4404 (1982); Dreesman, et al., Nature CA 022~1904 1998-10-16 W O 97/39029 PCT~US97106732 295:158-160 (1982); Prince, et al., Proc. Natl. Acad. Sci.
USA 79:579-582 (1982); Gerin, et al., Proc. Natl. Acad.
Sci. USA, 80:2365-2369 (1983); Ohnuma, et al., J. Immunol.
145:2265-2271 (1990); Manivel, et al., J. Immunol.
5 149:2082-2088 (1992)~.
The finding that synthetic peptides can mimic the a-determinant and elicit protective immunoresponses presents a new approach to HBV vaccine development. Although native or recombinant HBsAg based vaccines against HBV are wildly available, there is still an urgent need for the development of an appropriate vaccine for economical mass immunization. Chemically synthesized peptides, therefore, may have advantages in terms of cost and safety of HBV
vaccination programs. Indeed, such a strategy is being employed for the development of potential vaccines against HBV.
Several research groups [Bhatnagar, et al., Proc.
Natl. Acad. Sci. USA 79:4400-4404 (1982); Prince, et al., Proc. Natl. Acad. Sci. USA 79:579-582 (1982); Tam, ed., Synthetic ~e~tide: a~roaches to biolo~ical problems, Alan R. Liss Inc, New York, (1989)] demonstrated that a nonapeptide sequence (aa 139-147) of HBsAg represents an essential part of the a-determinant, eliciting antibodies crossreactive with both ad and ay subtype of HBsAg. It was further discovered that the cyclic version of the peptide (aa 139-147), in which there is a disulfide bond between Cysl39 and Cysl47 as shown in Figure 6, more closely resembles the native conformation of the a-determinant [Tam, ed., Synthetic ~eDtide: approaches to bioloaical problems, Alan R. Liss Inc, New York, (1989)]. The well defined loop structure and immunogenicity of the cyclic peptide (aa 139-147) make it an ideal candidate for a potential synthetic vaccine. However, recent research have established that a vaccine-induced escape mutant of HBV contains an amino acid substitution located in the loop region, with Glyl45 being substituted by Argl45 [Carman, et CA 022~1904 1998-10-16 W O 97/39029 PCT~US97/06732 al., Lancet 345:1406-1407 ~1990); Carman and Thomas, Gastroentroloov 102:711-719 (1992)]. As a result of the Gly-Arg 145 mutation, the HBV mutant escapes from the vaccine-induced immunity and is undetectable by monoclonal based immunoassay [Carman, et al., Lancet 345:1406-1407 (1990); Carman and Thomas, Gastroentrolo~y 102:711-719 (1992)]. Thus, the cyclic peptide (aa 139-147) has limited utility as a synthetic vaccine and diagnostic reagent due to the emergence of new HBV mutants.
Dreesman, et al. [Dreesman, et al. Nature 295:158-160 (1982)] identified another cyclic synthetic peptide (aa 122-137), in which there is a disulfide bond between Cysl24 and Cysl37, as shown in Figure 6, contributing to the a-determinant. However, the cyclic peptide showed much lower(i.e., 3 order lower) affinity than the native HBsAg [Ionescu-Matiu, et al., J. Immunol. 130:1947-lg52 (1983)].
In addition, as shown by the sequence alignment in Figure 5, there is an extensive sequence variation in the region covered by aa 124 to 137 among subtypes and mutants of HBV, indicating that the whole loop structure (aa 124-137) is less likely to be a common epitope shared by all subtypes of HBsAg.
The same peptide sequence (aa 124-147), but presented as an oligomerized form, was also identified as a conformational epitope contributing to the a-determinant [Manivel, et al., J. Immunol. 149:2082-2088 (1992);
European Patent Application WO 94/05698 (1994)]. This oligomerized peptide elicits an immune response that predominantly targets the a-determinant. Compared to the corresponding monomeric peptide, the oligomerized peptide is more immunogenic [Manivel, et al., J. lmmunol. 149:2082-2088 (1992)]. However, the exact structural feature of the oligomerized peptide is undefined. The undefined structure of the oligomerized peptide and the extensive variation of the peptide sequence limit its utility as a synthetic vaccine and a diagnostic reagent.
CA 022~l904 l998-l0-l6 Gerin et al. ~Gerin, et al., Proc. Natl. Acad. Sci.
USA 80:2365-2369 (1983)i Milich and Chisari, U.S. Patent No. 4,599, 230] demonstrated that a synthetic peptide (i.e., 5 aa 110-137) can elicit a subtype specific antibody response against HBsAg. However, because of the peptide subtype specificity, only the HBsAg's subtype from which a peptide is derived can be immune recognized. For instance, an antibody elicited by a peptide sequence derived from the ayw subtype of HBsAg, will not be able to recognize the three other subtypes: adr, awy, awr of HBV [Gerin, et al., Proc. Natl. Acad. Sci. USA 80:2365-2369 (1983)]. This is due to the fact that the peptide (aa 110-137) contains several subtype specific amino acids (see Figure 5) which mimic the subtype determinant more effectively than the a-determinant. The subtype specific immune response of the peptide sequence (aa 110-137) prevents its use as a general vaccine against all subtypes of HBV infections.
More recently, using two shorter peptides ~i.e., aa 115-129 and aa 123-136) of HBsAg, Ohnuma, et al. found that the peptide (aa 115-129) bears an epitope contributing to the a-determinant while the peptide (aa 123-136) represents mainly a subtype specific epitope [Ohnuma, et al. J.
25 Immunol. 145:2265-2271 (1990)]. Their studies showed that 30% of human serum samples from HBsAg-immunized individuals recognized the peptide (aa 115-129), indicating that the peptide (aa 115-129) is an immunodominant epitope.
However, similar to the longer peptide sequence (aa 110-30 137), the shorter peptide sequence (aa 115-129) contains the same subtype specific amino acids at position 117, 120, 122, 125, 126, 127 and 128. Therefore, on a structural basis, it is unknown why the shorter peptide (aa 115-129) mimics the a-determinant better than the longer peptide (aa 35 110-137).
In summary, several non-overlapping peptide sequences derived from the region 110-160 of HBsAg have been CA 022~1904 1998-10-16 W097/39029 PCT~S97/06732 identified as antigenic and immunogenic epitopes contributing to the a-determinant. However, due to subtype sequence variations of HBsAg and the emergence of HBV
mutants, there still existed a need to identify peptide epitopes contributing to the a-determinant with consensus - sequences shared by all subtypes of HBsAg including most HBV mutants. Such peptide epitopes will enhance the antigenicity and immunogenicity to the a-determinant, and the induced immune response will target all subtypes of HBsAg including most HBV mutants.
All U.S. patents and publications referred to herein are hereby incorporated in their entirety by reference.
SUMMARY OF THE INVENTION
The main object of the present invention is to provide a common peptide epitope contributing to the a-determinant of HBsAg that is shared by all subtypes of HBsAg including most HBV mutants.
