Title:  Vaccines containing ribavirin and methods of use thereof

United States Patent:  6,858,590

Issued:  February 22, 2005

Inventors:  Sallberg; Matti (Alvsjo, SE); Hultgren; Catharina (Stockholm, SE)

Assignee:  TRIPEP AB (Huddinge, SE)

Appl. No.:  929955

Filed:  August 15, 2001

Abstract

Compositions and methods for enhancing the effect of vaccines in animals, such as domestic, sport, or pet species, and humans are disclosed. More particularly, vaccine compositions comprising ribavirin and an antigen, preferably an antigen that has an epitope present in Hepatitis C virus (HCV), are disclosed for use in treating and preventing disease, preferably HCV infection.

Description of the Invention

FIELD OF THE INVENTION

The present invention relates to compositions and methods for enhancing the effect of vaccines in animals, such as domestic, sport, or pet species, and humans. More particularly, preferred embodiments concern the use of ribavirin as an adjuvant and compositions having ribavirin and an antigen.

BACKGROUND OF THE INVENTION

The use of vaccines to prevent disease in humans, farm livestock, sports animals, and household pets is a common practice. Frequently, however, the antigen used in a vaccine is not sufficiently immunogenic to raise the antibody titre to levels that are sufficient to provide protection against subsequent challenge or to maintain the potential for mounting these levels over extended time periods. Further, many vaccines are altogether deficient in inducing cell-mediated immunity, which is a primary immune defense against bacterial and viral infection. A considerable amount of research is currently focussed on the development of more potent vaccines and ways to enhance the immunogenicity of antigen-containing preparations. (See e.g., U.S. Pat. Nos. 6,056,961; 6,060,068; 6,063,380; and Li et al., Science 288:2219-2222 (2000)).

Notorious among such "weak" vaccines are hepatitis B vaccines. For example, recombinant vaccines against hepatitis B virus such as Genhevacb (Pasteur Merieux Serums et Vaccines, 58, Avenue Leclerc 69007 Lyon, France), Engerixb (Smith, Kline and Symbol French), and Recombivaxhb (Merck, Sharp, and Dhome) are effective only after at least three injections at 0, 30, and 60 or 180 days, followed by an obligatory booster after one year. (Chedid et al., U.S. Pat. No. 6,063,380). Additionally, many subjects receiving these vaccines respond poorly, if at all. Because many regions of the world are endemic for HBV infection, the poorly immunogenic character of existing HBV vaccines has become an extremely serious problem.

To obtain a stronger, humoral and/or cellular response, it is common to administer a vaccine in a material that enhances the immune response of the patient to the antigen present in the vaccine. The most commonly used adjuvants for vaccine protocols are oil preparations and alum. (Chedid et al., U.S. Pat. No. 6,063,380). A greater repertoire of safe and effective adjuvants is needed.

Nucleoside analogs have been widely used in anti-viral therapies due to their capacity to reduce viral replication. (Hosoya et al., J. Inf. Dis., 168:641-646 (1993)). ribavirin (1-.beta.-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a synthetic guanosine analog that has been used to inhibit RNA and DNA virus replication. (Huffman et al., Antimicrob. Agents. Chemother., 3:235 (1973); Sidwell et al., Science, 177:705 (1972)). ribavirin has been shown to be a competitive inhibitor of inositol mono-phosphate (IMP) dehydrogenase (IMPDH), which converts IMP to IMX (which is then converted to GMP). De Clercq, Anti viral Agents: characteristic activity spectrum depending on the molecular target with which they interact, Academic press, Inc., New York N.Y., pp. 1-55 (1993). Intracellular pools of GTP become depleted as a result of long term ribavirin treatment.

