|Title of Invention||
HEPATITIS C VIRUS VACCINE
|Abstract||The present invention relates to isolation of a novel Hepatitis C virus, more particularly, the present invention relates to a viral class Hepatitis C, polypeptides, polynucleotide, vaccine and antibodies derived there from.|
HEPATITIS C VIRUS VACCINE
FIELD OF INVENTION:
The present invention relates to isolation of a novel Hepatitis C virus. More particularly, the present invention relates to a viral class Hepatitis C, polypeptides, polynucleotide, vaccine and antibodies derived there from. BACKGROUND OF THE INVENTION:
Viral hepatitis, caused by the six hepatotropic viruses, viz, hepatitis A virus (HAV) hepatitis B virus (HBV), Hepatitis D virus (HDV), hepatitis E virus (HEV) and hepatitis G virus (HGV), represents a major health problem world wide. Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) are the major cause of devastating liver diseases all over the world. Recent estimates indicate that more than 500 million people appeared to have been infected by these liver-tropic viruses. With about 180 million people currently infected worldwide, HCV represents a daunting public health problem. Out of these, at least 15-20 million in India and about 4 million people in the USA suffer from chronic infection by HCV. In some countries like Egypt about 10 to 15 per cent of general population appears to carry HCV. More than 30 to 40% of the infected people develop liver cirrhosis and/or hepatocellular carcinoma after suffering with chronic infection for a decade or two and therefore HCV infection is considered to be a silent killer. Although interferon a in combination with ribavirin work well with some patients infected by some genotypes, more than 50% of the patients are refractory to such treatment.
Non-A, Non-B hepatitis (NANBH) are transmissible diseases that are believed to be viral induced, and that are distinguishable from other forms of viral-associated liver diseases, including that caused by the known hepatitis viruses. Viral hepatitis, caused by the hepatotropic viruses, viz, hepatitis A virus (HAV) hepatitis B. virus (HBV), Hepatitis D virus (HDV), hepatitis E virus (HEV) and hepatitis G virus (HGV), represents a major health problem world wide. Until recently there has been neither clarity nor agreement as to the identity or specificity of the antigen antibody systems associated with agents of NANBH. It is possible that NANBH is caused by more than one infectious agent and unclear what the serological assays detect in the serum of patients with NANBH.
In 1987, Houghton, et al. cloned the first virus definitively linked to NANBH.
Houghton et al. described there in the cloning of an isolate from a new viral class,
hepatitis C virus (HCV), the prototype isolate described therein being named "HCV1".
HCV is a Flavi-like virus, with an RNA genome. They described the production of
recombinant proteins from HCV sequences that are useful as Diagnostic reagents, as
well as polynucleotides useful in diagnostics hybridization assays and in cloning of
additional HCV isolates.
Hepatitis C virus (HCV) has emerged in recent years as the leading cause of worldwide
blood-transmitted chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. A
vaccine to prevent HCV infection has not yet been available any where in the world
and the existing antiviral treatments are ineffective in the majority of the HCV infected
Despite significant progress in the field of biotechnology, reliable diagnostic
procedures, an alternative animal model other than chimpanzee, efficient cell culture
systems that can support long-term replication of the virus and effective therapeutic
strategies are still lacking.
As with any disease, an accurate diagnosis of HCV infection is essential before patients
are counseled and treatment is initiated. Since, the identification and molecular
characterization of the HCV in 1989 by Choo and colleagues, a number of diagnostic
tests based on the detection of either the anti HCV antibodies or HCV-RNA by PCR in
patient sera have been developed.
Presently, a third generation ELISA that incorporates antigens from the Core, NS3,
NS4 and NS5 proteins of HCV, representing about 60% of the total amino acid
sequence of HCV polyprotein, is available in the market. Although, this ELISA is
significantly sensitive, a major drawback of this assay is that it fails to differentiate
between active and post infection cases. In addition to this, it is now well documented
that the commercially available third generation ELISA can not be used to detect all the
viral infections in Indian patients owing to genotype sequence variations.
It may be noted at this stage that the commercial 3rd EIA is based on genotype 1 (other
than Indian HCV strain) and genotype specific antibody response in this virus is now
Hepatitis, Cirrhosis and Hepatocellular carcinoma, caused by Hepatitis C Virus remain a global health problem and development of a vaccine to prevent this silent killer is of utmost priority. Every major country's goal is to produce a therapeutic vaccine for those 180 million people who are already infected by HCV and a preventive vaccine to eradicate future HCV infections. Just a few years ago, there was a lot of skepticism about the possibility of developing a viable vaccine for HCV. The situation, however, has changed in the last two to three years mainly because i) about 40 to 50% of the patients spontaneously recover from infection, implying that their immune system can fight off the virus; ii) infected chimpanzees (the only animal model available for HCV produced viremia) and convalescent humans are protected against the re-exposure; and iii) chronically infected patients improved their immune response and liver functions when the viral envelope protein El was administered as therapeutic vaccine. Luckily for Hepatitis B, there is a preventive vaccine available in most of the countries; thus, future infections can be prevented. A therapeutic vaccine for HBV to boost the immunity of the infected people may be forthcoming. Unfortunately such a vaccine is not available for HCV any where in the world. Because of the propensity of the virus to undergo genetic variation, resulting in the evolution of quasispecies, a vaccine developed in the western countries will not be effective in India. For that matter, a vaccine developed against the strain(s) prevalent in Northern India may not be effective in South India. Therefore, controlling HCV infection is a challenging task. With the recent breakthroughs in research and development on HCV, there is a lot of optimism now about the development of at least a therapeutic and a potential preventive vaccine. The major problem, however, is that a single vaccine may not be suitable for every country as there are several different genotypes. In India genotypes 1 and 3 are more prevalent, which are quite different from genotypes existing in other regions of the world. Therefore, our major goal is to make the vaccine candidate proteins, El and E2 for both genotypes in yeast and/or animal cells and test for their efficacy as therapeutic and preventive vaccines. We already know the sequence of these genotypes and we have also completed cloning of the genes encoding El and E2 proteins. Now, the major goal of this project is to make these proteins in large quantities, purify and characterize, and carry out human trials.
There is an ever-increasing demand for sensitive and accurate tests for detection and screening of Hepatitis Viral Carriers. There is also a need for effective vaccines and
therapeutic agents for preventing and treating viral hepatitis. Moreover, there is
tremendous genetic variation among existing strains from each country and thus
development of potential vaccines depend upon characterization of the strain(s) existing
among Indian population.
To overcome the problems associated with the prior art, the applicant has cloned and
sequenced the genome of a novel Indian strain of HCV. This sequence can be used to
develop HCV antigens, diagnostic kits and therapeutic vaccines.
OBJECTS OF THE INVENTION:
The principal objective of the present invention is to isolate a novel strain of Hepatitis
C Virus from a pool of Indian patients.
Another objective of the present invention is to characterize the novel strain of
Hepatitis C Virus.
Yet another objective of the present invention is to identify the polynucleotide sequence
for the novel strain of Hepatitis C Virus.
Still another objective of the present invention is to identify the polypeptide sequence
for the novel strain of Hepatitis C Virus.
Still another objective of the present invention is to identify the primers.
Still another objective of the present invention is to develop a therapeutic vaccine for
immunizing a subject with Hepatitis C infection.
Still another objective of the present invention is to develop a kit for identifying a
subject with Hepatitis C infection.
Still another objective of the present invention is to develop a method of diagnosing a
patient with Hepatitis C infection.
Still another objective of the present invention is to immunize a subject with Hepatitis
Still another object of the present invention is to develop an E.coli strain carrying HCV
STATEMENT OF THE INVENTION:
New isolates of HCV has been characterized from different parts of the world have been implicated as NANBH carriers. These isolates exhibit nucleotide and amino acid sequence heterogeneity with respect to the prototype isolate HCV1, in several viral
domains. It is believed that these distinct sequences are of in importance, particularly in
diagnostic assays and in vaccine development.
The invention relates to a novel class of Hepatitis C virus that has been isolated and
characterized from an Indian infected host. The entire genomic structure and the
nucleotide sequence of the novel HCV isolate have been deduced. The genome appears
to be single-stranded RNA comprising about 9442 nucleotides. When compared with
all known viral sequences, several distinct domains and sequences that are of much
importance clinically, particularly for diagnostic purposes and for vaccine development
have been observed. The said sequence has been deposited at the GenBank at accession
number AY651061. The said novel strain has been designated as Khajal.
Indian HCV isolate has been characterized from a chronic hepatitis C patient. Blood
was collected form this patient, the RNA and cDNA was isolated and the PCR reaction
was set up using specific primers. The PCR amplicons were cloned and sequenced.
This isolate exhibits nucleotide and amino acid sequence heterogeneity with respective
to prototype isolate in several viral domains. These distinct sequences are much in
importance, particularly in diagnostic assays and in vaccine development.