More specifically, the present invention encompasses an isolated or purified linear or cyclic peptide showing cross reactivity with anti-hepatitis B surface antigen (HBsAg) antiserum which comprises the "a" epitope of HBsAg.
The peptide is a 12 mer of the amino acid sequence corresponding to amino acid residues 117 to 128 of HBsAg.
This peptide may contain a disulfide bond between amino acid residues 121 and 124. This bond yields approximately an eight to ten fold increase in affinity as compared to the linear peptide.
The peptide may contain the C(K/R)TC motif and have the amino acid sequence Xlx2x3x4c~K/R)Tcxsx6x7xs wherein:
CA 022~1904 1998-10-16 x1 is selected from the group consisting of serine, glycine, alanine, valine and an aliphatic amino acid of from two to six carbon atoms;
X2 is selected from the group consisting of threonine, serine, alanine and glycine;
X3 is glycine or alanine;
X4 is selected from the group consisting of proline, serine and threonine;
Xs is selected from the group consisting of threonine, methionine, alanine, serine and glycine;
X6 is selected from the group consisting of threonine, serine, alanine, isoleucine and an aliphatic amino acid of from two to six carbon atoms;
X7 is selected from the group consisting of proline, leucine, threonine, serine, alanine and an aliphatic amino acid of from two to six carbon atoms; and X8 is selected from the group consisting of alanine, glycine, and valine.
Preferably, the peptide has the amino acid sequence STGPC(K/R)TCTT PA or AAGPC( K/R) TCATPA.
Furthermore, the present invention also includes a vaccine against hepatitis B comprising a pharmacologically effective dose of a cyclic peptide showing cross-reactivity with HBsAg antiserum which comprises the "a" epitope of HBsAg, wherein said peptide comprises amino acid residues 121-124 of HBsAg having a C(K/R~TC motif, and wherein said peptide is prepared by synthetic means and a pharmaceutically acceptable carrier. Preferably, the pharmaceutically acceptable carrier is alum. The peptide of the vaccine may further comprise a myristic acid residue added to the amino terminus.
Additionally, the present invention encompasses an antibody directed against the peptides of the invention as well as any fragments thereof. More specifically, it CA 022~1904 1998-10-16 includes those antibodies produced in response to peptides comprising residues 121-124 and having a C(K/R)TC motif.
The antibody may be either monoclonal or polyclonal.
Moreover, the present invention also includes a kit for detecting the presence of hepatitis B surface antigen or antibody in a test sample comprising a container containing a polypeptide comprising a cyclic peptide showing cross-reactivity with HBsAg antiserum which comprises the "a" epitope of HBsAg. The peptide comprises amino acid residues 121-124 of HBsAg having a C (K/R)TC
motif. The polypeptide may be prepared by synthetic means and may be attached to a solid phase.
Additionally, the present invention includes a method for detecting hepatitis B virus surface antigen (HssAg) in a test sample suspected of containing HBsAg comprising the steps of: a. contacting the test sample with an antibody or fragment thereof which specifically binds to a cyclic peptide showing cross-reactivity with HBsAg antiserum which comprises the "a" epitope of HBsAg, wherein the peptide comprises amino acid residues 121-124 of HBsAg having a C(K/R)TC motif, for a time and under conditions sufficient to allow the formation of antibody/antigen complexes; and b. detecting the complexes containing the antibody, wherein said antibody is producing by utilizing a polypeptide prepared by synthetic means. The antibody may be attached to a solid phase and may be monoclonal or polyclonal.
The invention also includes a method for detecting hepatitis B antibodies in a test sample suspected on containing these antibodies comprising the steps of:
a. contacting the test sample with a probe polypeptide wherein the polypeptide comprises a cyclic peptide showing cross-reactivity with HBsAg antiserum which comprises the - "a" epitope of HBsAg, wherein the peptide comprises amino acid residues 121-124 of HBsAg having a C (K/R)TC motif, for a time and under conditions sufficient to allow the CA 022~1904 1998-10-16 formation of antigen/antibody complexes; and b. detecting the complexes which contain the probe polypeptide said antibody. The probe polypeptide may be attached to a solid phase. This solid phase may be selected from the group 5 consisting of beads, microtiter wells, wall of test tube, nitrocellulose strips, magnetic beads and non-magnetic beads.
Furthermore, the present invention also includes a 10 method for producing antibodies to HBsAg comprising administering to an individual an isolated, immunogenic polypeptide or fragment thereof comprising a cyclic peptide showing cross-reactivity with HBsAg antiserum which comprises the "a" epitope of HBsAg, wherein the peptide comprises amino acid residues 121-124 of HBsAg having a C (K/R)TC motif, in an amount sufficient to produce an immune response. The polypeptide may be prepared by synthetic means.
The invention also includes a diagnostic reagent comprising a polypeptide or fragment thereof derived from hepatitis B surface antigen, wherein the polypeptide or fragment thereof comprises a cyclic peptide showing cross-reactivity with HBsAg antiserum which comprises the "a~
25 epitope of HBsAg, wherein the peptide comprises amino acid residues 121-124 of HBsAg having a C(K/R)TC motif. The polypeptide may be produced by synthetic means.
Further, the invention includes all of the above 30 entities and uses wherein the peptide may be, for example, amino acids 117-128 of HBsAg or any other length of an amino acid sequence or fragment of HBSAg provided the peptide contains amino acid residues 121-124 and thus the C (K/R)TC motif.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 ~A) represents binding of mAb H166 to rHBsAg subtype ac~ and ay with equal affinity in ELISA. (B) CA 022~1904 1998-10-16 W097/39029 PCT~S97/06732 represents inhibition of mAb H166 binding to rHBsAg (ay) by free rHBsAg (ay and ad). Degree of inhibition was determined using competitive ELISA as described in the examples.
Figure 2 represents inhibition of mAb H166 binding to rHBsAg (ay) by cyclic peptides derived from HBsAg sequence.
Degree of inhibition was determined using competitive ELISA
as described in the examples.
Figure 3 represents inhibition of mAb H166 binding to rHBsAg (ay) by the linear and the cyclic peptide I
(STGPCKTCTTPA). Degree of inhibition was determined using competitive ELISA as described in the Examples.
Figure 4 represents a plot of ~GmUt-wt at the various alanine substitution sites in peptide I derived from HBsAg.
Figure 5 represents a sequence alignment of the region from residues 101 to 160 of subtypes of HBsAg and HBsAg mutants.
The alignment was performed as described in the Examples.
All sequences are denoted by their Genbank accession codes (source). The original sequences and their authors can easily be retrieved from the NCBI (National Center for Biotechnology Information, Bethesda, MD) or from GCG
(Genetic Computer Group, Madison, WI) using the Genbank accession codes as keywords. The listed subtypes (SUBT) and genotypes (GTYPE) are from the original references. HBsAg mutants are denoted as 'mut' and several undefined subtypes of HBsAg sequences are labeled as 'nd'. The C(K/R)TC motif is shadowed.