In addition to antiviral activity, investigators have observed that some guanosine analogs have an effect on the immune system. (U.S. Pat. Nos. 6,063,772 and 4,950,647). ribavirin has been shown to inhibit functional humoral immune responses (Peavy et al., J. Immunol., 126:861-864 (1981); Powers et al., Antimicrob. Agents. Chemother., 22:108-114 (1982)) and IgE-mediated modulation of mast cell secretion. (Marquardt et al., J. Pharmacol. Exp. Therapeutics, 240:145-149 (1987)). Some investigators report that a daily oral therapy of ribavirin has an immune modulating effect on humans and mice. (Hultgren et al., J. Gen. Virol., 79:2381-2391 (1998) and Cramp et al., Gastron. Enterol., 118:346-355 (2000)). Nevertheless, the current understanding of the effects of ribavirin on the immune system is in its infancy.

SUMMARY OF THE INVENTION

It has been discovered that ribavirin can be used as an adjuvant to enhance or facilitate an immune response to an antigen. Embodiments of the invention described herein include "strong" vaccine preparations that comprise an antigen and ribavirin. Generally, these preparations have an amount of ribavirin that is sufficient to enhance or facilitate an immune response to the antigen. Other aspects of the invention include methods of enhancing or facilitating an immune response of an animal, including a human, to an antigen. By one approach, for example, an animal in need of a potent immune response to an antigen is identified and then is provided an amount of ribavirin together with the antigen. In some methods, the ribavirin and the antigen are provided in combination (e.g., in a single composition) and in others, the ribavirin and the antigen are provided separately. Several embodiments also concern the manufacture and use of compositions having ribavirin and an antigen.

Although the embodied compositions include ribavirin and virtually any antigen or epitope, preferred compositions comprise ribavirin and a hepatitis viral antigen or epitope. The antigen or epitope can be peptide or nucleic acid-based (e.g., a RNA encoding a peptide antigen or a construct that expresses a peptide antigen when introduced to a subject). Compositions having ribavirin and a peptide comprising an antigen or epitope from the hepatitis A virus (HAV) or a nucleic acid encoding said peptide are embodiments. Compositions having ribavirin and a peptide comprising an antigen or epitope from the hepatitis B virus (HBV) or a nucleic acid encoding said peptide are embodiments. HBV antigens that are suitable include, for example, hepatitis B surface antigen (HBsAg), hepatitis core antigen (HBcAg), hepatitis e antigen (HBeAg), and nucleic acids encoding these molecules. Still further, compositions having ribavirin and a peptide comprising an antigen or epitope from the hepatitis C virus (HCV) or a nucleic acid encoding said peptide are embodiments. Suitable HCV antigens include, but are not limited to, one or more domains of the HCV sequence (e.g., NS3 and/or NS4A) and nucleic acids encoding said molecules.

A new HCV sequence was also discovered. A novel NS3/4A fragment of the HCV genome was cloned and sequenced from a patient infected with HCV (SEQ. ID. NO.: 16). This sequence was found to be only 93% homologous to the most closely related HCV sequence. This novel peptide (SEQ. ID. NO.: 17) 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 are also embodiments of the present invention. A particularly preferred embodiment is a vaccine composition comprising ribavirin and the HCV peptide of SEQ. ID. NO.: 17 or a fragment thereof at least 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length (e.g., SEQ. ID. NO.: 25) or a nucleic acid encoding said peptide or fragments.

Additionally, it was discovered that truncated mutants and mutants of the NS3/4A peptide, which lack a proteolytic cleavage site, are highly immunogenic. These novel peptides (SEQ. ID. NOs.: 29-32 and 43-49) and fragments thereof at least 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length (e.g., SEQ. ID. NOs.: 26, 27, and 33-42), nucleic acids encoding these molecules, vectors having said nucleic acids, and cells having said vectors, nucleic acids, or peptides are also embodiments. A particularly preferred embodiment is a vaccine composition comprising ribavirin and at least one HCV peptide of SEQ. ID. NOs.: 29-32 and 43-49 or a fragment thereof at least 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length (e.g., SEQ. ID. NOs.: 26, 27, and 33-42) or a nucleic acid encoding said peptides or fragments.