In one aspect, the invention provides novel nucleotide sequences, obtained from the
novel HCV strain resulting polynucleotide, polypeptides and antibodies derived there
from. The invention also provides purified polypeptide sequences obtained from novel
isolate, said sequence being distinct from that of currently known HCV isolates. The
invention includes recombinant vectors comprising said sequences and host cells
transformed with such vectors.
Further, the invention provides probes derived from the HCV cDNA useful for
diagnose of the presence of HCV in samples, and to isolate naturally occurring variants
of the virus.
The invention also provides antibodies, both polyclonal and monoclonal, directed
against HCV epitopes contained within these polypeptide sequences are also useful for
diagnostic tests, as therapeutic agents, for screening of antiviral agents.
Also included within scope of the invention is an monoclonal antibody directed against
an HCV epitope and an anti-idiotype antibody comprising a region which mimics an
Another aspect of the invention relates to kit for detection of HCV comprising:
polynucleotides derived from novel HCV comprising a polynucleotide probe provided
in a suitable container; an I-ICV antigen comprising an antibody directed against the HCV antigen to be detect-ed, provided in a suitable container; antibodies directed against an HCV antigen comprising a polypeptide containing an HCV epitope present in the HCV antigen, provided in a suitable container.
Immunoassays are also included in the invention. These include an immunoassay for detecting an HCV antigen comprising incubating a sample suspected of containing an HCV antigen with a probe antibody directed against the HCV antigen to be detected under conditions which allow the formation of an antigen-antibody complex; and detecting an antigen-antibody complex containing the probe antibody. An immunoassay for detecting antibodies directed against an HCV antigen comprising incubating a sample suspected of containing anti-HCV antibodies with a probe polypeptide which contains an epitope of the HCV, under conditions which allow the formation of an antibody-antigen complex; and detecting the antibody-antigen complex containing the probe antigen.
Also included in the invention are vaccines for treatment of HCV infection comprising an immunogenic peptide containing an HCV epitope, or an inactivated preparation of HCV, or an attenuated preparation of HCV. These and other embodiments of the present invention will be readily apparent to those of ordinary skill in the art in view of the following description.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is in relation to a hepatitis C virus polynucleotide sequence set
forth in SEQ ID No. 1 (as shown in Fig.9) deposited at GenBank under accession
number under AY 651061.
Yet another embodiment of the present invention, wherein said sequence is isolated
from Indian patient pool.
The present invention relates to a hepatitis C virus polypeptide sequence set forth in
SEQ ID No. 2 (as shown in Fig. 10).
The present invention also relates to a polynucleotide sequence as set forth in SEQ ID
No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11,
SEQ ID No. 13, SEQ ID No. 15, or SEQ ID No. 17 encoding 5' untranslated region
(UTR), 3' untranslated region (UTR), Core protein, Envelope glycoprotein (El),
Envelop glycoprotein (E2) /Non-structural protein NS1, Non-structural protein NS2,
Non-structural protein NS3, Non-structural protein NS4, and Non-structural protein NS5 respectively.
The present invention further relates to a polypeptide sequence as set forth in SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, or SEQ ID No. 18 corresponding to Core protein, Envelope glycoprotein (El), Envelop glycoprotein (E2) /Non-structural protein NS1, Non-structural protein NS2, Non-structural protein NS3, Non-structural protein NS4, and Non-structural protein NS5 respectively.
The present invention further relates to a hepatitis C virus strain isolated from hepatitis C virus infected Indian patient pool.
The present invention furthermore relates to a pair of primers having sequence set forth in SEQ ID Nos. 19 and 20; SEQ ID Nos. 21 and 22; SEQ ID Nos. 23 and 24; SEQ ID Nos. 25 and 26; SEQ ID Nos. 27 and 28; SEQ ID Nos. 29 and 30; SEQ ID Nos. 31 and 32; SEQ ID Nos. 33 and 34; SEQ ID Nos. 35 and 36; SEQ ID Nos. 37 and 38; or SEQ ID Nos. 39 and 40.
The present invention farther relates to a vaccine for immunizing a subject against hepatitis C virus comprising at least one protein of a polypeptide sequence as set forth in SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, or SEQ ID No. 18 corresponding to Core protein, Envelope glycoprotein (El), Envelop glycoprotein (E2) /Non-structural protein NS1, Nonstructural protein NS2, Non-structural protein NS3, Non-structural protein NS4, and Non-structural protein NS5 respectively optionally along with a pharmaceutically acceptable vaccine adjuvants.
Yet another embodiment of the present invention, wherein the subject is a mammal including humans.
In still another embodiment of the present invention, wherein said vaccine adjuvants are selected from a group comprising mineral salts (aluminium hydroxide and aluminium or calcium phosphate gels, oil emulsions and surfactant based formulations (MF59, microfluidised detergent stabilized oil-in-water emulsion), particulate adjuvants (virosomes, polylactide co-glycolide, structured complex of saponins and lipids), microbial derivatives( natural and synthetic), endogenous human immunomodulators (hGM-CSF or hIL - 12, Immudaptin) and inert vehicles such as gold particles.
The present invention relates to a kit for identifying hepatitis C virus comprising at least one antigenic peptide selected from a polypeptide sequence as set forth in SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, or SEQ ID No. 18 corresponding to Core protein, Envelope glycoprotein (El), Envelop glycoprotein (E2) /Non-structural protein NS1, Non-structural protein NS2, Non-structural protein NS3, Non-structural protein NS4, and Non-structural protein NS5 respectively capable of reacting specifically with antibodies directed against said virus.
Yet another embodiment of the present invention, wherein said kit further comprises control standards and instructions for use of the kit.
The present invention relates to a method for detecting presence of hepatitis C virus comprises contacting sera with at least one antigenic polypeptide selected from a polypeptide sequence as set forth in SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, or SEQ ID No. 18 corresponding to Core protein, Envelope glycoprotein (El), Envelop glycoprotein (E2) /Non-structural protein NS1, Non-structural protein NS2, Non-structural protein NS3, Non-structural protein NS4, and Non-structural protein NS5 respectively wherein formation of an immunogenic complex confirms detection of said virus.
The present invention relates to a method of immunization against hepatitis C virus in a subject in need thereof, wherein said method comprises administering a pharmaceutical^ effective immunizing dose of the vaccine.
The present invention also relates to an E.coli strain TOP 10F'HCV-AY651061 having accession No. MTCC 5350, deposited at Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
Fig. 1: ORF of Hepatitis C Virus whole genome (9441 base pairs)
Fig. 2: Entire genome of Hepatitis C Virus
Fig. 3: SDS PAGE for the core protein
Fig. 4: Western Blot picture of the Core protein
Fig. 5: SDS PAGE for the NS3 protein
Fig. 6: SDS PAGE for the NS4 protein
Fig. 7: SDS PAGE for the NS5 protein
Fig. 8: Western blot analysis showing the presence of the NS5 protein
Fig. 9: Nucleotide sequence of HCV isolate-SEQ ID No. 1
Fig. 10: Polypeptide sequence of HCV isolates khajal, complete sequence -SEQ ID No.2
Fig: 11: Nucleotide comparison of HCV 1 5'UTR vs AY651061 5'UTR - SEQ ID
Fig: 12: Nucleotide comparison of HCVl CORE vs. AY651061 CORE - SEQ ID No.5
Fig: 13: Nucleotide comparison of HCVl El vs. AY651061 El- SEQ ID No.7
Fig: 14: Nucleotide comparison of HCVl E2/NS1 vs. AY651061 E2/NS1 - SEQ ID
Fig: 15: Nucleotide comparison of HCVl NS2 vs. AY651061 NS2 - SEQ ID No.ll
Fig: 16: Nucleotide comparison of HCVl NS3 vs. AY651061 NS3 - SEQ ID No.13
Fig: 17: Nucleotide Comparison of HCVl NS4 vs. AY651061 NS4 - SEQ ID No. 15
Fig: 18: Nucleotide Comparison of HCV 1 NS5 vs. AY651061 NS5 - SEQ ID No.17
Fig: 19: Nucleotide Comparison of HCVl 3'UTR vs. AY651061 3^UTR - SEQ ID
Fig: 20: Aminoacid Comparison of HCVl Core vs. AY651061 Core - SEQ ID No.6
Fig: 21: Aminoacid Comparison of HCVl El vs. AY651061 El - SEQ ID No.8
Fig: 22: Aminoacid Comparison of HCVl E2/NS1 vs. AY651061 E2/NS1- SEQ ID
Fig: 23: Aminoacid Comparison of HCVl NS2 vs. AY651061 NS2 - SEQ ID No.12
Fig: 24: Aminoacid Comparison of HCVl NS3 vs. AY651061 NS3 - SEQ H> No.14 Fig: 25: Aminoacid Comparison of HCVl NS4 vs. AY651061 NS4 - SEQ ID No. 16 Fig: 26: Aminoacid Comparison of HCVl NS5 vs. AY651061 NS5 - SEQ ID No. 18 Fig: 27: Sequence for primers having SEQ ID No. 19 to SEQ ID No. 40 The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA techniques and immunology, which are within the skill of the art. The term "hepatitis C virus" has been reserved by workers in the field for an unknown etiologic agent of NANBH. Accordingly, as used "hepatitis C virus" refers to an agent causative of NANBH, which was formerly referred to as NANBV and/or BB-NANBV from the class of the prototype isolate, HCVl described by Houghton et al. HCV is a Flavi-like virus. The morphology and composition of flavivirus particle are known, and
are discussed by Brinton (1986). Generally, with respect to morphology, Flaviviruses contain a central nucleocapsid surrounded by a lipid bilayer. Virions are spherical and have a diameter of about 40-50 nm. Their cores are about 25-30 nm in diameter. Along the outer surface of the virion envelope are projections that are about 5 -10 nm long with terminal knobs about 2 nm in diameter.
