Disclosed herein is the discovery of a novel hepatitis C virus (HCV) isolated from a human patient. Embodiments of the invention include HCV peptides, nucleic acids encoding said HCV peptides, antibodies directed to said peptides, compositions containing said nucleic acids and peptides, as well as, methods of making and using the aforementioned compositions including, but not limited to, diagnostics and medicaments for the treatment and prevention of HCV infection.

SUMMARY OF THE INVENTION

A new HCV sequence was discovered. A novel NS3/4A fragment of the HCV genome was cloned and sequenced from a patient infected with HCV (SEQ. ID. NO.: 1). This sequence was found to be only 93% homologous to the most closely related HCV sequence. Embodiments include this novel peptide (SEQ. ID. NO.: 2) and fragments thereof at least 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length, nucleic acids encoding these molecules, vectors having said nucleic acids, and cells having said vectors, nucleic acids, or peptides. The NS3/4A nucleic acid, fragments thereof and corresponding peptides are immunogenic. Accordingly, preferred embodiments include vaccine compositions comprising the HCV peptide of SEQ. ID. NO.: 2 or a fragment thereof at least 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length (e.g., SEQ. ID. NOs.: 14 and 15) or a nucleic acid encoding said peptide or fragments.

Mutants of the NS3/4A peptide were also created. Some mutants are truncated versions of the NS3/4A peptide (e.g., SEQ. ID. NOs.: 12 and 13) and others lack a proteolytic cleavage site (e.g., SEQ. ID. NOs.: 3-11). These molecules and the nucleic acids encoding them are also immunogenic. These novel peptides (SEQ. ID. NOs.: 3-13) and fragments thereof at least 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length (e.g., SEQ. ID. NOs.: 15-26), nucleic acids encoding these molecules, vectors having said nucleic acids, and cells having said vectors, nucleic acids, or peptides are embodiments of the invention. A particularly preferred embodiment is a vaccine composition comprising at least one HCV peptide of SEQ. ID. NOs.: 3-11 or a fragment thereof at least 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length (e.g., SEQ. ID. NOs.: 16-26) or a nucleic acid encoding said peptides or fragments.

Methods of making and using the compositions described herein are also embodiments of the invention. In addition to methods of making the embodied nucleic acids and peptides, other embodiments include methods of making vaccine compositions that can be used to treat or prevent HCV infection. Some methods are practiced, for example, by mixing an adjuvant with a peptide or nucleic acid antigen (e.g., an HCV peptide or HCV nucleic acid, as described above so as to formulate a single composition (e.g., a vaccine composition). Preferred methods involve the mixing of ribavirin with an HCV gene or antigen disclosed herein.

Preferred methods of using the compositions described herein involve providing an animal in need of a potent immune response to HCV with a sufficient amount of one or more of the nucleic acid or peptide embodiments described herein. By one approach, for example, an animal in need of potent immune response to HCV (e.g., an animal at risk or already infected with HCV) is identified and said animal is provided an amount of NS3/4A (SEQ. ID. NO.: 2), a mutant NS3/4A (SEQ. ID. NOs.: 3-13), a fragment thereof (e.g., SEQ. ID. NOs.: 14-26) or a nucleic acid encoding said molecules that is effective to enhance or facilitate an immune response to the hepatitis viral antigen. Additional methods are practiced by identifying an animal in need of a potent immune response to HCV and providing said animal a composition comprising a peptide comprising an antigen or epitope present on SEQ. ID. NOs.: 2-27 or a nucleic acid encoding said peptide. Particularly preferred methods involve the identification of an animal in need of an potent immune response to HCV and providing said animal a composition comprising an amount of HCV antigen (e.g., NS3/4A (SEQ. ID. NO.: 2)), mutant NS3/4A (SEQ. ID. NOs.: 3-13), a fragment thereof at least 3, 4-10, 10-20, 20-30, 30-50 amino acids in length (e.g., SEQ. ID. NOs.: 14-26) or a nucleic acid encoding one or more of these molecules) that is sufficient to enhance or facilitate an immune response to said antigen. In some embodiments, the composition described above also contains an amount of ribavirin that provides an adjuvant effect.

DETAILED DESCRIPTION OF THE INVENTION

A novel nucleic acid and protein corresponding to the NS3/4A domain of HCV was cloned from a patient infected with HCV (SEQ. ID. NO.: 1). A Genebank search revealed that the cloned sequence had the greatest homology to HCV sequences but was only 93% homologous to the closest HCV relative (accession no AJ 278830). This novel peptide (SEQ. ID. NO.: 2) and fragments thereof at least 3-20 amino acids in length (e.g., 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length) (e.g., SEQ. ID. NOs.: 14 and 15), nucleic acids encoding these molecules, vectors having said nucleic acids, and cells having said vectors, nucleic acids, or peptides are embodiments of the invention.

Mutants of the novel NS3/4A peptide were created. It was discovered that truncated mutants (e.g., SEQ. ID. NOs.: 12 and 13) and mutants that lack a proteolytic cleavage site (SEQ. ID. NOs.: 3-11), are highly immunogenic. These novel peptides (SEQ. ID. NOs.: 3-13) and fragments thereof at least 3-20 amino acids in length (e.g., 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length) (e.g., SEQ. ID. NOs.: 16-26), nucleic acids encoding these molecules, vectors having said nucleic acids, and cells having said vectors, nucleic acids, or peptides are also embodiments of the invention.