Furthermore, compositions having a mixture of the antigens above are embodiments of the invention. For example, some compositions comprise a HBV antigen, a HAV antigen, and ribavirin or a HBV antigen, a HCV antigen, and ribavirin or a HAV antigen, a HCV antigen, and ribavirin or a HBV antigen, a HAV antigen, a HCV antigen, and ribavirin. Other embodiments comprise ribavirin and a nucleic acid encoding a mixture of the antigens described above. Some embodiments also include other adjuvants, binders, emulsifiers, carriers, and fillers, as known in the art, including, but not limited to, alum, oil, and other compounds that enhance an immune response.

Methods of making and using the compositions described herein are also aspects of the invention. Some methods are practiced by mixing ribavirin with a peptide or nucleic acid antigen (e.g., an HAV, HBV, HCV antigen) so as to formulate a single composition (e.g., a vaccine composition). Preferred methods involve the mixing of ribavirin with an HCV antigen that has an epitope present on one or more domains of HCV (e.g., NS3 and/or NS4A).

Preferred methods of using the compositions described herein involve providing an animal in need with a sufficient amount of ribavirin and a hepatitis viral antigen (e.g., HBV antigen, HAV antigen, HCV antigen a nucleic acid encoding one of these antigens or any combination thereof). By one approach, for example, an animal in need of potent immune response to a hepatitis viral antigen (e.g., an animal at risk or already infected with a hepatitis infection) is identified and said animal is provided an amount of ribavirin and a hepatitis viral antigen (either in a single composition or separately) that is effective to enhance or facilitate an immune response to the hepatitis viral antigen. Preferably, an animal in need of a potent immune response to HCV is identified and said animal is provided a composition comprising ribavirin and a peptide comprising an antigen or epitope present on SEQ. ID. NO.: 1, 6, 7, or 17 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 ribavirin and an amount of an HCV antigen (e.g., NS3/4A (SEQ. ID. NO.: 17), mutant NS3/4A SEQ. ID. NOs.: 29-32 and 43-49, or a fragment thereof at least 3, 4-10, 10-20, 20-30, or 30-50 amino acids in length (e.g., SEQ. ID. NOs.: 25-27, and 33-42) or a nucleic acid encoding one or more of these molecules) that is sufficient to enhance or facilitate an immune response to said antigen.

DETAILED DESCRIPTION OF THE INVENTION

It has been 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, or parasite) enhance and/or facilitate an animal's immune response to the antigen. That is, it was discovered that ribavirin is an 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.

Several compositions (e.g., vaccines) that comprise ribavirin and an antigen or epitope are described herein. Vaccine formulations containing ribavirin, for example, can vary according to the amount of ribavirin, the form of ribavirin, and the type of antigen. The antigen can be a peptide or a nucleic acid (e.g., a RNA encoding a peptide antigen or a construct that expresses a peptide antigen when introduced into a subject). Preferred compositions comprise ribavirin and a hepatitis viral antigen (e.g., HAV antigen, HBV antigen, HCV antigen, a nucleic acid encoding these molecules, or any combination thereof). In particular, at least one HCV antigen or an epitope present on SEQ. ID. NO.: 1 or a nucleic acid encoding said HCV antigen are desired for mixing with ribavirin to make said compositions. That is, some embodiments include, but are not limited to, compositions comprising ribavirin and a peptide comprising SEQ. ID. NO.: 1, or a fragment thereof having at least 2500, 2000, 1600, 1200, 800, 400, 200, 100, 50, 10, or 3 consecutive amino acids of SEQ. ID. NO.: 1. Additional embodiments concern compositions comprising ribavirin and a nucleic acid encoding SEQ. ID. NO.: 13 or a fragment thereof having at least 9, 12, 15, 20, 30, 50, 75, 100, 200, 500 consecutive nucleotides of SEQ. ID. NO.: 13.