In one of the embodiment, the HCV genome is comprised of RNA. It is known that RNA containing viruses have relatively high rates of spontaneous mutation, i.e., reportedly on the order of 10'3 to 10'4 per incorporated nucleotide. Therefore, there are multiple strains, which may be virulent or avirulent, within the HCV class of species. It is believed that the genome of HCV isolates is comprised of a single ORF of approximately 9,000 nucleotides to approximately 12,000 nucleotides encoding a polyprotein similar hydrophobic and antigenic character to that of HCVl. In addition, the genome is believed to be a positive stranded RNA.
Yet another embodiment comprises of isolates of HCV comprise epitopes that are immunologically cross-reactive with epitopes in the HCVl genome. At least some of these are epitopes unique to HCV when compared to other known Flaviviruses. The uniqueness of the epit6pe may be determined by its immunological reactivity with anti-HCV antibodies and lack of immunological reactivity with antibodies to other Flavivirus species. Methods for determining immunological reactivity are known in the art for example by radioimmunoassay by ELISA assay by hemagglutination, and several examples of suitable techniques for assays are provided. In still another embodiment it is expected that the overall homology of HCV isolates and HCVl genomes at the nucleotide level probably will be about 40% or greater, probably about 60% or greater, and even more probably about 80% to about 90% or greater. In addition that there are many corresponding contiguous sequences of at least 13 nucleotides that are fully homologous. The correspondence between the sequence from a new isolate and the HCVl sequence can be determined by techniques known in the art. For example they can be determined by a direct comparison of the sequence information of the polynucleotide from the new isolate and HCVl sequences. Alternatively homology can be determined by hybridization of the poly nucleotides under conditions which form stable duplexes between homologous regions.
In still another embodiment the evolutionary relationship strains or isolates of HCV the putative HCV strains or isolates are identifiable by their homology at the polypeptide level. Thus, new HCV isolates are expected to be more than about 40% homologous, probably more than about 70% homologous, and even more probably more than about 80% homologous, and possibly even more than about 90% homologous at the polypeptide level. The techniques for determining amino acid sequence homology are known in the art. For example, the amino acid sequence may be determined directly and compared to the sequences provided. Alternatively the nucleotide sequence of the genomic material of the putative HCV may be determined, the amino acid sequence encoded therein can be determined and the corresponding regions compared. In still another embodiment the non-structural core and envelope domains of the polyprotein have been predicted for HCV1. The "C", or core, polypeptide is believed to be 6[.coded from the 5* terminus to about nucleotide 345 of HCV1. The putative "E". or envelope, domain of 19CV1 is believed to be encoded from about nucleotide 346 to about nucleotide 1050. Putative NS1 or nonstructural one domain, is thought to be encoded from about nucleotide 1051 to about nucleotide 1953. For the remaining domains, putative NS2 is thought to be encoded from about nucleotide 1954 to about nucleotide 3018, putative NS3 from about nucleotide 3019 to about nucleotide 4950, putative NS4 from about nucleotide 4951 to about nucleotide 6297, and putative NS5 from about nucleotide 6298 to the 3' terminus respectively.
In still another embodiment the portions of the cDNA sequences derived from HCV are useful as probes to diagnose the presence of virus in HCV infected imdividuals, and to isolate naturally occurring variants of the virus. These cDNAs also make available polypeptide sequences of HCV antigens encoded within the HCV genome(s) and permits the' production of polypeptides which are useful as standards or reagents in diagnostic tests and are components as vaccines. Antibodies, including for example both polyclonal and monoclonal, directed against HCV epitopes contained within these polypeptide sequences are also useful for diagnostic tests, as therapeutic agents, for screening of antiviral agents, and for the isolation of the NANB virus agent which these cDNA's derive. In addition, by utilizing probes derived from these cDNS's it is possible to isolate and sequence other portions of the HCV genome, thus giving rise to additional
probes and polypeptides which are useful in the diagnosis and/or treatment, both prophylactic therapeutic, NANBH.
In still another embodiment it is with respect to polynucleotides, some aspects of the invention are: a purified HCV polynucleotide; a recombinant HCV polynucleotide; a recombinant polynucleotide comprising a sequence derived from an HCV genome or from HCV cDNA; a recombinant polynucleotide encoding -an epitope of HCV; a recombinant vector containing the any of the above recombinant polynucleotides, and a host cell transformed with any of these vectors.
In still another embodiments of the invention: a recombinant expression system comprising an open reading frame (ORF) of DNA derived from an HCV genome or from HCV cDNA, wherein the ORF is operably linked to a control sequence compatible with a desired host, a cell transformed with the recombinant expression system, and a polypeptide produced by the transformed cell. Still other aspects of the invention are: a preparation of polypeptides from the purified HCV; a purified HCV polypeptide; a purified polypeptide comprising an epitope which is immunologically identifiable with an epitope contained in HCV. In still another embodiment invention immunoassays are also included. These include an immunoassay for detecting an HCV antigen comprising incubating a sample suspected of containing an HCV antigen with a probe antibody directed against the HCV antigen to be detected under conditions which allow the formation of an antigen-antibody complex; and detecting an antigen-antibody complex containing the probe antibody. An immunoassay for detecting antibodies directed against an HCV antigen comprising incubating a sample suspected of containing anti-HCV antibodies with a probe polypeptide which contains an epitope of the HCV, under conditions which allow the formation of an antibody-antigen complex; and detecting the antibody-antigen complex containing the probe antigen.
In still another embodiment the term polypeptide is used referring to a polymeric form of nucleotide of any length, either ribonucleotides or deoxyribonucleotides. It also includes the known types of modifications, for example, labels which are known in the art, methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as for example, those
with unchanged linkages (e.g., methyl phosphates, phosphotriesters, phosphoamidates, carbamates etc.) and with charged linkages. "Purified polypeptide" refers to a composition comprising a specified polypeptide that is substantially free of other components, such composition typically comprising at least about 70% of the specified polypeptide, more typically at least about 80%,90% or even 95% to 99% of the specified polypeptide.
In still another embodiment the "Recombinant host cells", "host cells", "cells", "cell lines", "cell cultures", and other such terms denote microorganisms or higher eukaryotic cell lines cultured as unicellular entities that can be, or have been, used as recipients for a recombinant vector or other transfer DNA, and include the progeny of the original cell which has been transformed. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent due to natural, accidental, or deliberate mutation.
In still another embodiment the term "replicon" is any genetic element, e.g., a plasmid, a chromosome, a virus, a cosmid, etc. that behaves as an autonomous unit of polynucleotide replication within a cell: i.e., capable of replication under its own control. A "cloning vector" is a replicon that can transform a selected host cell and in which another polynucleotide segment is attached, so as to bring about the replication and/or expression of the attached segment. Typically, cloning vectors include plasmids, virus (e.g., bacteriophage vector) and cosmids. An "integrating vector" is a vector that does not behave as a replicon in a selected host cell, but has the ability to integrate into a replicon (typically a chromosome) resident in the selected host to stably transform the host. An "expression vector" is a construct that can transform a selected host cell and provides for 30 expression of a heterologous coding sequence in the selected host. Expression vectors can be either a cloning vector or an integrating vector. In still another embodiment the "coding sequence" is a polynucleotide sequence which is transcribed into mANA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5 -terminus and a translation stop codon at the y -terminus. A coding sequence can include, but is not limited to mRNA, cDNA and recombinant polynucleotide sequences. "Control sequence" refers to polynucleotide regulatory sequences which are necessary to effect the expression of
coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism. In prokaryotes, control sequences generally include promoter, ribosomal binding site and terminators. In eukaryotes generally control sequences include promoters, terminators and, in some instances, enhancers. The term "control sequences" is intended to include, at a minimum, all components the presence of which are necessary for expression and may also include additional advantageous components. "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
In still another embodiment an open reading frame or ORF is a region of a polynucleotide sequence which encodes a polypeptide: this region may represent a portion of a coding sequence or a total coding sequence.
In still another embodiment the term 'immunologically cross-reactive' refers to two or more epitopes or polypeptides that are bound by the same antibody. Cross-reactivity can be determined by any of a number of immunoassay techniques, such as a competition assay. As used, the term "antibody" refers to a polypeptide or group of polypeptides which comprise at least one epitope. An "antigen binding site" is formed from the folding of the variable domains of an antibody molecule(s) to form three-dimensional binding sites with an internal surface Shape and charge distribution complementary to the features of an epitope of an antigen, which allows specific binding to form an antibody-antigen complex. An antigen binding site may be formed from a heavy- and'or light-chain domain (VH and VL. respectively), which form hypervariable loops which contribute to antigen binding.
