Drug – bio-affecting and body treating compositions – Nonspecific immunoeffector – per se ; or nonspecific...
Reexamination Certificate
2003-04-15
2004-06-22
Mosher, Mary E. (Department: 1648)
Drug, bio-affecting and body treating compositions
Nonspecific immunoeffector, per se ; or nonspecific...
C424S232100
Reexamination Certificate
active
06752996
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the use of B2L and PP30 proteins of a Parapox virus as a vaccine adjuvant.
BACKGROUND OF THE INVENTION
Cutaneous Parapoxvirus ovis causes recruitment of epidermal dendritic cells to the infection site in sheep and subsequent cell-mediated immunity (Lear et al., Eur. J. Dermatol. 6, 135-40, 1996; Haig et al., Comp Immun. Microbiol. Infect. Dis. 20 197-204, 1997). Attenuated Parapox viruses can be used to induce Paradox-specific immunity. U.S. Pat. No. 6,162,600. In addition, the highly attenuated strain D1701 (Baypamun HK®) is used as a non-specific immunomodulator (Buttner et al., Immunol. Microbiol. Infect. Dis. 16, 1-10, 1993) to promote immunity to heterologous pathogens.
Attenuation of Parapox virus, however, is time-consuming, taking from 100 to 200 culture passages; according to WO 95/22978, it takes from three to five years to perform each 100 passages, depending on the species of virus used. Attenuation can, therefore, “encompass a period lasting from ten to twenty years.” See WO 95/22978, page 9.
WO 95/22978 discloses the use of combinations of two or more individual Parapox virus components as “multipotent paramunity inducers” for use as adjuvant therapy for tumors and the prevention of metastases. The components can be individual polypeptides or detached envelopes of poxviruses. WO 95/22978, however, does not disclose any particular viral polypeptides other than the viral fusion protein and adsorption protein. Moreover, WO 95/22978 teaches that the disclosed paramunity inducers have virtually no immunogenic properties.
There is a need in the art for simple, effective vaccine adjuvants that can be used to enhance immune responses against tumors and dysplastic lesions and against exogenous pathogens.
SUMMARY OF THE INVENTION
It is an object of the invention to provide reagents and methods for modifying immune responses to administered antigens. This and other objects of the invention are provided by one or more of the embodiments described below.
One embodiment of the invention provides a method of enhancing an immune response to a vaccine composition. The method involves administering to a subject in need thereof (a) an effective amount of a B2L viral envelope protein of a Parapox virus and (b) a vaccine composition comprising an active component. The adjuvant B2L viral envelope protein thereby enhances the immune response to the vaccine composition.
Another embodiment of the invention provides a pharmaceutical composition comprising a B2L viral envelope protein of a Parapox virus and a vaccine composition comprising an active component.
Yet another embodiment of the invention provides a pharmaceutical composition comprising a nucleic acid molecule encoding a B2L viral envelope protein of a Parapox virus and a vaccine composition comprising an active component.
Thus, the invention provides pharmaceutical compositions and methods using B2L protein to modify immune responses to administered antigens.
DETAILED DESCRIPTION OF THE INVENTION
The invention is based on the ability of a Parapox viral envelope protein termed “B2L” to act as an adjuvant, i.e., to augment or otherwise modify a subject's immune response to an administered antigen and/or an active component of a vaccine. As noted below, the descriptions herein are also applicable to a second Parapox immunostimulatory polypeptide, PP30, and references herein to B2L are understood to encompass both proteins, absent contrary implication. Administered antigens include, but are not limited to, cells expressing tumor antigens, attentuated or killed pathogens and antigenic components thereof or nucleic acids encoding the antigenic components.
Both antibody and cellular immune responses can be modified. B2L protein is particularly useful as an adjuvant for poorly immunogenic tumor antigens and subunit vaccines, such as those useful for preventing and/or treating flu, tuberculosis, respiratory syncytial virus, anthrax and HIV.
B2L is the second open reading frame in the BamHl B fragment of the Orf virus genome (Sullivan et al., Virology 202, 968-73,1994). Prior work teaches that, as the activity of epitopes responsible for antigen-specific immunization decrease, adjuvant activity of the preparations increases. See WO 95/22978, page 4. B2L is an immunogenic protein; in fact, B2L protein is one of a few Orf virus proteins to which a strong antibody response can be mounted in sheep. Sullivan et al., 1994. Thus, it is surprising that purified B2L protein itself has adjuvant activity.
B2L proteins for use in the compositions and methods described herein are those of the Parapoxvirus genus, such as Orf virus (OV), particularly the Parapox ovis strains NZ2, NZ7, NZ10, and D1701. Orf viruses are reviewed in Robinson & Balassu, Vet. Bull 51, 771, 1981; Robinson & Lyttle, in Binns & Smith, eds., recombinant poxviruses, Chapter 9, pp. 306-17, CRC Press, Boca Raton, 1992. An amino acid sequence for the B2L protein of OV NZ2 is disclosed in Sullivan et al., Identification and characterization of an orf virus homologue of the vaccinia virus gene encoding the major envelope antigen p37K, Virology 202 (2), 968-73, 1994, and is shown in SEQ ID NO:2. A coding sequence for SEQ ID NO:2 is shown in SEQ ID NO:1. The amino acid sequences of the B2L proteins obtained from D1701 and NZ2 are highly conserved. The amino acid sequence of the D1701 protein is shown in SEQ ID NO:4. A coding sequence for SEQ ID NO:4 is shown in SEQ ID NO:3.
Purified B2L protein is separated from other compounds that normally associate with the B2L protein in the virus, such as other envelope components. A preparation of purified B2L protein is at least 80% pure; preferably, the preparations are 90%, 95%, or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis.
Purified B2L protein for use in compositions and methods of the invention can be purified from Parapox viruses or from cells infected by the viruses, by recombinant DNA methods, and by chemical synthesis. Purification methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis.
B2L protein can be expressed recombinantly, after insertion of B2L coding sequences into an expression vector that contains the necessary elements for the transcription and translation of the inserted coding sequence. Maintenance of orf viruses in culture is disclosed in WO 97/37031. A preferred system for maintaining and expressing B2L protein is HKB11 cells transfected with B2L in a vector such as a p2ToP, pCEP4, or pcDNA3.1 vector (Invitrogen). Recombinantly produced B2L protein can be secreted into the culture medium and purified. Methods for producing proteins recombinantly are well known to those skilled in the art.
A B2L protein also can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrifield, J. Am. Chern. Soc. 85, 2149-2154, 1963; Roberge et al., Science 269, 202-204,1995). Protein synthesis can be performed using manual techniques or by automation. Optionally, fragments of a B2L protein can be separately synthesized and combined using chemical methods to produce a full-length molecule.
“B2L protein” as used herein includes both functional portions of B2L and full-length or partial biologically active B2L variants. Biologically active variants (i.e., variants that possess adjuvant activity) comprise amino acid substitutions, insertions, and/or deletions with respect to the amino acid sequences shown in SEQ ID NOS:2 or 4. Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine. Biologically
Johnston Stephen A.
McGuire Michael J.
Board of Regents , The University of Texas System
Mosher Mary E.
Osman Richard Aron
LandOfFree
Use of parapox PP30 protein to modify immune responses to... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Use of parapox PP30 protein to modify immune responses to..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Use of parapox PP30 protein to modify immune responses to... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3321222