Figure 6 illustrates the proposed structures of the HBsAg a determinant. (A) Amino acids 124-147 of H3sAg form two putative loops via the disulfide bridges between cysteines at 124-137 and 139-147 [Bhatnagar, et al., Proc. Natl.
Acad. Sci. USA 79: 4400-4404, (1982); Dreesman, et al., Nature 295:158-160, ~1982); Brown, et al., J. Immunol CA 022~1904 1998-10-16 W O 97/39029 PCTrUS97/06732 Methods, 72: 41-48, (1984)]. (B) The C(K/R)TC motif forms a loop structure via the disulfide bridging between Cysl21 and Cysl24.
DETAILED DESCRIPTION OF THE INVENTION
The subject invention relates to an isolated peptide corresponding to amino acid residues 117 to 128 of hepatitis B surface antigen (HBsAg) and uses thereof. The peptide is an antigenic epitope contributing to the a determinant and may be used as a diagnostic reagent for the detection of hepatitis B virus (HBV) and in the production of vaccines against HBV. Further, the invention relates to the C(K/R)TC motif (aa 121-124) within the epitope which is the main binding site for recognition by anti-a monoclonal antibody. This motif was discovered to be a common epitope shared by all subtypes of HBsAg including most HBV mutants.
The present invention also encompasses peptides and kits containing this motif.
Initially, the peptides of the invention were identified by using an anti-a monoclonal antibody (H166).
The peptide sequence corresponding to amino acid residues 117 to 128 of HBsAg specifically binds to this anti-a monoclonal antibody. In particular, the peptide specifically inhibits the native protein HBsAg binding to the anti-a monoclonal antibody with a IC50 (50% inhibition concentration) in the range of 10-6 to 10-7 M, and the maximum inhibition by the peptide is about 80% (Figure 2 and Figure 3). Therefore, the peptide (aa 117-128) contains an antigenic epitope contributing to the a-determinant.
In addition to the peptides noted above, a previously unidentified disulfide bond between residues Cysl21 and Cysl24 of HBsAg, as shown by Figure 6B, is disclosed by the present invention. As stated in the background, disulfide bond formation among the cysteine residues in the a-CA 022~1904 1998-10-16 W097/39029 PCT~S97/06732 determinant is crucial in defining the structure of the a-determinant. Formation of a correct disulfide bond will enhance the ability of the peptide to mimick the a-determinant, resulting in increased binding affinity of the peptide for anti-a monoclonal antibody. Indeed, compared to the native protein HBsAg, the cyclic form of the peptide (aa 117-128) is only 20-fold less effective, whereas the linear form of the peptide is 160-fold less effective in the inhibition of the anti-a monoclonal antibody binding to HBsAg. Therefore, the loop structure constrained by the disulfide bond between Cysl21 and Cysl24 represents more closely the native conformation of the a-determinant.
It is well known in the art that appropriate restriction of the conformational freedom of synthetic peptides lead to their enhanced performances.
Conformational restriction is usually achieved by crosslinking amino acids within the synthetic peptide. The simplest way to achieve this is to cyclize the peptide via disulfide bridges. There are numbers of examples in which the procedure has improved the antigenic and/or immunogenic properties of synthetic peptides (Kennedy, et al., J.
Virol. 46:653-655 (1983), Ferguson, et al., Virolo~v 143:505-515 (1985)). Similarly, formation of the disulfide bond between Cysl21 and Cysl24 imparts an eight fold increase in affinity to the linear peptide (aa 117-128) for immune recognition by the anti-a monoclonal antibody (F igure 3).
Similar to the previously identified peptide sequences (aa 110-137 and aa 115-129), both linear and cyclic peptide sequences (aa 117-128) of the present invention contain the same subtype specific amino acids at position 117, 120, 122, 125, 126, 127 and 128. The effect of these subtype specific amino acids on the peptides mimicking the a-determinant is unknown. In order to identify the most critical residues in the peptide (aa 117-128) for mimicking the a-determinant, a set of alanine-substituted analogs of CA 022~l904 l998-l0-l6 W O 97/3gO29 PCT~US97/06732 the peptide (aa 117-128), which represent single amino acid substitutions by alanine, were used. Using the alanine-substituted analogs of the peptide (aa 117-128), three residues Cysl21, Thrl23 and Cysl24 were found to be the most critical residues for the anti-a monoclonal antibody recognition (see Example VII). Substitution of alanine for any one of the three residues completely eliminated the peptide (aa 117-128) binding to the anti-a monoclonal antibody (see Table I and Figure 4). Minor effects (i.e., a less than 10 fold decrease or increase in binding affinity) were observed for the remaining residues in the sequence (aa 117-128) (see Table I and Figure 4). These results indicate that the CXTC motif, where position 122 can be any amino acid as represented by the x residue (see Summary section), is the most important structural feature for antibody recognition and for mimicking the a-determinant. In other words, it is the CXTC motif that contributes to the a-determinant, not the remaining residues in the peptide sequence.
Furthermore, sequence analysis indicated that the CXTC
motif is highly conserved among 100 subtypes and mutants of HBsAg isolates. As shown by the sequence alignment (see Figure 5), although there are many sequence variations in the region of residues 101 to 160, the CXTC motif is fully conserved in all of the sequences shown in Figure 5. In addition, the residue at position 122 is relatively conserved; it is either Lys or Arg in all of the sequences shown in Figure 5. Also, the alanine-substituted alalogs showed that keeping Lys or Arg at position 122 imparted approximately a ten-fold increase in affinity (see Table I). Therefore, using both alanine-substituted alalogs and computational sequence analysis, the CXTC, or more accutately the C(K/R)TC motif was identified as the key element or the essential core epitope within the peptides ~aa 117-128 or aa 115-129) for mimiking the a-determinant.
Sequence analysis also indicated that the C(K/R)TC motif is a common epitope shared by all subtypes of HBsAg including CA 022~1904 1998-10-16 W097/39029 PCT~S97/06732 most HBV mutants. Thus, the conservation of the C(K/R)TC
motif explained on a structural basis why immunization with one subtype of HBsAg can confer protection against HBV
infection with all subtypes. Since all subtypes contain the C(K/R)TC motif, immunization with one subtype will induced neutralizing antibodies targeting the C(K/R)TC
motif. Thus, the antibodies will target all HBsAg subtypes as long as they contain the C(K/R)TC motif.