In still another embodiment the term "Epitope" refers to an antibody binding site usually defined by a polypeptide, but also by non-amino acid haptens. An epitope could comprise 3 amino acids in a spatial conformation which is unique to the epitope, generally an epitope consists of at least 5 such amino acids and more usually consists of at least 8-10 such amino acids. "Antigen-antibody complex" refers to the complex formed by an antibody that is specifically bound to an epitope on an antigen. "Immunogenic polypeptide" refers to a polypeptide that elicits a cellular and/or humoral immune response in a mammal whether alone or linked to a carrier in the
presence or absence of an adjuvant. "Polypeptide" refers to a polymer of amino acids and does not refer to a specific length of the molecule. Thus peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art both naturally occurring and non-naturally occurring. "Transformation", as used refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion, for example, direct uptake, transduction, f-mating or electroporation. The exogenous polynucleotide may be maintained as a non-integrated vector, for example a plasmid or alternatively, may be integrated into the host genome. A "transformed" host cell refers to both the immediate cell that has undergone transformation and its progeny that maintain the originally exogenous polynucleotide. "Treatment" as used refers to prophylaxis and/or therapy. "Sense strand" refers to the strand of a double-stranded DNA molecule that is homologous to a mRNA transcript thereof. The "anti-sense strand" contains a sequence which is complementary to that of the "sense strand". In still another embodiment an "Antibody-containing body component" refers to a component of an individual's body which is a source of the antibodies of interest. Antibody-containing body components are known in the art, and include but are not limited to, whole blood and components thereof, plasma, serum, spinal fluid, lymph fluid, the external sections of the respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, white blood cells, and myelomas. "Purified HCV" isolate refers to a preparation of HCV particles which has been isolated from the cellular constituents with which the virus is normally associated, and from other types of viruses which may be present in the infected tissue. The techniques for isolating viruses are known to those of skill in the art, and include, for example centrifugation and affinity chromatography. In still another embodiment an HCV "particle" is an entire virion, as well as particles which are intermediates in virion formation. HCV particles generally have one or more HCV proteins associated with the HCV nucleic acid. "Probe" refers to a polynucleotide which forms a hybrid structure with a sequence in a target polynucleotide, due to complementarity of at least one region in the probe with a region In the target.
"Biological sample" refers to a sample of tissue or fluid isolated from an individual, including but not limited to, for example whole blood and components thereof, plasma, serum, spinal fluid, lymph fluid. The external sections of the skin respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs and also samples of in vitro cell culture constituents (including but not limited to conditioned medium resulting from the growth of cells in cell culture medium, putatively virally infected cells, recombinant cells, and cell components).
In still another embodiment the present invention pertains to the isolation and characterization of a newly discovered isolate of HCV Indian isolate (AY651061), its nucleotide sequence, protein sequences and resulting polynucleotides, polypeptides and antibodies derived. Isolate Indian isolate (AY651061) is novel in its nucleotide and amino acid sequences and is believed to characteristic of HCV isolates from Indonesia. In still another embodiment the nucleotide sequences derived from Indian isolate (AY651061) is useful as probes to diagnose the presence of virus in samples, and to isolate other naturally occurring variants of the Virus. These nucleotide sequences also make available polypeptide sequences of HCV antigens encoded within the Indian isolate (AY651061) genome and permits the production of polypeptides which are useful as standards or reagents in diagnostic tests and/or as components of vaccines.Antibodies both polyclonal and monoclonal directed against HCV epitopes contained within these polypeptide sequences are also useful for diagnostic tests, as therapeutic agents, for screening of antiviral agents, and for the isolation of the NANBH virus. In addition, by utilizing probes derived from the sequences disclosed herein it is possible to isolate and sequence other portions of the Indian isolate (AY651061) genome, thus giving rise to, additional probes and polypeptides which are useful in the diagnosis and/or treatment, both prophylactic and therapeutic, of NANB Hepatitis.
In still another embodiment the availability of the Indian isolate (AY651061) nucleotide sequences enable the construction of polynucleotide probes and polypeptides useful in diagnosing NANBH due to HCV infection and in screening blood donors as well as donated blood and blood products for infection. The Indian isolate (AY651061) sequences also allow the design and production of HCV specific polypeptides which are useful as diagnostic reagents for the presence of antibodies raised during NANBH. Antibodies to purified polypeptides derived from the Indian
isolate (AY651061) sequences may also be used to detect viral antigens in infected individuals and in blood.
The knowledge of the Indian isolate (AY651061) sequences also enables the design and production of polypeptides which may be used as vaccines against HCV and also for the production of antibodies, which in turn may be used for protection against the disease, and/or for therapy of HCV infected individuals, Moreover, the disclosed Indian isolate (AY651061) sequences enable further characterization of the HCV genome. Polynucleotide probes derived from these sequences, as well as from the HCV genome may be used to screen cDNA libraries for additional viral cDNA sequences.
The Indian isolate (AY651061) polynucleotide sequences, the polypeptides derived and the antibodies directed against these polypeptides, are useful in the isolation and identification of the BBNANBV agent(s). For example, antibodies directed against HCV epitopes contained in polypeptides derived from the Indian isolate (AY651061) sequences may be used in processes based upon affinity chromatography to isolate the virus. Alternatively, the antibodies may be used to identify viral particles isolated by other techniques. The viral antigens and the genomic material within the isolated viral particles may then be further characterized.
The information obtained from further sequencing of the Indian isolate (AY651061) genome, as well as from further characterization of the Indian isolate (AY651061) antigens and characterization of the genomes enable the design and synthesis of additional probes and polypeptides and antibodies which may be used for diagnosis, for prevention, and for therapy of HCV induced NANB Hepatitis and for screening for infected blood and blood-related products.
In still another embodiment the DNA encoding the desired polypeptide, whether in fused or mature form, and whether or no containing a signal sequence to permit secretion may be ligated into expression vectors suitable for any convenient host. Both eukaryotic and prokaryotic host systems are presently used in forming recombinant polypeptides, and a summary of some of the more common control systems and host cell is given below. The polypeptide produced in such host cells is then isolated from lysed cells or from the culture medium and purified to the extent needed for its intended use. Such recombinant or synthetic HCV polypeptides can be used as diagnostics or those which give rise to neutralizing antibodies may be formulated into vaccines.
Antibodies raised against these polypeptides can also be used as diagnostics or for passive immunotherapy. In addition, antibodies to these polypeptides are useful 'for isolating and identifying HCV particles.
In still another embodiment the observed relationship of the putative polyproteins of HCV and the Flaviviruses allows a predictor of the putative domains of the HCV "nonstructural" (NS) proteins. The locations of the individual NS proteins in the putative Flavivirus precursor polyprotein are fairly well-known. Moreover, these also coincide with observed gross fluctuations in the hydrophobicity profile of the polyprotein. It is established that NS5 of Flaviviruses encodes the virion polymerase, and that NSI corresponds with a complement fixation antige which has been shown to be an effective vaccine in animals. Recently, it has been shown that a flavivir protease function resides in NS3. Due to the observed similarities between HCV and the Flaviviruses deductions concerning the approximate locations of the corresponding protein domains and functions in the HCV polyprotein are possible. The expression of polypeptides containing these domains in a variety of recombinant host cells including, for example, bacteria, yeast, insect and vertebrate cells, should give rise to important immunological reagents which can be used for diagnosis, detection and vaccines. In still another embodiment although the non-structural protein region of the putative polyproteins of the HCV isolate described herein and of Flaviviruses appears to be generally similar, there is less similarity between the putative structural regions which are towards the N-terminus. In this region, there is a greater 'divergence in sequence, and in addition the hydrophobic profile of the two regions show less similarity. This "divergence" begins in the N-terminal region of the putative NSI domain in HCV and extends to the presumed N-terminus. Nevertheless it is still possible to predict the approximate locations of the putative nucleocapsid (N-terminal basic domain) and E (generally hydrophobic) domains within the HCV polyprotein.
In still another embodiment from these predictions it may be possible to identify approximate regions of the HCV polyprotein that could correspond with useful immunological reagents. For example, the E and NSI proteins of Flaviviruses are known to have efficacy as protective vaccines. These regions, as well as some which are shown to be antigenic in the HCV1, for example those within putative NS3, C, and NS5, etc. should also provide diagnostic reagents.