The peptides and nucleic acids described above are useful as immunogens, which can be administered alone or in conjunction with an adjuvant. Preferred embodiments include compositions that comprise one or more of the nucleic acids and/or peptides described above and an adjuvant. That is, some of the vaccine embodiments described herein comprise an adjuvant and the novel NS3/4A peptide (SEQ. ID. NO.: 2) or a fragment thereof at least 3-20 amino acids in length (e.g., 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length) (e.g., SEQ. ID. NOs.: 14 and 15) or a nucleic acid encoding one or more of these molecules. Additional vaccine embodiments comprise an adjuvant and one or more of the NS3/4A mutant peptides (SEQ. ID. NOs.: 3-13) or a fragment thereof at least 3-20 amino acids in length (e.g., 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length) (e.g., SEQ. ID. NOs.: 16-26) or a nucleic acid encoding one or more of these molecules.

It was also discovered that compositions comprising ribavirin and an antigen (e.g., a molecule containing an epitope of a pathogen such as a virus, bacteria, mold, yeast, parasite) enhance and/or facilitate an animal's immune response to the antigen. That is, it was discovered that ribavirin is a very effective "adjuvant," which for the purposes of this disclosure, refers to a material that has the ability to enhance or facilitate an immune response to a particular antigen. The adjuvant activity of ribavirin was manifested by a significant increase in immune-mediated protection against the antigen, an increase in the titer of antibody raised to the antigen, and an increase in proliferative T cell responses.

Accordingly, compositions (e.g., vaccines and other medicaments) that comprise ribavirin and one or more of the peptides or nucleic acids described herein are embodiments. These compositions can vary according to the amount of ribavirin, the form of ribavirin, as well as the sequence of the HCV nucleic acid or peptide.

Also embodied are methods of making and using the compositions above. Some methods involve the making of nucleic acids encoding NS3/4A, mutant NS34A, fragments thereof at least 9-30 consecutive nucleotides in length (e.g., 9, 12, 15, 18, 21, 24, 27, or 30 consecutive nucleotides in length), peptides corresponding to said nucleic acids, constructs comprising said nucleic acids, and cells containing said compositions. Preferred methods, however, concern the making of vaccine compositions comprising the newly discovered NS3/4A fragment or an NS3/4A mutant (e.g., a truncated mutant or a mutant lacking a proteolytic cleavage site), or a fragment thereof of at least three amino acids in length or a nucleic acid encoding one or more of these molecules. Preferred fragments for use with the methods described herein include SEQ. ID. NOs.: 12-27. The compositions described above can be made by providing an adjuvant (e.g., ribavirin), providing an HCV antigen (e.g., a peptide comprising an HCV antigen such as (SEQ. ID. NOs.: 2-11) or a fragment thereof such as, SEQ. ID. NOs.: 12-26 or a nucleic acid encoding one or more of said peptides), and mixing said ribavirin and said HCV antigen so as to formulate a composition that can be used to enhance or facilitate an immune response in a subject to said antigen.

Methods of enhancing or facilitating the immune response of an animal, including humans, to an antigen are also desired. Such methods can be practiced, for example, by identifying an animal in need of a potent immune response to HCV and providing said animal a composition comprising one or more of the nucleic acids or peptides above and an amount of adjuvant (e.g., ribavirin) that is effective to enhance or facilitate an immune response to the antigen/epitope. In some embodiments, the antigen and the adjuvant are administered separately, instead of in a single mixture. Preferably, in this instance, the ribavirin is administered a short time before or a short time after admininstering the antigen. Preferred methods involve providing the animal in need with ribavirin and NS3/4A (e.g., SEQ. ID. NO.: 2), a mutant NS3/4A (e.g., SEQ. ID. NOs.: 3-13), a fragment thereof of at least 3-20 amino acids in length (e.g., 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length) (e.g., SEQ. ID. NOs.: 14-26) or a nucleic acid encoding said molecules.

Other embodiments of the invention concern methods of treating and preventing HCV infection. By one approach, an immunogen comprising one or more of the HCV nucleic acids or peptides described herein are used to prepare a medicament for the treatment and/or prevention of HCV infection. By another approach, an individual in need of a medicament that prevents and/or treats HCV infection is identified and said individual is provided a medicament comprising ribavirin and an HCV antigen such as NS3/4A (e.g., SEQ. ID. NO.: 2) or a mutant NS3/4A (e.g., SEQ. ID. NOs.: 3-13), a fragment thereof of at least 3-20 amino acids in length (e.g., 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length) (e.g., SEQ. ID. NOs.: 14-26) or a nucleic acid encoding one or more of these molecules. The section below discusses the discovery of the novel NS3/4A, the creation of NS3/4A mutants, and the characterization of these nucleic acids and peptides corresponding thereto.

NS3/4A and NS3/4A Mutants

A novel nucleic acid and protein corresponding to the NS3/4A domain of HCV was cloned from a patient infected with HCV (SEQ. ID. NOs.: 1 and 2). A Genebank search revealed that the cloned sequence had the greatest homology to HCV sequences but was only 93% homologous to the closest HCV relative (accession no AJ 278830). A truncated mutant of the novel NS3/4A peptide and NS3/4A mutants, which lack a proteolytic cleavage site, were also created. It was discovered that these novel peptides and nucleic acids encoding said peptides were potent immunogens that can be mixed with adjuvants (e.g., ribavirin) so as to make a composition that provides a recipient with a potent immune response to HCV.
 

Claim 1 of 84 Claims

1. A method of inducing an immune response comprising: (a) identifying a subject in need of an immune response to hepatitis-C virus (HCV); and (b) providing said subject a composition that comprises a purified or isolated nucleic acid comprising at least 100 consecutive nucleotides of SEQ ID NO. 1 or the complement thereof, wherein said nucleic acid comprises a sequence that encodes the NS3 domain or an immunogenic fragment thereof, whereby an immune response specific to a peptide encoded by said nucleic acid is induced.

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