The highly conserved C(K/R)TC motif and its antigenic nature permit the use of the peptide (aa 117-128) or synthetic peptides containing the C(K/R)TC motif as synthetic vaccines. Since the loop structure of the C(K/R)TC motif is well constrained by the disulfide bond between Cysl21 and Cys 124, synthetic peptides containing this motif will mimic the native structure of the a-determinant on HBsAg and elicit neutralizing antibodies that target the C(K/R)TC motif on all subtypes and most mutants of HBsAg. Furthermore, the finding that the high binding affinity can be maintained by the C(K/R)TC motif establishes the feasibility of using the C(K/R)TC motif in tracer epitopes for the detection of HBsAg in a homogeneous immunoassay format. These potential utilities are described below.
Vaccine Pre~aration The peptides of the invention can serve as synthetic vaccines by conjugating the peptides to immunogenic carriers. Suitable carriers include proteins, polysaccharides such as latex functionalized sepharose, agarose, cellulose, cellulose beads, and polymeric amino acids such as polyglutamic acid and polylysine. Examples of protein substrates or carriers include serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, and yet other proteins known to those skilled in the art.
CA 022~l904 l998-l0-l6 WO 97~9029 PCTrUS97/06732 Conjugation methods are known in the art and include but are not limited to using N-succinimidyl-3-(2-pyridylthio)propionate (SPDP) and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC). Either the amino or the carboxyl terminal of the peptides disclosed here can be modified by adding a cysteine residue. These reagents create a disulfide linkage between themselves and peptide cysteine residues on one protein and an amide linkage through the epsilon-amino on a lysine, or other free amino group in the other. A variety of such disulfide/amide-forming agents are known. Other bifunctional coupling agents form a thioester rather than a disulfide linkage. Many of these thio-ether-forming agents are commercially available and are known to those of ordinary skill in the art. The carboxyl group of the peptides also can be activated by combining them with succinimide or 1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt, and the conjugation of the peptides to carriers can be achieved by the formation of an amide bond.
Typically, such synthetic vaccines are prepared as injectables, either as liquid solutions or suspensions;
solid forms suitable for solution in or suspension in liquid prior to injection also may be prepared. The preparation may be emulsified or the protein may be encapsulated in liposomes. The active immunogenic ingredients often are mixed with pharmacologically acceptable excipients which are compatible with the active ingredient. Suitable excipients include but are not limited to water, saline, dextrose, glycerol, ethanol and the like; combinations of these excipients in various amounts also may be used. The vaccine also may contain small amounts of auxiliary substances such as wetting or emulsifying reagents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine. For example, such adjuvants can include aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-CA 022~1904 1998-10-16 W097t3~29 PCT~S97/06732 acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred to as nor-MDP), N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'2'-dipalmitoyl-sn-glycero-3-hydroxphosphoryloxy)-ethylamine (CGP 19835A, also referred to as MTP-PE), and RIBI (MPL + TDM+ CWS) in a 2%
squalene/Tween-80~ emulsion.
The vaccines usually are administered by intravenous or intramuscular injection. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include but are not limited to polyalkylene glycols or triglycerides. Such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10%, preferably, about 1% to about 2%.
oral formulation include such normally employed excipients as, for example pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions may take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 10% to about 95% of active ingredient, preferably about 25% to about 70%.
Vaccines are administered in a way compatible with the dosage formulation, and in such amounts as will be prophylactically and/or therapeutically effective. The quantity to be administered generally is in the range of about 5 micrograms to about 250 micrograms of antigen per dose, and depends upon the subject to be dosed, the capacity of the subject~s immune system to synthesize antibodies, and the degree of protection sought. Precise amounts of active ingredient re~uired to be administered also may depend upon the ~udgment of the practitioner and may be unique to each subject. The vaccine may be given in a single or multiple dose schedule. A multiple dose is one in which a primary course of vaccination may be with one to CA 022~l904 lsss-l0-l6 W 097/39029 PCTrUS97/06732 ten separate doses, followed by other doses given at subsequent time intervals required to maintain and/or to reinforce the immune response, for example, at one to four months for a second dose, and if required by the individual, a subsequent dose or doses after several months. The dosage regimen also will be determined, at least in part, by the need of the individual, and be dependent upon the practitioner~s judgment.
Pre~aration of antibodies a~ainst the C(K/R)TC motif The peptides prepared as described herein are used to produce antibodies against the C(K/R)TC motif, either polyclonal or monoclonal. The peptide could be the sequence aa 117-128 of HBsAg, it also could be any peptide as long as it contains C(K/R)TC motif (aa 121-124). When preparing polyclonal antibodies, a selected mammal (for example, a mouse, rabbit, goat, horse or the like) is immunized with the peptide-carriers disclosed herein.
Serum from the immunized animal is collected after an appropriate incubation period and treated according to known procedures. If serum containing polyclonal antibodies to the C(K/R)TC motif contains antibodies to other antigens, the polyclonal antibodies can be purified by, for example, immunoaffinity chromatography. Techniques for producing and processing polyclonal antibodies are known in the art and are described in, among others, Mayer and Walker, eds., Immunochemical Methods In Cell and Molecular BioloaY, Academic Press, London (1987).
Antibodies specifically against the C(K/R)TC motif also may be obtained from a mammal previously immunized with HBsAg.
An example of a method for purifying antibodies specific to the C(K/R)TC motif from serum of an individual immunized with HBsAg using affinity chromatography is provided herein.
Monoclonal antibodies directed against the C(K/R)TC
motif also can be produced hy one skilled in the art. The CA 022~1904 1998-10-16 general methodology for producing such antibodies is well-known and has been described in, for example, Kohler and Milstein, Nature 256:494 (lg75) and reviewed in J.G.R.
Hurrel, ed., Monoclonal Hvbridoma Antibodies: Techniaues and A~lications, CRC Press Inc., soca Raton, FL (1982), as well as that taught by L. T. Mimms et al., Viroloov 176:604-619 (1990). Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus.
See also, M. Schreier et al., Hybridoma Techniques, Scopes (1980) Protein Purification, Principles and Practice, 2nd Edition, Springer-Verlag, New York (1984); Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas (1981);
Kennet et al., Monoclonal Antibodies (1980). Examples of uses and techniques of monoclonal antibodies are well known to those of skill in the art.
Monoclonal and polyclonal antibodies thus developed, directed against the C(K/R)TC motif, are useful in diagnostic and prognostic applications as well as in passive immunotherapy. Monoclonal antibodies especially can be used to produce anti-idiotype antibodies. These anti-idiotype antibodies are immunoglobulins which carry an ~linternal image~ of the antigen of the infectious agent against which protection is desired. See, for example, A.
Nisonoff et al., Clin. Immunol. Immuno~ath. 21:397-406 (1981), and Dreesman et al., J. Infect. Dis. 151:761 (1985). Techniques for raising such idiotype antibodies are known in the art and exemplified, for example, in Grych et al., Nature 316:74 (1985); MacNamara et al., Science 226:1325 (1984); and Uytdehaag et al., J. Immunol. 134:1225 (1985). These anti-idiotypic antibodies also may be useful for treatment of HBV infection.