In still another embodiment the immunogenicity of the HCV sequences may also be enhanced by preparing the sequences fused to or assembled with particle-forming proteins. In addition, all of the vectors prepared include epitopes specific to HCV having various degrees of immunogenicity such as, for example, the pre-S peptide. Thus, particles constructed from particle forming protein which includes HCV sequences are immunogenic with respect to HCV and particle-form protein. In still another embodiment therapeutic vaccine may be prepared from one or more immunogenic polypeptides derived from Indian isolate (AY651061). The observed homology between HCV and Flaviviruses provides information concerning the polypeptides which are likely to be most effective as vaccines as well as the regions of the genome in which they are encoded. The general structure of the Flavivirus genome is discussed in Rice et al. (1986) in THE VIRUSES: THE TOGAVIRIDAE AND FLAVIVIRIDAE (Series eds. Fraenkel-Conrat and Wagner. Vol eds. Schlesinger and Schlesinger. Plenum Press). It is known that major neutralizing epitopes for Flaviviruses reside in the E (envelope) protein. Roehrig (1986) in THE VIRUSES: THE TOGAVIRIDAE AND FLAVIVIRIDAE, (Series eds. Fraenkel-Conrat and Wagner, Vol eds. Schlesinger and Schlesinger, Plenum Press). The corresponding HCV E gene and polypeptide encoding region may be predicted, based upon the homology to Flaviviruses. Thus, vaccines may be comprised of recombinant polypeptides containing epitopes of HCV E. These polypeptides may be expressed in bacteria, yeast, or mammalian cells, or alternatively may be isolated from viral preparations. It is also anticipated that the other structural proteins may also contain epitopes which give rise to protective anti-HCV antibodies. Thus, polypeptides containing the epitopes of E, C, and M may also be used, whether singly or in combination, in HCV vaccines. In still another embodiment it has been shown that immunization with NS1 (nonstructural protein 1), results in protection against yellow fever. Schlesinger et al (1986) J. Virol. 60 :115.3. This is true even though the immunization does not give rise to neutralizing antibodies. Thus, particularly since this protein appears to be highly conserved among Falviviruses, it is likely that HCV NS1 will also be protective against HCV infection. Moreover, it also shows that nonstructural proteins may provide protection against viral pathogenicity, even if they do not cause the production of neutralizing antibodies.
In still another embodiment multivalent vaccines against HCV may be comprised of one or more epitopes from one or more structural proteins, and/or one or more epitopes from one or more non structural proteins.
In still another embodiment the vaccines may be comprised of, for example, recombinant HCV polypeptides and/or polypeptides isolated from the virions. In particular, vaccines are contemplated comprising one or more of the following HCV proteins, or subunit antigens derived therefrom: E, NS1, C, NS2, NS3, NS4 and NS5. Particularly preferred are vaccines comprising E and/or NS1, or subunits thereof. In addition, it may be possible to use inactivated HCV in vaccines: inactivation may be by the preparation of viral lysates, or by other means known in the art to cause inactivation of Flaviviruses, for example, treatment with organic solvents or detergents, or treatment with formalin. Moreover, vaccines may also be prepared from attenuated HCV strains or from hybrid viruses such as vaccinia vectors known in the art [Brown et al. Nature 319:549-550].
In still another embodiment the proteins may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as for example hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like. Generally, it is expected that the HCV genome sequence will be present in serum of infected individuals at relatively low levels i.e., at approximately 10 - 10 chimp infectious doses (CID) per ml. This level may require that amplification techniques be used in hybridization assays.
In still another embodiment the Flavivirus model for HCV allows predictions regarding the likely location of diagnostic epitopes for the virion structural proteins. Similarly, domains of the nonstructural proteins are expected to contain important diagnostic epitopes (e.g., NS5 encoding a putative polymerase: and NS1 encodes a putative complement-binding antigen). Recombinant polypeptides, or viral polypeptides, which include epitopes from these specific domains may be useful for the detection of viral
antibodies in infections blood donors and infected patients. Moreover, these antibodies may be extremely useful in detecting acute-phase donors and patients. In still another embodiment the antigenic regions of the putative polyprotein can be mapped and identified by screening the antigenicity of bacterial expression products of HCV cDNAs which encode portions of the polyprotein. Other antigenic regions of HCV may be detected by expressing the portions of the HCV cDNAS in other expression systems, including yeast systems and cellular systems derived from insects and vertebrates. In addition, studies giving rise to an antigenicity index and hydrophobicity/hydrophilicity profile give rise to information concerning the probability of a region's antigenicity. Efficient detection systems may include the use of panels of epitopes. The epitopes in the panel may be constructed into one or multiple polypeptides.
In still another embodiment kits suitable for immunodiagnosis and containing the appropriate labeled reagents are constructed by packaging the appropriate materials, including the polypeptides of the invention containing HCV epitopes or antibodies directed against HCV epitopes in suitable containers along with the remaining reagents materials required for the conduct of the assay (e.g., wash buffers, detection means like labeled anti human Ig, labeled anti-HCV, or labeled HCV antigen), as well as a suitable set of assay instructions.
The Indian isolate (AY651061) nucleotide sequence information described herein may be used to gain information on the sequence of the HCV genomes, and for identification and isolation of additional HCV isolates related to this isolate. This information, in turn, can lead to additional polynucleotide probes, polypeptides derived from the HCV genome, and antibodies directed against HCV epitopes which would be useful for the diagnosis and/or treatment of HCV caused NANB Hepatitis The current standard-of-care therapy for chronically infected HCV patients is a combination of pegylated IFN and ribavirin, which is costly, lengthy (6-12 months), associated with significant side effects and results in sustained viral response in only -50% of patients. In patients infected with genotype 1, the most common form, response rates are even lower. With an estimated 170 million HCV carriers worldwide, it is clearly important to develop better therapeutic options. With our increasing knowledge of the virus encoded enzymes and genetic elements vital to the life-cycle of HCV, much attention is now being focused on the development of HCV protease,
replicase, helicase, antisense, silencing RNA and other specific inhibitors. However, preliminary data have directly linked responses to IFN-a and ribavirin with pretreatment titers of viral antibodies (presumed to be against the envelope glycoproteins), peripheral TH cell responses to the HCY core and other antigens, as well as to intrahepatic CDS+ CTL responses to the virus. Total pretreatment CDS-f T-cell counts in the liver have also been correlated with sustained responses to standard-of-care therapy. Therefore, it may be possible to boost such immune responses in patients by appropriate vaccination and thereby improve the response rate to the standard-of-care therapy. Such immunotherapy may also help control the emergence of escape mutants that would be predicted to arise from any future use of HCV protease or replicase inhibitors, for example, given the extreme fluidity and heterogeneity of the HCV genome.
In still another embodiment provides many therapeutic vaccine trials are planned or are already in progress and use diverse delivery methods and formulations but little information is available about their efficacy at present. What is known, however, is that use of an alum-adjuvanted recombinant gpEl antigen was able to boost humoral and cellular immune responses to gpEl in viraemic patients, providing encouragement that vaccination can increase immune responses in pre-existing carriers. It remains to be seen whether boosting viral-neutralizing antibody titres or broad CD4+ TH responses or broad CDS' 'I-cell responses will have the greatest impact on reducing viral load and in the response to antiviral therapy. But, as may be the case for optimal prophylaxis, boosting of these immune responses may be ideal for immunotherapy. In still another embodiment the general techniques used in extracting the genome from a virus preparing and probing a cDNA library, sequencing clones, constructing expression vectors, transforming cells, performing immunological assays such as radioimmunoassays and ELISA assays, for growing cells in culture, and the like are known in the art and laboratory manuals are available describing these techniques. However, as a general guide the following sets forth some sources currently available for such procedures, and for materials useful in carrying them out. In still another embodiment both prokaryotic and eukaryotic host cells may be used for expression of desired coding sequences when appropriate control sequences which are compatible with the designated host are used. Among prokaryotic hosts, E. coli is most frequently used. Transfer vectors compatible with prokaryotic hosts are commonly
derived from, for example, pBA322. a plasmid containing operons conferring ampicillin and tetracycline resistance, and the various pUC vectors, which also contain sequences conferring antibiotic resistance markers. These markers may be used to obtain successful transform ants by selection. The foregoing systems are particularly compatible with E. coli : if desired, other prokaryotic hosts such as strains of Bacillus or Pseudomonas may be used, with corresponding control sequences. In still another embodiment a vector construction employs techniques which are known in the art. Site-specific DNA cleavage is performed by treating with suitable restriction enzymes under conditions which generally are specified by the manufacturer of these commercially available enzymes. The cleaved fragments may be separated using polyacrylamide or agarose gel electrophoresis techniques, according to the general procedures found in Methods in Enzymology (1980) 65 :499-560. Sticky ended cleavage fragments may be blunt ended using E. coli DNA polymerase I (Klenowfln the presence of the appropriate deoxynucleotide triphosphates (dNTPS) present in the mixture. Treatment with SI nuclease may also be used, resulting in the hydrolysis of any single stranded DNA portions.
In still another embodiment ligations are carried out using standard buffer and temperature conditions using T4 DNA ligase and ATP; sticky end ligations require less ATP and less ligase than blunt end ligations. When vector fragments are used as part of a ligation mixture, the vector fragment is often treated with bacterial alkaline phosphatase (BAP) or calf intestinal alkaline phosphatase to remove the 5* -phosphate and thus prevent religation of the vector; alternatively, restriction enzyme digestion of unwanted fragments can be used to prevent ligation .. Ligation mixtures are transformed into suitable cloning posts, such as E. coli , and successful transform ants selected by, for example, antibiotic resistance, and screened for the correct construction.