Other embodiments include a composition (e.g., a vaccine) that comprises ribavirin and a specific fragment of SEQ. ID. NO.: 1, wherein said fragment corresponds to a particular domain of HCV. Some embodiments, for example, comprise a fragment of HCV corresponding to amino acids 1-182, 183-379, 380-729, 730-1044, 1045-1657, 1658-1711, 1712-1971, or 1972-3011 of SEQ. ID. NO.: 1. Compositions comprising ribavirin and a nucleic acid encoding one or more of these fragments are also embodiments of the invention.

Additionally, a novel HCV sequence was discovered. 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.: 16). 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.: 17) 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 are also embodiments of the invention. Further, some of the vaccine embodiments described herein comprise ribavirin and this novel NS3/4A peptide or a fragment thereof at least 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length (e.g., SEQ. ID. NO.: 25) or a nucleic acid encoding one or more of these molecules.

Mutants of the novel NS3/4A peptide were also created. It was discovered that truncated mutants (e.g., SEQ. ID. NO.: 29) and mutants, which lack a proteolytic cleavage site, are highly immunogenic. These novel peptides SEQ. ID. NOs.: 29-32 and 43-49 and fragments thereof at least 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length (e.g., SEQ. ID. NOs.: 26, 27, and 33-42), nucleic acids encoding these molecules, vectors having said nucleic acids, and cells having said vectors, nucleic acids, or peptides are also embodiments. Furthermore, some of the compositions described herein comprise ribavirin and at least one of the mutant HCV peptides described above or a fragment thereof at least 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length. Other vaccine embodiments comprise ribavirin and a nucleic acid (e.g., DNA) encoding one or more of the peptides described above.

Methods of making and using the compositions above are also embodiments. For example, the compositions described above can be made by providing ribavirin, providing an antigen (e.g., a peptide comprising an HCV antigen or a nucleic acid encoding said peptide), and mixing said ribavirin and said antigen so as to formulate a composition that can be used to enhance or facilitate an immune response in a subject to said antigen. Preferred methods entail mixing a preferred antigen or epitope (e.g., a peptide comprising SEQ. ID. NO.: 1, 6, 7, or 17 or specific fragments thereof, such as amino acids 1-182, 183-379, 380-729, 730-1044, 1045-1657, 1658-1711, 1712-1971, 1972-3011 of SEQ. ID. NO.: 1 and nucleic acids encoding these molecules) with ribavirin. Other antigens or epitopes can also be mixed with ribavirin including, but not limited to, fragments of SEQ. ID. NO.: 1 that have at least 2500, 2000, 1600, 1200, 800, 400, 200, 100, 50, 10, or 3 consecutive amino acids and nucleic acids encoding these fragments. Particularly preferred methods 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 four amino acids in length or a nucleic acid encoding one or more of these molecules.

Methods of enhancing or facilitating the immune response of an animal, including humans, to an antigen are embodiments of the invention. Such methods can be practiced, for example, by identifying an animal in need of a potent immune response to an antigen/epitope and providing said animal a composition comprising the antigen/epitope and an amount of ribavirin that is effective to enhance or facilitate an immune response to the antigen/epitope. In some embodiments, the ribavirin and the antigen 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 a hepatitis antigen (e.g., HAV antigen, HBV antigen, HCV antigen, a nucleic acid encoding these molecules, or any combination thereof). Some of these methods involve HCV antigens, such as a peptide comprising SEQ. ID. NO.: 1, or a fragment thereof having at least 2500, 2000, 1600, 1200, 800, 400, 200, 100, 50, 10, or 3 consecutive amino acids of SEQ. ID. NO.: 1. Additional methods involve compositions comprising ribavirin and a nucleic acid encoding SEQ. ID. NO.: 13 or a nucleic acid encoding one or more of the fragments discussed above.