CA 022~1904 1998-10-16 W O 9713gO29 rcTrusg7/o6732 Immunoassa~ and Diaanostic Kits Both the peptides of the present invention and the antibodies raised against the C(K/T)TC motif are useful in immunoassays to detect the presence of HBsAg or HBV in biological test samples. The design of these immunoassays is subject to variation, and a variety of these are known in the arti a variety of these have been described herein.
Examples of assays which utilize labels as the signal generating compound and those labels are described herein.
Signals also may be amplified by using biotin and avidin, enzyme labels or biotin anti-biotin systems.
One of the competitive assay formats using solid phase can be designed which utilize the signal labeled ( with radioactive isotope, such as 1125, or with an enzyme, or with biotin) synthetic peptides detailed herein and a monoclonal or polyclonal antibodies directed against the C(K/T)TC motif. In the assay format to detect the presence of HBsAg in a human test sample, a known amount of signal labeled peptides is first added to the human test sample, then the human test sample containing certain amount signal labeled peptides is contacted and incubated with a solid phase coated with a monoclonal or polyclonal antibodies directed against the C(K/T)TC motif. If HBsAg and HBV
particles are present in the test sample, they will compete with the labeled peptides binding to the monoclonal or polyclonal antibodies on the solid surface. After removal of unbound materials and peptides by washing the solid phase, the amount of peptides bound by the antibodies can be determined by determining the level of radioactivity or by adding an enzyme substrate or by adding the anti-biotin conjugate following the addition of substrate. ~he reduced signal is proportional to the amount of HBsAg and HBV
particles in the human test sample.
CA 022~l904 l998-l0-l6 In fluorescence polari~ation immunoassay (FPIA), which is a homogeneous competitive assay,; i.e. it does not reguire wash or separation steps, synthetic peptides, particularly the C(K/R)TC motif, of the present invention may be used as tracer epitopes for the detection of HBsAg.
Using synthetic peptides as tracer epitopes in FPIAs of high molecular weight proteins is known in the art and is described in, among others, Geysen, et al., J. Immunol.
Methods 102:259-274 (1987); Houghten, et al., Nature 354:84-86 (1991); Lam, et al., Nature 354:82-83 (1991);
U.S. Patent No. 4,833, 092. In the FPIA, peptides of the present invention covalently linked to labeling group (such as fluorescein) will be used as a tracer; and an antibody (monoclonal or polyclonal) against the C(K/R)TC motif will be used to form a complex with the peptide epitope tracer.
The system containing the complex of the epitope tracer and the antibody then can be used to detect and quantitate the presence of HBsAg in human test sample by monitoring the change of fluorescence polarization.
The present invention can be illustrated by the use of the following non-limiting examples: ' EXAMPLES
EXAMPLE I
PEPTIDE SYNTHESIS
All Fmoc protected amino acid and reagents used for peptide synthesis were purchased from Applied Biosystem (Foster City, CA) or Rainin Instrument Co. Inc.
(Emeryville, CA) Monoclonal antibody (mAb) H166 [Peterson, et al., J. Immunol. 132: 920927 (1984)] was obtained from the Abbott Monoclonal Antibody Development Group, Abbott Park, Illinois. Recombinant HBsAg [Mimms, et al., J Virol.
Methods 25:211-232, (1989)], subtype ad and ay, was obtained from Abbott Rare Reagent Development Group (Abbott Park, IL). Alkaline phosphates-conjugated goat anti-mouse CA 022~1904 1998-10-16 W O 97t39029 PCTrUS97/06732 IgG and IgM were purchased from Pierce Chemical Co., (Rockford,IL).
All peptides were synthesized by the stepwise solid-phase method of Merrifield ~Merrifield, J. Am. Chem. Soc.
85:2149-2154, (1963)] on an Applied siosystem 431A
Synthesizer (Foster City, CA) or a Rainin Symphony Synthesizer (Emeryville, CA) using standard Fmoc (9-fluorenylmethoxycarbonyl) chemistry. Peptides were cleaved and deprotected with a mixture of 82% trifluoroacetic acid (TFA), 5% phenol, 5% H2O, 5% thioanisole and 2.5%
ethanedithiol for 2-4 hours at 25~C and precipitated by addition of cold ether. Crude peptides were purified by reverse phase HPLC using a 5-50% acetonitrile gradient containing 0.1% TFA. The homogeneity and identity of the purified peptides were confirmed by electro-spray mass spectrometry. All peptides were determined to be at least 95% pure. Cyclic peptides formed by intramolecular disulfide bridging were synthesized using the air oxidation method [Tam, et al., Proc. Natl. Acad. Sci. USA 83: 8082, (1986)]. Briefly, purified linear peptides were dissolved in 0.1 mM Tris-HCl buffer (pH 8.4) at a concentration of 0.5 mg/ml and stirred at 25 C for varying periods of time exposed to air. Cyclization was monitored by HPLC. Cyclic peptides were purified as described above and formation of intramolecular disulfide bonds was confirmed by electro-spray mass spectra.
EXAMPLE II
DETERMINATION OF AFFINITY
Enzyme-linked immunoassays (ELISA~ were used to evaluate the affinity of monoclonal antibody H166 to the rHssAg, subtype (ad and ay, 5 ~g/ml) in 0.2 M carbonate-bicarbonate buffer (pH 9.0) and blocked with 1% ssA in Pss buffer (pH 7.4). Twofold dilutions of H166, starting concentration (1 ~g/ml), were subsequently added (100 ~l/well) and the ad and ay. Briefly, microtiter plates were coated with ~l/well of rHBsAg 100 plates were incubated for 1 hour at 25 C. The wells were washed with CA 022~1904 1998-10-16 W 097139029 PCTrUS97/06732 PBS buffer containing 0.5% Tween 20, and 100 ~Ll/well of alkaline phosphates-conjugated goat antimouse Ig at 5000 fold dilution was added and incubated for 1-2 hr at 25 C.
After washing, 100 ~l/well of p-nitrophenyl phosphate substrate was added, and the absorbance was measured at 405 nm using an automated microtiter plate reader (Molecular Devices Corp., Menlo Park, CA). ELISA results were used to fit an analog of the Michaelis-Menten equation [Langone and Van Vunakis eds, Methods in Enz~moloov, Academic Press, San Diego, (1986)]:
[Ag--A~] = [Ag--Ab]ma~. x [Ab] + K
Where [Ag-Ab] is the antigen-antibody complex concentration, [Ag-Ab] maX is the maximum complex concentration, [Ab] is the antibody concentration, and Kd is the dissociation constant. During the curve fitting, the [Ag - Ab] was substituted with the value of OD405mm at the given concentration of antibody. Both Kd and [OD405nm]max~ which corresponds to the [Ag - Ab]~aX, were treated as fitted parameters.