In still another embodiment the present invention describes the cloning of the Indian isolate (AY651061) nucleotide sequences. Blood sample was used as a source of HCV virions was found to be positive in an anti HCV antibody assay. The HCV isolate from these samples were named Indian isolate (AY651061). The infectivity of the blood sample containing the Indian isolate (AY651061) isolate was confirmed by a prospective study of blood transfusion recipients. Dr. CM. Habibullah from the Department of Gastroenterology at Owaisi Hospital, Hyderabad, India collected blood
from patients who have contracted post-transfusion non-A, non-B hepatitis. He also collected blood samples from the respective blood donors of these patients. Next, these samples were assayed for antibodies to the 3rd EIA and blood from one of the donors was found to be positive.
In still another embodiment isolation of the RNA from the blood samples began by pelleting virions in the blood sample by ultracentrifugation [Bradley, OW, McCaustland, K.A., Cook E.H., Schable. CA, Ebert. JW. and Maynard, J.E. (1985) Gastroenterology 88, 773-779]. RNA was then extracted from the pellet by the guanidinium/cesium chloride method [Maniatis T., Fritsch, E.F., and Sambrook J. (1982) "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory. Cold Spring Harbor] and further purified by 35 phenol/chloroform extraction in the presence of urea, [Berk. A.J. Lee.F.. Harrison, T. Williams, J. and Sharp, P.A. (1979) Cell 17, 935-944]. Eleven pairs of Synthetic oligonucleotide primers were designed from the 5^UTR, C, El. E2, P7, NS2, NS3, NS4 and N55 domains of the nucleotide sequence of Indian isolate (AY651061) to isolate fragments from AY051292 and HCV 1 genome. The first set of primers was to isolate the sequence from the 5'UTR and a bit of core, the second set was core, third set envelope domain, fourth set envelope domain, fifth set of primers were to isolate a fragment which overlapped the putative envelope and non-structural one, NS1 domains, sixth set wasNS2 domain, seventh set of primers was NS3, eighth set of primers was NS4, ninth set of primers NS5A, Tenth set of Primers were a part of NS5B, eleventh set of primers were a part of NS5B and 3'UTR. The sequences for the various primers are shown in Fig: 27.
In still another embodiment about 1 \ig of the anti sense primers, was added to 10 units of reverse transcriptase(Promega) to synthesize cDNA fragments from the isolated RNA as the template. The cDNA fragments were then amplified by a standard polymerase chain reaction [Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn G.T., Erlich, H.A., and Arnheim, N. (1985) Science 230 , 1350-1354]- after 1 ng of the appropriate sense primers was added. The cDNA fragments amplified by the PCR method were gel isolated and cloned pGMT EASSY vector. Clones which contain the fragments of the viral domains were successfully constructed. From the PCR reaction of the Indian isolate (AY651061), three independent clones from each region, C/E, E, E1NS1, NS3, and NS5, have been sequenced by the dideoxy chain termination method. Sequence from all regions has been isolated from the Indian isolate (AY651061).
However, there is heterogeneity between clones containing sequence from the same
region. Consequently, a consensus sequence was constructed for each of the domains.
These differences may be explained as artifacts which occur randomly during the PCR
amplification [Saiki, R.K., Scharf, S., Faloona,' F., Mullis, K.B., Horn, G.T., Erlich.
HA, and Arnheim, N. (1985) Science 230, 1350-1354. Another explanation is that more
than one virus genome is present in the plasma of a single healthy carrier and that these
genomes are heterogeneous at the nucleotide level.
In still another embodiment, it was determined how many of these nucleotide
differences would lead to amino acid changes, using the sequence from the NS3
domain of the HCV 1 isolate as an example. Out of the five nucleotide differences,
three fall on the third position' of the amino acid codon and do not change the amino
acid sequence. Both of the remaining two nucleotide changes fall on the first position
of the amino acid codon and generate amino acid changes of threonine to alanine and
proline to alanine all of which are small, neutral amino acid residues. Similarly, when
analyzing the nucleotide differences in other domains, many silent and conserved
mutations are found. These results suggest that nucleotide sequences of the HCV
genomes in the plasma of a single healthy donor are heterogeneous at the nucleotide
level. In addition, once the consensus sequences for each of the fragments were
compiled each sequence was compared to the HCV 1 isolate in Figures 11 through 19.
The invention is further elaborated with the help of following example(s). However,
these example(s) should not be construed to limit the scope of the invention.
Selection and HCV RNA isolation from HCV infected patient:
A single chronic Hepatitis C virus infected patient was selected for sequence and
characterization of complete genome of hepatitis C virus. Twenty ml of intravenous
blood was collected and serum was separated and stored at -70°C.
HCV RNA isolation: HCV RNA was isolated from the serum by the guanidinium
isothicynate (GITC) acid -phenol method (Chomeczynski and Saachi 1987).
200(xl of serum was mixed with 500 \xl lysis buffer (4M GITC, 0.75 M Sodium acetate,
0.5% Sarkosyl, 0.1M p-meracapto ethanol), SOyi of 3M sodium acetate (Ph 5.2), 500 \il
water saturated phenol and 200 \il cholorofrom : Iso amyl alcohol (24:lv/v).
The tubes were vortexed in each step and finally kept in ice for 15min. The tubes were
centrifuged at 12000 rpm for 30 min in refrigerated centrifuge.
The aqueous phase (approx. 500jil) was collected very carefully, mixed with equal
volume of isopropanol and kept at -70°C over night.
The over night kept solutions were centrifuged at 12000 rpm for 30 min at 4°C and
pellets were washed with 1ml of 70% ethanol. The tubes were dried in heat block at
55°C for 10 min and each pellet was re-suspended in 20pl DEPC treated water.
Many sets of primers were designed to construct cDNA followed by PCR. (The list of
primers is attached)
Example: 2 cDNA Synthesis:
cDNA synthesis was carried out by adding 6jil of the isolated HCV RNA to a final 20
jj.1 of reaction mix composed of 40 picomoles of reverse primer, 4 jul of 5X reverse
transcriptase buffer, l|al of lOmM dNTPs, 7p.l of DEPC-treated water, 100U of reverse
transcriptase (MMLV supplied by promega). The mixture was incubated at 42°C for 60
min. The reaction was terminated by heating at 70°C for 15 min, and then chilled the
mixture on ice.
Polymerase chain reaction:
For amplification by PCR, a 50 jil mixture containing JO |il of the c DNA, IX PCR buffer, ljil lOmM dNTPs, 1.5(il MgC12, 2U Taq polymerase and 1 jil of each forward and reverse primers was denatured at 95 °C for 3 minutes and amplified for 36 cycles under the following conditions:94°C for 1 minutes (denaturation), 54°C for 1.5 minutes ( annealing), and 72°C for 2 minutes (extension), followed by a final extension at 72°C for 5 minutes. PCR products were analyzed on 2% agarose gels followed by staining with ethidium bromide and visualized under a UV illuminator. A 100 bp ladder (Promega, Madison, WI) was used as a size marker.
The PCR product was purified by Quiagen PCR clean up kit as per the protocol described by the supplier (Quiagen, Germany). Example: 3 Cloning of the PCR product:
The purified PCR product was ligated with pGEM-T easy vector. The ligation mix included 2X rapid ligation buffer, T4 DNA ligase (3 Weiss units/|al), PCR product and the final reaction volume made upto 10|il with deionized water. The reaction mixture was incubated at 16°C overnight.
