Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14311—Parvovirus, e.g. minute virus of mice
  • C12N2750/14322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S424/00—Drug, bio-affecting and body treating compositions
  • Y10S424/818—Viral vaccine for canidae or mustelidae, e.g. dogs, foxes, minks

Definitions

• Parvoviruses are characterized as small animal DNA viruses consisting of an isometric protein capsid and a short molecule of single-stranded DNA. Although parvoviruses have been recovered and isolated from various animals, there had been no definite isolation of a pathogenic canine parvovirus until now (Siegl, The Parvoviruses, Springer-Verlay, New York 1976). Bachmann et al. include the dog as a possible parvovirus host in a report detailing the characteristics of parvoviruses in general (Bachmann et al., Intervirology ⁇ Q: in press, 1978). In 1970, Binn et al. reported the recovery and characterization of a "minute virus of canines" (Binn et al., Infect. Immun.
• the principal object of this invention is to provide a vaccine for the protection of dogs against canine parvoviral disease.
• Another object of the present invention is to provide a method of preparing such a vaccine from a strain of parvovirus that can be propagated in primary cells derived from canine and feline species as well as in several established cell lines from various species.
• the isolate recovered is Cornell type strain 780916 (referred to as CPV916) .
• the strain was recovered from the feces and intestinal tract samples of composite specimens submitted for diagnosis from the Argonne National Laboratories on September 9, 1978. Aggregates of the canine parvovirus (CPV) were detected by electron microscopy.
• the fecal material was partially purified by differential ultracentrifugation and diluted in Medium 199 plus Gentamicin (50 r ⁇ g/ml).
• Various non-oncogenic cell cultures were inoculated with this material at the time of cell passage.
• CPV916 The results of cell passage are shown in Table I.
• CPV 916 strain was used as seed for vaccine preparation.
• a non-inhibitory medium opti ⁇ al for each cell type. Infected cells were allowed to grow out, together with uninoculated controls, and were incubated at about 35°C - 37°C for 5 days in a serum medium (for example, MEM-KL0% FCS). Cells then were incubated for an additional two days in a non-serum medium (for example, MEM or Eagles + LAH o.5%).
• a serum medium for example, MEM-KL0% FCS
• a non-serum medium for example, MEM or Eagles + LAH o.5%
• the cells were frozen at -70°C and thawed rapidly three times to disrupt the cells. Ultrasonic treatment may also be used. Culture fluids were clarified by centrifugation at 500 x g for 20 minutes. At this point, the virus harvests were tested for infectivity and hemagglutination (HA) titers.
• the virus grown in MLCL (CCL-64) cells had an infectivity of 10 5.0 TCD 50 /ml.
• HA titers were greater than 1024.
• the virus grown in canine renal cells had infectivity of 10 5.5 TCD 50 /ml and HA of 2048.
• the virus was inactivated by adding formalin (37% formaldehyde), 1:400, to the clarified tissue culture in a tightly stoppered bottle with a magnetic stirrer. The mixture was placed in an incubator at 37oC and mixed well for 48 hours. It was found that the vaccine in the crude form caused no local or systemic reactions in the dogs. Concentrations of formaline, ranging from 0.05 to 0.25 percent, have been found acceptable. Other methods of inactivation, e.g. beta-propio lactone, also may be used in appropriate concentrations by those skilled in the art.
• the vaccine Since the vaccine will be unsatisfactory if any virus is present, it must be tested for residual virus. For this purpose, 0.1 ml of stock 35% sodium bisulfite, diluted 1:2 in PBS, was added to each 5 ml of vaccine. The formalin was removed by dialyzing against PBS, pH 7.2 for 24 hours. To test for virus, 0.5 ml amounts were inoculated into 10 tubes and incubated for 8 days. The presence of virus in inoculated test cultures was tested by hemagglutination tests or fluorescent antibody (FA) tests for viral growth. None was detected.
• FA fluorescent antibody
• isoagglutinins in the canine test serum were greater than 1: 80 it was found necessary to first absorb the serum with 0.1 ml of 50% packed PRC. Serum dilutions commenced at 1:80 and 2-fold serial dilutions then were made using 0.025 ml microdiluters. Antigen (0.025 ml) diluted to contain 4-8 units of HA was added and the mixtures were incubated for one hour at room temperature. The PRC suspension was added, mixed, and the test was incubated at 2oC - 4oC for 2-4 hours before reading. The highest dilution of serum inhibiting HA by 4-8 units of virus (usually 8 units) was considered the endpoint. The titer was expressed as the reciprocal of the endpoint dilution.
• Vaccine trials The vaccine was administered as an aqueous suspension either in adjuvant (Alhydrogel; AL(OH) 3 ) or without adjuvant. A dose of 1 cc was given intramuscularly (IM). A second dose was given 9-10 days later, after the first antibody response was noted. Antibody responses were measured by HA-HI tests, as described above. Experimental specific pathogen-free beagle dogs were challenged intravenously (IV) ten days later with 10 4.0 TCD 50 of virulent CPV. Because the experimental disease is milder than the field cases, in most instances, criteria for illness were elevated termperatures and/or relative lymphopenia, the latter being the most consistent sign.
• Example 1 Example 1
• peripheral white blood cell and lymphocyte counts of the vaccinated and control dogs are shown in Table 4. None of the vaccinated animals developed elevated termperatures, or other signs, in contrast to the control animals. The most prominent and consistent sign in the experimental controls was a reduction in circulating lymphocytes (relative lymphopenia), even when leukopenia was not marked.
• the Cornell type strain 780916 (CPV 916) is on deposit and can be obtained from the James A. Baker Institute for Animal Health, New York State College of Veterinary Medicine at Cornell University, Ithaca, New York. This same strain is also on deposit with the American Type Culture Collection, Rockville, Maryland, U.S.A. under accession number ATCC VR-2006.
• the isolation of the CPV 916 strain of parvovirus and its propagation in primary cells and in cell lines of various species such as mink lung cells and canine kidney cells represent a distinct advance in the art.
• the isolation of CPV 916 is not only the first definite isolation and propagation in vitro of a novel and distinct pathogenic canine parvovirus, but it supplies a method of vaccinating dogs against virulent CPV.

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• Gastroenterology & Hepatology (AREA)
• General Health & Medical Sciences (AREA)
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• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Virology (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)

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• 1978
  • 1978-12-20 US US05/971,296 patent/US4193991A/en not_active Expired - Lifetime
• 1979
  • 1979-12-19 CA CA000342265A patent/CA1138772A/en not_active Expired
  • 1979-12-20 WO PCT/US1979/001112 patent/WO1980001243A1/en not_active Ceased
  • 1979-12-20 NZ NZ192477A patent/NZ192477A/xx unknown
  • 1979-12-20 JP JP55500272A patent/JPS6330287B2/ja not_active Expired
  • 1979-12-20 DE DE19792953417 patent/DE2953417C2/de not_active Expired
  • 1979-12-20 NL NL7920193A patent/NL7920193A/nl unknown
  • 1979-12-20 AU AU54056/79A patent/AU532290B2/en not_active Ceased
  • 1979-12-20 GB GB8025768A patent/GB2048071B/en not_active Expired
• 1980
  • 1980-07-01 EP EP80900176A patent/EP0020743A1/en not_active Withdrawn
  • 1980-08-19 SE SE8005824A patent/SE423312B/sv not_active IP Right Cessation

Non-Patent Citations (8)

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