CA 022~1904 1998-10-16 W O 97/39029 PCTrUS97/06732 EXAMPLE III
COMPETITIVE ELISAs Competitive ELISAs were used to identify the potential antigenic determinants and to compare their affinity through the determined IC50s. Microtiter plates were coated with rHBsAg and blocked with ~SA as described above.
Aliquots of peptides or rHBsAg competitors (50 ~l/well) at varying dilutions were added together with 100 ~l of H166 at 0.4 ~l/ml, and incubated for 1 hour at 25'C. The wells were washed with phosphate buffered saline (PBS) containing 0.5% Tween 20, and alkaline phosphatase-conjugated goat anti-mouse Ig at 5000 fold dilution (100 ~l/well) was added. The plates were incubated for 1-2 hr at 25 C, washed, and 100 ~l/well of p-nitrophenyl phosphate substrate was added, and the absorbance was measured at 405 nm. The percent inhibition of mAb H166 binding to rHBsAg by peptides was calculated according to the 'following equation:
. . OD40swith peptide - bkgd Inhlbltlon% = 100 x 1- . .
OD40swlthout peptlde-bkgd where the bkgd is the absorbance from the well without coating of rHBsAg.
EXAMPLE IV
SEOUENCE ANALYSIS
Nucleotide sequences corresponding to ~BsAg coding sequences were obtained from Genbank (release 89.0, 6/95) and EMBL (release 42.0, 3/95). The nucleotide sequences were translated into protein sequences and analyzed using programs from the Wisconsin Genetic Computer Group sequence analysis package (GCG, Version 8.0). Multiple sequence alignment of protein sequence was performed using progressive pairwise alignment (PILEUP, GCG), and sequences were displayed using the program PRETTY ( GCG). Final alignment and editing were performed manually.
CA 022~1904 1998-10-16 W O 97/39029 PCTrUS97/06732 EXAMPLE V
IDENTIFICATION OF PEPTIDE EPITOPE CONTRIBUTING TO THE A-DETERMINANT
Monoclonal antibody H166 is specific for the a-determinant, recognizing nine different subtypes of HBsAg as observed by Peterson et al.[Peterson, et al., J.
Immunol. 132: 920927 (1984)]. In order to quantitatively compare the affinity of mAB H166 for different HBsAg subtypes, the apparent dissociation constants (Kd) derived from the fitting curves for both subtype ad and ay of rHBsAg were determined via ELISA. As shown in Figure lA, mAb H166 binds the ad and ay subtypes with equal affinity.
The Kd derived from the fitting curves for both subtypes ad and ay, is approximately 0.9 ~g/ml, corresponding to lx10-8M. Competitive ELISAs were also used to evaluate the crossreactivity of mAb H166 with subtype ad and ay .
Figure lB shows the inhibition curves using free ad and ay subtypes as competitors. It is clear that both subtypes inhibit mAb H166 binding to rHBsAg with similar affinities.
The IC50s for subtype ay and ad are 0.026 ~M and 0.030 ~M
respectively. Thus, H166 recognizes a common epitope shared by the ad and ay subtypes of HBsAg.
Since H166 is a monoclonal anti-a antibody, it can be used to screen for an epitope that contributes to the a -determinant of HBsAg. Identifying the epitope of H166 will help to define the a determinant, or part of the a-determinant, on HBsAg. To identify the H166 epitope, three cyclic peptides derived from the extracellular hydrophilic region of HBsAg were synthesized. The peptide sequences are shown in Figure 2. Although the cyclic peptides II (aa 124-137) and III (aa 139-147) are known to be a major part of the a-determinant [Bhatnagar, et al. Proc. Natl. Acad.
Sci. USA 79:4400-4404 (1982); Dreesman, et al. Nature, 295:158-160 (1982)] only cyclic peptide I (aa 117-128~
binds specifically to H166. As shown in Figure 2, the cyclic peptide I inhibits 70% of HBsAg binding to the H166 -CA 022~l904 l998-l0-l6 W 097/39029 PCTrUS97/06732 at a concentration of 2 mM. Therefore, it is clear that peptide I represents the epitope of H166. It is interesting to note that peptide II exhibits about 20%
inhibition at a concentration of 2 mM, probably because part of its se~uence (aa 124-128) overlaps with peptide I
(aa 117-128).
EXAMPLE VI
AFFINITY OF LINEAR VS CYCLIC PEPTIDE I (aa 117-128) To further characterize the identified epitope of mAb H166, the IC50 of the cyclic peptide I (aa 117-128) was compared to the IC50s of its linear sequence and the native protein, rHBsAg (subtype ad), using competitive ELISA. As shown in Figure 3, the IC50 of cyclic peptide I is 0.54 ~1, only 20 fold less than the IC50 of the native protein, rHBsAg (0. 026~1M). The high potency of the cyclic peptide indicates that it contains the essential part of the epitope on HBsAg recognized by the mAb. More importantly, the IC50 of the linear peptide (4.0 ~M) is 8 fold higher than the IC50 of the cyclic peptide, although both peptides exhibit similar inhibitions at higher concentration (>0.1 mM). These result indicate that the cyclic peptide I
contains a micro conformation that is closer to the native structure of the a determinant on HBsAg compared to the linear peptide I.
An experiment was performed to verify that the sulfhydryl groups of the linear peptide I (aa 117-128) remained in a reduced form under the experimental conditions. Linear peptide I was first incubated with H166 in PBS buffer for 1 hour, and the mixture was analyzed by electro-spray mass spectrometry. No trace of the oxidized molecular ion was observed in the mass spectra, confirming that no oxidation of the sulfhydryl groups occurred during the time course of the ELISA. These results indicate that the linear peptide can be recognized by the mAb H166, although the cyclic form is the favorable confirmation.
CA 022~1904 1998-10-16 W097/39029 PCT~S97/06732 EXAMPLE VII
RESIDUES IN PEPTIDE I (aa 117-128) CRITICAL FQR BINDING
AFFINITY
The cyclic peptide I (aa 117-128) contains a critical residue at position 122 that differentiates the d and y subtypes. For the d subtype, position 122 is Lys, and Arg for the y subtype [Okamoto, et al. J. Virol. 61: 3030-3034, (1987)]. The insensitivity of mAb H166 to the subtype-specific residue implies this residue is not critical for the binding affinity. To prove this hypothesis and to identify the critical residues for mAb H166 recognition, a set of alanine-substituted analogs of peptide I, which represent single amino acid substitutions by alanine, were synthesized. Inhibition of H166 binding to HBsAg by the alanine-substituted peptides were determined using competitive ELISA. Table 1 shows the alanine-substituted peptide sequences and their determined IC50s.
Substitutions of Cysl21 and Cysl24 with Ala almost completely eliminate specific binding to H166. IC50s could not be obtained for these two peptides (121C and 124C) because no concentration dependent inhibition curves were observed in the concentration range 2.9x10-9 to 2.9x10-4 M.