Competent cells were prepared by Picking a single bacterial colony from a plate that has been incubated for 16-20 hours at 37°C. Transfer the colony into 100 ml of LB broth in a 1 liter flask. Incubate the culture for 3 hours at 37°C with vigorous agitation, monitoring the growth of the culture. As a guideline, 1 OD600 of a culture of E.Coli strain TOP 10 F contains ~ 109 bacteria/ml. Transfer the bacterial cells to sterile, disposable, ice-cold 50 ml polypropylene tubes. Cool the cultures to 0°C by storing the tube on ice for 10 minutes. Recover the cells by centrifugation at 5000 rpm for 10 minutes at 4°C. Decant the medium from the cell pellets. Stand the tubes in an inverted position on a pad of paper towels for 1 minute to allow the last traces of media to drain away. Resuspend each pellet by swirling or gentle vortexing in 30 ml of ice cold CaCl2 (CaCl2.2H20 (1M)) solution and kept on ice for one hour. Recover the cells by centrifugation at 5000 rpm for 10 minutes at 4°C. Decant the medium from the cell pellets. Stand the tubes in an inverted position on a pad of paper towels for 1 minute to allow the last traces of media to drain away. Resuspend the pellet by swirling or gentle vortexing in 2 ml of ice - cold 0.1 M CaCb for each 50 ml of original culture. At this point, either use the cells directly for transformation or dispense into aliquots contain each 200(il and freeze at -70°C. To transform the CaCfe- treated cells, thaw cells on ice for 15 minutes. Add DNA (no more than 50 ng in a volume of 10 ul or less) to each tube. Mix the contents of the tube by swirling gently. Store the tubes on ice for 30 minutes. Transfer the tubes to a rack placed in a preheated 42°C circulating water bath. Store the tubes in the rack for exactly 90 seconds. Do not shake the tube. Rapidly transfer the tubes to an ice bath. Allow the cells to chill for 1-2 minutes. Add 800 \i\ of LB medium to each tube. Incubate the cultures for 45 minutes in a water bath set at 37°C to allow the bacteria to recover and to express the antibiotic resistance markers encoded by the plasmid. Recover the cells by centrifugation at 5000 rpm for 5 minutes, resuspend each pellet by swirling or gentle vortexing in 100^1 of LB medium, IM IPTG and X-gal for blue white screening. Transfer the appropriate volume of transformed competent cells on LB agar medium containing the appropriate antibiotic. Store the plates at room temperature until the liquid has been absorbed. Invert the plates and incubate at 37°C. Transformed colonies should appear in 12-16 hours. Remove the plates from the incubator and store them for several hours at 4° C to allow blue color to develop. Identify colonies carrying recombinant plasmids, colonies that carry wild -type plasmids contain active p - Galactosidase. These colonies are pale blue in the
center and dense blue at their periphery. Colonies that carry recombinant plasmids do not contain active β - Galactosidase .These colonies are creamy white or eggshell blue , sometimes with a faint blue spot in the center . Select and culture colonies carrying recombinant plasmids.
Plasmid DNA was prepared by Alkaline Lysis method. Inoculate 5 ml of LB medium containing the appropriate antibiotic with a single colony of transformed bacteria. Incubate the culture overnight at 37°C with vigorous shaking. Pour 1.5 ml of the culture into a microfuge tube. Centrifuge at maximum speed for 30 seconds at 4°C in a microfuge. Store the unused portion of the original culture at 4°C.When centrifugation is complete, remove the medium by aspiration, leaving the bacterial pellet as dry as possible. Resuspend the bacterial pellet in 100μl of ice-cold Alkaline lysis solution I (50mM glucose, 25mM Tris-Cl (pH 8.0), l0mM EDTA (pH 8.0) by vigorous vortexing. Add 200μl of freshly prepared Alkaline Lysis Solution II (0.2N NaOH, 1% (w/v) SDS) to each bacterial suspension. Close the tube tightly, and mix the contents by inverting the tube rapidly five times. Do not vortex! Store the tube on ice. Add 150μl o ice - cold Alkaline lysis solution III (. Close the tube and disperse Alkaline lysis solution III (5M potassium acetate, glacial acetic acid) through the viscous bacterial lysate by inverting the tube several times. Store the tube on ice for 3-5 minutes, centrifuge the bacterial lysate at maximum speed for 5 minutes at 4 °C in a microfuge. Transfer the supernatant to a fresh tube. Add an equal volume of phenol; chloroform. Mix organi and aqueous phases by vortexing and then centrifuge the emulsion at maximum speed for 2 minutes at 4°C in a microfuge. Transfer the aqueous upper layer to a fresh tube. Precipitate nucleic acids from the supernatant by adding 2 volumes of ethanol at room temperature, Mix the solution by vorterxing and then allow the mixture to stand for 2 minutes at room temperature. Collect the precipitated nucleic acids by centrifugation at maximum speed for 5 minutes at 4°C in a microfuge. Remove the supernatant by gently. Stand the tube in an inverted position on a paper towel to allow all of the fluid to drain away. Add 1 ml of 70% ethanol to the pellet and invert the closed tube several times. Recover the DNA by centrifugation at maximum speed for 2 minutes at 4°C in a microfuge. Again remove all of the supernatant by gently and store the open tube at room temperature until the ethanol has evaporated and no fluid is visible in the tube ( 5-10 minutes). Dissolve the nucleic acids in 50μl of TE (pH 8.0).
Vortex the solution gently for a few seconds. Store the DNA solution at - 20°C. All the clones were digested with Eco Rl to excise the fragment and were checked on the gel for confirmation analysis. The gel picture shows us the results of the clones of all AY651061.
In compliance with the requirements of full disclosure, an E.coli strain carrying the HCV genome has been deposited in Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India. The deposit made is of the Escherichia coli strain TOP10F'HCV-AY651061 under the accession No. MTCC 5350.
Detection of Antibody to HCV has become the principal method for the diagnosis of HCV infection in individuals with chronic hepatitis and for the screening of blood donors. The original assay based upon the recombinant proteins derived from NS4 showed non-specificity and insensitivity, the more recently developed assays that use recombinant proteins from the core and NS3 regions of the HCV genome (second generation) and the NS5 region of the HCV genome (third generation) have proved to be more effective.
HCV can be classified into at least into six major genotypes, whose nucleotide and inferred amino acid sequences over the whole genome differ by approximately 30%. Significant antigenic differences have been documented and form the basis of their classification into serotypes. We wanted to measure serological reactivities to the individual component antigens core, NS3, NS4 and NS5. ORF of Hepatitis C Virus whole genome (9441 base pairs) is shown in Fig. 1.
The entire genome of Hepatitis C virus genotype (as shown in Fig. 2) predominant in India was cloned, sequenced and submitted to GenBank (Accession Number AY651061). DNA fragments of all the four antigens viz., Core, NS3, NS4 and NS5 used in the 3rd generation diagnostic kits were cloned into pET21 vectors and expressed in E.coli BL21DE3 strain. Example: 4 CORE:
Cloning and Characterization: The sequencing encoding the core protein is highly conserved among all the Hepatitis C viral subtypes and is localized to nucleotides 342 to 915. The corresponding protein has 191 amino acids and with a molecular weight of about 22 kDa. The coding sequence of core was amplified by Polymerase Chain
Reaction (PCR) using gene specific primers. The forward primer contains a BamHI site and the reverse primer contains an EcoRsite. The amplified 567 bp DNA fragment was then inserted between the BamHI and EcoRI sites of the expression vector pET21a (Fig.2a). This DNA was transferred in to E.coli BL21(DE3) cells and individual clones expressing high levels of core were selected. The core sequence was confirmed by sequencing.
Protein purification; E.coli cells expressing core were induced by isopropyl-thiogalactoside (IPTG) and pelleted by centrifugation. The pellet was resuspended in lysis buffer and the inclusion bodies were isolated as described (Sambrook et al., 2001). Following solubilization of inclusion bodies with detergents, the protein was purified to homogeneity either by preparative electro-elution or ion exchange chromatography. Purity of the protein was assessed by SDS- polyacrylamide gel electrophoresis (PAGE) (Fig. 3) SDS PAGE gel for the core protein, and a western blot picture of Core protein is shown in Fig. 4. Example: 5 NS3:
Cloning and Characterization: The DNA fragment encoding the 271 amino acid NS3 (amino acids from 1192 to 1463) was amplified by PCR and cloned in to pET21b at the Sail site under the T7 promoter (Fig. 2b). The clone was verified by DNA sequencing and then introduced into expression host E.coli BL21(DE3).
Protein Purification: The bacterial clone carrying NS3 gene was induced by IPTG, collected cells by centrifugation and inclusion bodies were prepared as described for Core. Following solubilization of inclusion bodies the protein was purified to homogeneity either by preparative gel electro-elution or ion exchange chromatography. Purity of NS3 protein was checked by SDS-PAGE (Fig. 5). Example: 6 NS4:
Cloning and Characterization: The DNA sequence encoding the NS4 region (nucleotides 5246 to 6015) was amplified by PCR using the gene specific forward and reverse primers. Thus amplified 765 bp DNA is digested with BamHI and NotI at 5" and 3' ends respectively and inserted into the BamH I and NotI sites in pET21a (+) vector (Fig. 2c) and transferred into E.coli BL21(DE3) cells. The clone expressing highest levels of NS4 was selected and the DNA was sequenced. It should be pointed out that in this clone about of 83 amino acids at the COOH-end was missing. Subsequently we cloned this additional sequence also to give to produce full length
protein (-305 amino acids). It should be pointed out that both truncated and the full
length proteins are equally efficient in detecting positive patients' sera.
Protein purification: Standard purification protocol described above for Core and NS3
was used to purify NS4 to homogeneity which was verified by SDS-PAGE (Fig. 6)
Example: 7 NS5: Cloning and Characterization
The NS5A region extends from nucleotides 6281 to 7403 (374 amino acids and mass of
about 45 kDa. The region was amplified by PCR using the gene specific primers
containing Ndel and BamHI sites in the forward and reverse primers, respectively. The
amplified 1100 bp DNA was digested with Ndel and BamH I and inserted at the Nde I
and Bam HI sites of pET21a (+) vector (Fig.2d), which was then transferred into E.coli
BL21(DE3) cells. The bacterial clone carrying the NS5 gene was confirmed by DNA
Protein purification: Induction, isolation of inclusion bodies and solubilization were
as described for Core and NS3. Purity was checked by SDS-PAGE (Fig. 7) SDS PAGE
gel for the NS5 protein, Western blot analysis showing the presence of the NS5 protein
is shown in Fig.8.
