Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
2001-05-14
2003-11-04
Housel, James (Department: 1648)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C435S069100, C530S300000
Reexamination Certificate
active
06642359
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to polypeptides useful for diagnosis and both preventive and prophylactic treatment of feline infectious peritonitis virus disease. More specifically, the invention relates to new recombinant feline coronavirus S proteins and fusion proteins.
BACKGROUND OF THE INVENTION
Feline Infectious Peritonitis (FIP) is a highly lethal disease in both wild and domestic cats, occurring predominantly in young animals although cats of all ages are susceptible. Symptoms of FIP may include anemia, neutrophilia, increased concentrations of immunoglobulin and/or fibrinogen, renal damage as indicated by high levels of urea and creatinine, and disseminated intravascular coagulation.
Previous attempts to develop an effective FIPV vaccine have been largely unsuccessful. Administration of traditional inactivated whole virus vaccines have actually predisposed cats to the development of FIP and produced a more rapid and fulminating disease after challenge. Cats vaccinated with an avirulent strain of FIPV were more readily infected than non-immunized cats and animals immunized with a sublethal dose of virulent FIPV showed inconsistent protection from challenge [Pedersen and Black,
Am. J. Vet. Res.,
44:229-234 (1983)].
Immunization of cats with other antigenically related coronaviruses has also not been successful. In most experiments, the administration of TGEV, CCV and human coronavirus 229E has neither sensitized nor protected cats [Woods and Pedersen,
Vet. Microbiol.,
4:11-16 (1979); Toma et al,
Rec. Med. Vet.,
155:788-803 (1979); Barlough et al,
Can. J. Comp. Med.,
49:303-307 (1985); Barlough et al,
Lab. Anim. Sci.,
34:592-597 (1984); Stoddart et al,
Res. Vet. Sci.,
45:383-388 (1988)].
Recently, a temperature-sensitive FIPV (TS-FIPV) vaccine has been developed which, when administered intranasally, is efficacious and safe upon FIPV challenge [Christianson et al,
Arch. Virol.,
109:185-196 (1989)]. This vaccine has limited efficacy when administered subcutaneously, but appears to be effective against homologous and heterologous strains. Generally, intranasal administration is not preferred because the dosage amount is less quantifiable than other routes.
There remains a need for effective diagnostic, therapeutic and protective compositions for use in diagnosing, treating, and vaccinating animals against FIPV and serologically related infections.
SUMMARY OF THE INVENTION
In one aspect, the invention provides protein and peptide fragments of a feline coronavirus S gene. These peptides may be expressed recombinantly or synthetically and are useful as diagnostic, therapeutic or vaccinal components. In one embodiment, the feline coronavirus S-derived peptides fall within the range of amino acid numbers 1 to about 1454 of the S genomes of a variety of FIPV strains and 1 to about 1454 of the FECV S genome, or smaller peptide fragments therein. In a preferred embodiment, the feline coronavirus S-derived peptides fall within the range of amino acid numbers 1 to about 748 of the S genes of the FIPV strains or 1 to about 748 of the FECV S genome [SEQ ID NO: 32]. More particularly, peptides falling within the range of about amino acid #94 to about amino acid #223 of the FIPV or FECV S genomes are desirable. In a particularly preferred embodiment, the feline coronavirus S-derived peptides are found to be within the range of amino acid #97-222 of the FIPV or FECV S genomes. In still another embodiment, peptides falling within the range of about amino acids #121 to about amino acid #180 of the FIPV or FECV genome are disclosed.
Peptide fragments of the invention are capable of distinguishing between FIPV and FECV, or different strains of FIPV when used in diagnostic assays, such as enzyme linked immunosorbant assays (ELISA) or Western Blots. These peptides may also be used as antigens to screen cat sera for the presence of antibody or to generate antibodies capable of distinguishing between FIPV and FECV, or different strains of FIPV.
In another aspect, the present invention provides nucleotide sequences from FIPV and FECV within the regions of nucleotide #1 to about #4365 and #1 to about #2246, which encode the above-described peptides, or which flank the above-described peptide-encoding sequences. These nucleotide sequences are capable of distinguishing between the FIPV and FECV S genomes, when they are used in diagnostic assays as PCR primers or hybridization probes.
Another aspect of the invention provides novel recombinant FIPV or FECV S fusion proteins. The feline coronavirus S-derived peptides of the present invention may be fused with a selected protein which confers a desired advantage upon recombinant expression of the S peptide. For example, the fusion partner may be a protein which is highly expressed in the desired host cell system or which is characterized by a high degree of secretion. The fusion partner may also be a signal sequence or a sequence which enhances the stability of the S-derived peptide in a selected host cell system. In one embodiment of this aspect, peptides derived from the S gene of feline coronavirus are fused with the N-terminal 52 amino acids of galactokinase (GalK).
In another aspect the present invention provides a diagnostic reagent composition which comprises an FIPV S-derived peptide or fusion protein of the present invention, optionally associated with a detectable label. Such diagnostic reagents may be used to assay for the presence of FIPV or FECV in cats using standard assay formats.
In a similar aspect the present invention provides a diagnostic reagent composition which comprises a nucleotide sequence encoding or flanking an FIPV S-derived peptide or fusion protein of the present invention, the DNA sequence being optionally associated with a detectable label. Such diagnostic reagents may be used to assay for the presence of FIPV or FECV in cats in hybridization assays or in the PCR technique.
In still another aspect of the present invention, the S-derived peptides and/or the S-derived fusion proteins may be utilized as the active component in vaccines to protect animals against infection with FIPV or FECV. A vaccine composition includes an effective amount of an FIPV or FECV S-derived peptide or fusion protein of the present invention capable of stimulating immunity against one or more virulent feline coronaviruses and a carrier suitable for internal administration. Additionally, characterization of the immune response to these peptides and proteins may also suggest other region(s) of the FIPV or FECV sequences which should be included in vaccines.
In yet a further aspect, the present invention provides a pharmaceutical composition for the treatment of FIPV or FECV infection comprising a therapeutically effective amount of a FIPV or FECV S-derived peptide or fusion protein of the invention and a pharmaceutically effective carrier.
In still another aspect, the invention provides a diagnostic kit which may be used by veterinarians to identify cats which are uninfected or which have been exposed to FECV or native FIPV. The kit will also allow the identification of cats which have been vaccinated against these diseases. Such a kit may also allow one to distinguish between different strains of FIPV, or to identify cats at advanced stages of FIPV infection. The kit may be comprised of PCR primers of this invention selected from the S gene nucleotide sequences; a selected FIPV S-derived peptide or fusion protein; primers, peptides and fusion proteins of related or similar viruses, and primers, peptides and fusion protein-encoding regions from a “consensus” sequence as described below.
In a further aspect, the invention provides a method of using the PCR S-derived primers and/or the S-derived peptides and fusion proteins of this invention to identify previously exposed and naive cats, as well as to differentiate exposure to FIPV from exposure to other related coronaviruses. Another diagnostic method of this invention permits the use
Jones Elaine V.
Klepfer Sharon R.
Miller Timothy J.
Pfeiffer Nancy E.
Reed Albert Paul
Hill Myron G.
Housel James
Pfizer Inc.
Scully Scott Murphy & Presser
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