Both peptides (121C and 124C) exhibited only 20% inhibition at the highest peptide concentration (3 mM).
Under the assay conditions, the IC50 is approximately inversely proportional to the binding affinity constant Ka [Cheng, et al., Biochem. Pharmacol. 22: 3099. (1973);
Munson, et al., ~n~l. Biochem. 107:220-239, (1980)]. Thus, the differences in binding free energy between alanine-substituted peptide and the wild type peptide (~Gmut-wt) were derived based on the equation:
~Gm~t-WI = - RT lnlc5ow~
where R is the gas constant and T is the temperature.
Figure ~ shows the plot of ~GmUt-wt at the alanine-substitution site. It is very clear that Cysl21, Thrl23 CA 022~1904 1998-10-16 W097/39029 PCT~S97/06732 and Cysl24 are the most critical residues for H166 recognition, since alanine substitutions at these three residues cause substantial loss in binding energy (>4.5 kcal/mol). Minor effects (0.5-1.5kcal/mol) were observed for the remaining residues, including residue 122. These results indicate that the CXTC motif is the main binding site or the essential core epitope of H166 on HBsAg where position 122 can be any amino acid as represented by the X
residue. These results also explain why mAb H166 binding is insensitive to the ad and ay subtype-specific residue.
Since H166 recognition only requires the CXTC motif, it will recognize all subtypes of HBV as long as the CXTC
motif is conserved in their HBsAg sequences.
EXAMPL E VI I I
SEOUENCE ALIGNMENT OF HBsAa In order to evaluate the conservation of the CXTC
motif in all subtypes of HBsAg and HBsAg mutants, analysis of 100 HBsAg sequences derived from human HBV genomes retrieved from the Genbank and EMBL databases was performed. The retrieved nucleotide sequences were translated into protein sequences and aligned using PILEUP.
From the sequence analysis, 43 unique sequences in the extracellular hydrophilic region from residues 101 to 160 HBsAg were identified and the sequence alignment is shown in Figure 5. Although there are many sequence variations in the entire region, it is very clear that the CXTC motif is fully conserved in all retrieved sequences regardless of the genotypes or subtypes. The alignment also shows that residue 122 is relatively conserved; it is either Lys or Arg in all sequences shown in Figure 5. From the sequence analysis results, it can be concluded that the CXTC, or more accurately the C(K/R)TC motif, is a common epitope shared by all subtypes of HBsAg including HBsAg mutants.
Claims (2)
1. An isolated linear or cyclic peptide showing cross reactivity with anti-hepatitis B surface antigen (HBsAg) antiserum which comprises the "a" epitope of HBsAg, wherein said peptide is a 12 mer of the amino acid sequence corresponding to amino acid residues 117 to 128 of HBsAg.
2. The peptide of claim 1 wherein said peptide is cyclic and contains a disulfide bond between amino acid residues 121 and 124, said bond yielding approximately an eight to ten fold increase in affinity as compared to the linear peptide.
3. The peptide of claim 2 wherein said peptide contains the C(K/R)TC motif and has the amino acid sequence X1X2X3X4C(K/R)TCX5X6X7X8 wherein:
X1 is selected from the group consisting of serine, glycine, alanine, valine and an aliphatic amino acid of from two to six carbon atoms;
X2 is selected from the group consisting of threonine, serine, alanine and glycine;
X3 is glycine or alanine;
X4 is selected from the group consisting of proline, serine and threonine;
X5 is selected from the group consisting of threonine, methionine, alanine, serine and glycine;
X6 is selected from the group consisting of threonine, serine, alanine, isoleucine and an aliphatic amino acid of from two to six carbon atoms;
X7 is selected from the group consisting of proline, leucine, threonine, serine, alanine and an aliphatic amino acid of from two to six carbon atoms; and X8 is selected from the group consisting of alanine, glycine, and valine.
4. The peptide of claim 3 wherein said peptide has the amino acid sequence STGPC(K/R)TCTTPA.
5. The peptide of claim 3 wherein said peptide has the amino acid sequence AAGPC(K/R)TCATPA.
6. A vaccine against hepatitis B comprising a pharmacologically effective dose of a cyclic peptide showing cross-reactivity with HBsAg antiserum which comprises the "a" epitope of HBsAg, wherein said peptide comprises amino acid residues 121-124 of HBsAg having a C(K/R)TC motif, and wherein said peptide is prepared by synthetic means and a pharmaceutically acceptable carrier.
7. A kit for detecting the presence of hepatitis B
surface antigen or antibody in a test sample comprising a container containing a polypeptide comprising a cyclic peptide showing cross-reactivity with HBsAg antiserum which comprises the "a" epitope of HBsAg, wherein said peptide comprises amino acid residues 121-124 of HBsAg having a C(K/R)TC motif, and wherein said polypeptide is prepared by synthetic means.
8. A method for detecting hepatitis B virus surface antigen (HBsAg) in a test sample suspected of containing HBsAg comprising the steps of:
a. contacting said test sample with an antibody or fragment thereof which specifically binds to a cyclic peptide showing cross-reactivity with HBsAg antiserum which comprises the "a" epitope of HBsAg, wherein said peptide comprises amino acid residues 121-124 of HBsAg having a C(K/R)TC motif, for a time and under conditions sufficient to allow the formation of antibody/antigen complexes; and b. detecting said complexes containing said antibody, wherein said antibody is producing by utilizing a polypeptide prepared by synthetic means.
9. A method for detecting hepatitis B antibodies in a test sample suspected on containing said antibodies comprising the steps of:
a. contacting said test sample with a probe polypeptide wherein said polypeptide comprises a cyclic peptide showing cross-reactivity with HBsAg antiserum which comprises the "a" epitope of HBsAg, wherein said peptide comprises amino acid residues 121-124 of HBsAg having a C(K/R)TC motif, for a time and under conditions sufficient to allow the formation of antigen/antibody complexes; and b. detecting said complexes which contain the probe polypeptide said antibody.
10. A method for producing antibodies to HBsAg comprising administering to an individual an isolated, immunogenic polypeptide or fragment thereof comprising a cyclic peptide showing cross-reactivity with HBsAg antiserum which comprises the "a" epitope of HBsAg, wherein said peptide comprises amino acid residues 121-124 of HBsAg having a C(K/R)TC motif, in an amount sufficient to produce an immune response, wherein said polypeptide is produced by utilizing a polypeptide prepared by synthetic means.
11. A diagnostic reagent comprising a polypeptide or fragment thereof derived from hepatitis B surface antigen, wherein said polypeptide of fragment thereof comprises a cyclic peptide showing cross-reactivity with HBsAg antiserum which comprises the "a" epitope of HBsAg, wherein said peptide comprises amino acid residues 121-124 of HBsAg having a C(K/R)TC motif, wherein said polypeptide is produced by synthetic means.