A total number of 532 patients were screened for HCV infection. A total of 218 patients
were found to be positive by RT-PCR. Among the 218 samples, 211 were positive by
3rd EIA where as all the 218 were positive by the Core, NS3, NS4, NS5 proteins
derived from our Indian isolate (AY651061). Further competitive analysis for each
antigen showed the following results:
About 98 samples were positive by all the four proteins and 38 samples were reactive
with core, NS3, NS4, but were not picked by NS5. Similarly, 29 samples were not
detected by NS4 and NS5 and 12 samples were not detected by core protein EIA. 10
samples were not picked by NS5 and Core, 16 samples were not picked by NS4, 8
samples were not picked by NS4 and core.
The interesting observation of the present analysis was that 7 samples were not picked
by 3rd EIA but were picked by our purified proteins. A total of 4 samples were picked
by core and NS3, 2 samples were picked by core, NS3 and NS4, and 1 sample was
picked by core. About 315 samples were negative by all the methods that were used.
The findings of significant antigenic variability of antigens used for serological screening will form the basis for a number of future investigations. It will be possible to carryout screening of our population infected with genotype 1. This may reveal the frequency with which anti-HCV samples are being missed therefore, assays developed from purified proteins from our isolate may be more effective for the detection of antibody elicited by infection with a specific genotype that is more prevalent. Our recent progress on HCV gave us a lot of new information on the genetic variation of the strains floating in Indian population. This will provide the basis for developing an effective vaccine. Example: 8 Transformation:
About 10 μl of ligation mixture was added to the competent E.coli ToplOF' cells and incubated on ice for 20 minutes. Following a 90 sec incubation at 42°C, 1000μl of LB medium was added to the tube and incubated for 45 min at 37°C. Cells were centrifuged at 5000 rpm for 5 min, dissolved the pellet in 100μl LB medium and plated on a LB plate with appropriate antibiotic and IPTG/X-gal. Plates were incubated overnight at 37°C. Colonies and selected and grown in a 5 ml LB broth overnight at 37°C with shaking. DNA was isolated from a 1.5 ml culture in 1.5 ml Eppendoff using
Quiagen plasmid DNA kit. After plasmid DNA isolation, DNA was digested with
EcoRI enzyme to check the insert and the DNA was sent for sequencing.
Example: 9 DNA sequencing:
The cloned HCV sequences were amplified using Ml3 primers both forward and
reverse. The re-amplified PCR products were sequenced by the direct sequencing
method in an automated sequencer (ABI, Applied bio systems, Foster city, CA, USA).
Sequences of all clones of HCV genome were analyzed and checked with NCBI Blast
software program. All the sequences from different clones of HCV genome were joined
using chromas and chromas-pro software programs. This complete Indian isolate of
HCV genome was submitted to GENBANK.
Example: 10 Comparative analysis of new isolate (Indian Strain: AY 651061) with
the prototype HCV1:
The HCV genome organized into several regions which code for various viral proteins
- 5" Untranslated region (UTR), core gene, genes for two envelope glycopreoteins (El
& E2/NS1) genes for seven nonstructural proteins (NS2, NS3A,NS3B, NS4A, NS4B,
NS5A & NS5B) and 3"UTR. The nucleotide comparison of various viral proteins are
shown in Fig. 11 to Fig. 19, and aminoacid comparison with various viral proteins are
shown in Fig. 20 to Fig. 26
The complete genome of new Indian isolate shows 82.9% nucleotide homology with
prototype HCV1 strain.
The 5'UTR is highly conserved among all the strains, 99.1% nucleotide homology was
observed in new Indian isolate compared HCV1.
The new Indian isolate of HCV Core gene was showing 97.6% nucleotide homology
compared with prototype HCV1.
The new Indian isolate of HCV El gene was showing 80.8% nucleotide homology
compared with prototype HCV1.
The new Indian isolate of HCV E2/NS1 gene was showing 84.7% nucleotide homology
compared with prototype HCV1.
The new Indian isolate of HCV NS2 gene was showing 81.9% nucleotide homology
compared with prototype HCV1.
The new Indian isolate of HCV NS3 gene was showing 82.4% nucleotide homology
compared with prototype HCV1.
The new Indian isolate of HCV NS4 gene was showing 79.3% nucleotide homology
compared with prototype HCV1.
The new Indian isolate of HCV NS5 gene was showing 80% nucleotide homology
compared with prototype HCV1.
The new Indian isolate of HCV S'UTR gene was showing 81.5% nucleotide homology
compared with prototype HCV.
The complete genome of new Indian isolate shows 86.3% amino acid homology with
prototype HCV1 strain.
The new Indian isolate of HCV Core gene was showing 98.4% amino acid homology
compared with prototype HCV1.
The new Indian isolate of HCV El gene was showing 80.5% amino acid homology
compared with prototype HCV1.
The new Indian isolate of HCV E2/NS1 gene was showing 86.2% amino acid
homology compared with prototype HCV1.
The new Indian isolate of HCV NS2 gene was showing 80.7% amino acid homology
compared with prototype HCV1.
The new Indian isolate of HCV NS3 gene was showing 91.4% amino acid homology
compared with prototype HCV1.
The new Indian isolate of HCV NS4 gene was showing 87% amino acid homology
compared with prototype HCV1.
The new Indian isolate of HCV NS5 gene was showing 83.1% amino acid homology
compared with prototype HCV1.
A polynucleotide sequence as set forth in SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, or SEQ ID No. 17 encoding 5' untranslated region (UTR), 3' untranslated region (UTR), Core protein, Envelope glycoprotein (El), Envelop glycoprotein (E2) /Non-structural protein NS1, Non-structural protein NS2, Non-structural protein NS3, Non-structural protein NS4, and Non-structural protein NS5 respectively.
A polypeptide sequence as set forth in SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, or SEQ ID No. 18 corresponding to Core protein, Envelope glycoprotein (El), Envelop glycoprotein (E2) /Non-structural protein NS1, Non-structural protein NS2, Non-structural protein NS3, Non-structural protein NS4, and Non-structural protein NS5 respectively.
A hepatitis C virus strain isolated from hepatitis C virus infected Indian patient pool.
A pair of primers having sequence set forth in SEQ ID Nos. 19 and 20; SEQ ID
Nos. 21 and 22; SEQ ID Nos. 23 and 24; SEQ ID Nos. 25 and 26; SEQ ID Nos.
27 and 28; SEQ ID Nos. 29 and 30; SEQ ID Nos. 31 and 32; SEQ ID Nos. 33
and 34; SEQ ID Nos. 35 and 36; SEQ ID Nos. 37 and 38; or SEQ ID Nos. 39
A vaccine for immunizing a subject against hepatitis C virus comprising at least
one protein according to claim 2 optionally along with a pharmaceutical^
acceptable vaccine adjuvants.
A vaccine as claimed in claim 5, wherein the subject is a mammal including
A vaccine as claimed in claim 5, wherein said vaccine adjuvants are selected
from a group comprising mineral salts, oil emulsions and surfactant based
formulations, particulate adjuvants, microbial derivatives, endogenous human
immunomodulators and inert vehicles such as gold particles.
A kit for identifying hepatitis C virus comprising at least one antigenic peptide
selected from claim 2 capable of reacting specifically with antibodies directed
against said virus.
A kit as claimed in claim 8, wherein said kit further comprises control standards
and instructions for use of the kit.
A method for detecting presence of hepatitis C virus comprises contacting sera
with at least one antigenic polypeptide of claim 2, wherein formation of an
immunogenic complex confirms detection of said virus.
A method of immunization against hepatitis C virus in a subject in need thereof,
wherein said method comprises administering a pharmaceutically effective
immunizing dose of the vaccine as claimed in claim 5.
A hepatitis C virus polypeptide sequence set forth in SEQ ID No. 2.
An E.coli strain TOP10F'HCV-AY651061 having accession No. MTCC 5350,
deposited at Microbial Type Culture Collection and Gene Bank (MTCC),
The Polynucleotide sequences, the polypeptide sequences, the hepatitis virus
strain, the pair of primers, the vaccine, the kit, the method for detecting hepatitis
C virus, the method of immunization against hepatitis C Virus and the E.coli
strain carrying HCV genome as herein described with reference to examples.
|Indian Patent Application Number||1922/CHENP/2007|
|PG Journal Number||31/2010|
|Date of Filing||07-May-2007|
|Name of Patentee||SUDERSHAN BIOTECH LTD|
|Applicant Address||4-100 C/9 & C/10, DOCTORS COLONY, L.B. NAGAR , HYDERABAD 500 074, ANDHRA PRADESH INDIA|
|PCT International Classification Number||C12N 15/51|
|PCT International Application Number||PCT/IN2006/000191|
|PCT International Filing date||2006-06-07|