Some preferred methods, for example, concern the use of a composition (e.g., a vaccine) that comprises ribavirin and a peptide comprising SEQ. ID. NO.: 1 or a specific fragment thereof, which corresponds to an HCV domain including, but not limited to, a peptide comprising amino acids 1-182, 183-379, 380-729, 730-1044, 1045-1657, 1658-1711, 1712-1971, or 1972-3011 of SEQ. ID. NO.: 1. Particularly preferred methods concern the use of a vaccine composition comprising the NS3/4A fragment of SEQ. ID. NO.: 17 or the mutant NS3/4A (e.g., SEQ. ID. NOs:. 29-32 and 43-49), which lack a proteolytic cleavage site, or a fragment thereof of at least 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length (e.g., SEQ. ID. NOs.: 26, 27, and 33-42). Compositions comprising ribavirin and a nucleic acid encoding these fragments can also be used with the methods described herein.

Other embodiments concern methods of treating and preventing HCV infection. By one approach, ribavirin and an HCV antigen or epitope 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 or epitope, preferably an epitope present on SEQ. ID. NO.: 1, more preferably a fragment of SEQ. ID. NO.: 1 having at least 2500, 2000, 1600, 1200, 800, 400, 200, 100, 50, 10, or 3 consecutive amino acids or most preferably a fragment of SEQ. ID. NO.: 1 such as 1-182, 183-379, 380-729, 730-1044, 1045-1657, 1658-1711, 1712-1971, or 1972-3011 or a nucleic acid encoding SEQ. ID. NO.: 1 or said fragments above. Particularly preferred methods concern the use of a vaccine composition comprising ribavirin and the NS3/4A fragment of SEQ. ID. NO.: 17 or the mutant NS3/4A, which lacks a proteolytic cleavage site (e.g., SEQ. ID. NOs.: 29-32 and 43-49) or a fragment thereof of at least 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length (e.g., SEQ. ID. NOs.: 25-27, and 33-42) or a nucleic acid encoding one or more of these molecules. The section below discusses the use of ribavirin as an adjuvant in greater detail.

Ribavirin

The compositions described herein can be manufactured in accordance with conventional methods of galenic pharmacy to produce medicinal agents for administration to animals, e.g., mammals including humans. Ribavirin can be obtained from commercial suppliers (e.g., Sigma and ICN). Ribavirin and/or the antigen can be formulated into the vaccine with and without modification. For example, the ribavirin and/or antigen can be modified or derivatized to make a more stable molecule and/or a more potent adjuvant. By one approach, the stability of ribavirin and/or an antigen can be enhanced by coupling the molecules to a support such as a hydrophilic polymer (e.g., polyethylene glycol).

Many more ribavirin derivatives can be generated using conventional techniques in rational drug design and combinatorial chemistry. For example, Molecular Simulations Inc. (MSI), as well as many other suppliers, provide software that allows one of skill to build a combinatorial library of organic molecules. The C2.Analog Builder program, for example, can be integrated with MSI's suite of Cerius2 molecular diversity software to develop a library of ribavirin derivatives that can be used with the embodiments described herein.

By one approach, the chemical structure of ribavirin is recorded on a computer readable medium and is accessed by one or more modeling software application programs. The C2.Analog Builder program in conjunction with C2Diversity program allows the user to generate a very large virtual library based on the diversity of R-groups for each substituent position, for example. Compounds having the same structure as the modeled ribavirin derivatives created in the virtual library are then made using conventional chemistry or can be obtained from a commercial source.

The newly manufactured ribavirin derivatives are then screened in assays, which determine the extent of adjuvant activity of the molecule and/or the extent of its ability to modulate of an immune response. Some assays may involve virtual drug screening software, such as C2.Ludi. C2.Ludi is a software program that allows a user to explore databases of molecules (e.g., ribavirin derivatives) for their ability to interact with the active site of a protein of interest (e.g., RAC2 or another GTP binding protein). Based upon predicted interactions discovered with the virtual drug screening software, the ribavirin derivatives can be prioritized for further characterization in conventional assays that determine adjuvant activity and/or the extent of a molecule to modulate an immune response.

Claim 1 of 14 Claims

What is claimed is:

1. An immunogenic composition comprising nucleic acid molecule encoding a viral antigen polypeptide and ribavirin for co-administration thereof.

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