12. An antibody directed against said peptide of
2. The peptide of claim 1 wherein said peptide is cyclic and contains a disulfide bond between amino acid residues 121 and 124, said bond yielding approximately an eight to ten fold increase in affinity as compared to the linear peptide.
3. The peptide of claim 2 wherein said peptide contains the C(K/R)TC motif and has the amino acid sequence X1X2X3X4C(K/R)TCX5X6X7X8 wherein:
X1 is selected from the group consisting of serine, glycine, alanine, valine and an aliphatic amino acid of from two to six carbon atoms;
X2 is selected from the group consisting of threonine, serine, alanine and glycine;
X3 is glycine or alanine;
X4 is selected from the group consisting of proline, serine and threonine;
X5 is selected from the group consisting of threonine, methionine, alanine, serine and glycine;
X6 is selected from the group consisting of threonine, serine, alanine, isoleucine and an aliphatic amino acid of from two to six carbon atoms;
X7 is selected from the group consisting of proline, leucine, threonine, serine, alanine and an aliphatic amino acid of from two to six carbon atoms; and X8 is selected from the group consisting of alanine, glycine, and valine.
4. The peptide of claim 3 wherein said peptide has the amino acid sequence STGPC(K/R)TCTTPA.
5. The peptide of claim 3 wherein said peptide has the amino acid sequence AAGPC(K/R)TCATPA.
6. A vaccine against hepatitis B comprising a pharmacologically effective dose of a cyclic peptide showing cross-reactivity with HBsAg antiserum which comprises the "a" epitope of HBsAg, wherein said peptide comprises amino acid residues 121-124 of HBsAg having a C(K/R)TC motif, and wherein said peptide is prepared by synthetic means and a pharmaceutically acceptable carrier.
7. A kit for detecting the presence of hepatitis B
surface antigen or antibody in a test sample comprising a container containing a polypeptide comprising a cyclic peptide showing cross-reactivity with HBsAg antiserum which comprises the "a" epitope of HBsAg, wherein said peptide comprises amino acid residues 121-124 of HBsAg having a C(K/R)TC motif, and wherein said polypeptide is prepared by synthetic means.
8. A method for detecting hepatitis B virus surface antigen (HBsAg) in a test sample suspected of containing HBsAg comprising the steps of:
a. contacting said test sample with an antibody or fragment thereof which specifically binds to a cyclic peptide showing cross-reactivity with HBsAg antiserum which comprises the "a" epitope of HBsAg, wherein said peptide comprises amino acid residues 121-124 of HBsAg having a C(K/R)TC motif, for a time and under conditions sufficient to allow the formation of antibody/antigen complexes; and b. detecting said complexes containing said antibody, wherein said antibody is producing by utilizing a polypeptide prepared by synthetic means.
9. A method for detecting hepatitis B antibodies in a test sample suspected on containing said antibodies comprising the steps of:
a. contacting said test sample with a probe polypeptide wherein said polypeptide comprises a cyclic peptide showing cross-reactivity with HBsAg antiserum which comprises the "a" epitope of HBsAg, wherein said peptide comprises amino acid residues 121-124 of HBsAg having a C(K/R)TC motif, for a time and under conditions sufficient to allow the formation of antigen/antibody complexes; and b. detecting said complexes which contain the probe polypeptide said antibody.
10. A method for producing antibodies to HBsAg comprising administering to an individual an isolated, immunogenic polypeptide or fragment thereof comprising a cyclic peptide showing cross-reactivity with HBsAg antiserum which comprises the "a" epitope of HBsAg, wherein said peptide comprises amino acid residues 121-124 of HBsAg having a C(K/R)TC motif, in an amount sufficient to produce an immune response, wherein said polypeptide is produced by utilizing a polypeptide prepared by synthetic means.
11. A diagnostic reagent comprising a polypeptide or fragment thereof derived from hepatitis B surface antigen, wherein said polypeptide of fragment thereof comprises a cyclic peptide showing cross-reactivity with HBsAg antiserum which comprises the "a" epitope of HBsAg, wherein said peptide comprises amino acid residues 121-124 of HBsAg having a C(K/R)TC motif, wherein said polypeptide is produced by synthetic means.
12. An antibody directed against said peptide of
claim 2.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US63542896A | 1996-04-18 | 1996-04-18 | |
| US08/635,428 | 1996-04-18 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2251904A1 true CA2251904A1 (en) | 1997-10-23 |
Family
ID=24547753
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002251904A Abandoned CA2251904A1 (en) | 1996-04-18 | 1997-04-18 | An antigenic epitope of the a determinant of hepatitis b surface antigen and uses thereof |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0906337A2 (en) |
| JP (1) | JP2000514643A (en) |
| CA (1) | CA2251904A1 (en) |
| WO (1) | WO1997039029A2 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0919568A1 (en) * | 1997-12-01 | 1999-06-02 | Sorin Diagnostics S.r.l. | Escape mutant of the surface antigen of hepatitis B virus |
| EP1111388A1 (en) * | 1999-12-23 | 2001-06-27 | Universiteit Gent | Interaction between CD14 and HBV components |
| US7563873B2 (en) | 2004-06-07 | 2009-07-21 | Novartis Vaccines And Diagnostics, Inc. | Rabbit monoclonal antibodies to hepatitis B surface antigens and methods of using the same |
| JP4948783B2 (en) * | 2005-05-18 | 2012-06-06 | シスメックス株式会社 | Anti-HBs monoclonal antibody |
| KR101944263B1 (en) * | 2012-06-11 | 2019-02-01 | 시아먼 유니버시티 | Polypeptides and antibodies for treating hbv infection and related diseases |
| WO2017059878A1 (en) | 2015-10-07 | 2017-04-13 | Humabs Biomed Sa | Antibodies that potently neutralize hepatitis b virus and uses thereof |
| KR102358577B1 (en) | 2017-02-17 | 2022-02-04 | 시아먼 유니버시티 | Peptide carriers presenting target polypeptides and uses thereof |
| PE20240080A1 (en) * | 2020-06-08 | 2024-01-16 | Hoffmann La Roche | ANTI-HBV ANTIBODIES AND METHODS OF USE |
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| AU579148B2 (en) * | 1984-03-09 | 1988-11-17 | Scripps Clinic And Research Foundation | Synthetic hepatitis b virus vaccine including both t cell and b cell determinants |
| JPH045300A (en) * | 1990-04-20 | 1992-01-09 | Tetsuo Nakamura | Viral hepatitis type b vaccine and antibody specific to hbs antigen |
| GB9325772D0 (en) * | 1993-12-16 | 1994-02-16 | Smithkline Beecham Biolog | Novel compounds |
-
1997
- 1997-04-18 JP JP09537434A patent/JP2000514643A/en active Pending
- 1997-04-18 WO PCT/US1997/006732 patent/WO1997039029A2/en not_active Application Discontinuation
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| WO1997039029A3 (en) | 2001-06-28 |
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