Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Virus or component thereof
Reexamination Certificate
2001-04-23
2004-04-20
Mosher, Mary E. (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Virus or component thereof
C435S235100, C435S236000
Reexamination Certificate
active
06723325
ABSTRACT:
BACKGROUND OF THE INVENTION
Variola virus, the causative agent of smallpox, is a member of the Orthopoxvirus genus, which also includes monkeypox, cowpox, and vaccinia viruses. The disease caused by variola major strains is characterized by a low infectious dose (10-100 virions), long incubation period (averaging 12 days), fever, constitutional symptoms, rash progressing to a pustular stage, death in up to 30% of those affected, and facial scarring in survivors. The disease is spread person-to-person via the respiratory route by contact (droplets) and, possibly, by aerosol.
Smallpox was one of the most important causes of morbidity and mortality worldwide throughout the first half of the 20
th
century. However, in part because of the lack of animal reservoir for the virus, the systematic use of a vaccine (live, attenuated vaccinia virus) was highly effective in fighting this disease. Indeed, between 1967-1977, a global program of smallpox eradication resulted in the elimination of the natural disease (Fenner et al., WHO, Geneva, p. 1460, 1988). Because of the absence of smallpox and the risk of vaccine-associated adverse events, routine vaccination of children, hospital personnel, and military personnel has ceased, and only persons working with vaccinia and related viruses in the laboratory are currently immunized. Thus, a substantial portion of the world's population has no immunity to smallpox. The remaining population has little residual immunity, as vaccine immunity lasts only 5 years after primary vaccination and less than 20 years after revaccination. The eradication of smallpox and the cessation of vaccination have, thus, created vulnerability in the population to covert attack or biowarfare employing variola virus. Should such an event occur, epidemic spread would be unchecked by an immune barrier in the population (Anon. (Editorial), Lancet 353:1539, 1999; Henderson, Science 283:1279-1282, 1999; Henderson et al., J.A.M.A. 281:2127-2137, 1999).
Because of the uncertainties surrounding smallpox eradication, vaccine was stockpiled for emergency use. In the United States, for example, 155,000 vaccine vials (nominally 15.5 million doses) produced by Wyeth Laboratories were originally stockpiled under the control of the Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, U.S. At a meeting of the National Vaccines Advisory Committee in January 1999, the CDC reported on the status of the national smallpox vaccine repository. At that time, of the 15.5 million doses held by Wyeth, 3.4 million doses had failed quality control testing and 10.3 million were beyond the expiration date specified by the last control test for extended dating, leaving 1.7 million doses that met release specifications (LeDuc, Presentation to the National Vaccines Advisory Committee, Washington D.C., Jan. 11-12, 1999). In addition to the limited supply, the vaccine is packaged in 100 dose vials, which restricts distribution and increases the likelihood of wastage during an emergency.
In addition to the U.S. stockpile, there is a supply of vaccine (Lister, Elstree strain) stored at the National Institute of Public Health, Bilthoven, Netherlands, and certain other countries have supplies of smallpox vaccine, which at the time of eradication may have included up to 300 million doses. However, similar problems of stability in storage have reduced this supply to less than 50 million doses (Henderson, Science 283:1279-1282, 1999).
SUMMARY OF THE INVENTION
The invention provides stable strains of vaccinia virus that are isolated from cultured cells in which Dryvax® has been propagated, and which have characteristics that make them suitable for use as human vaccines against smallpox. The invention also provides methods of generating these strains and methods of using them to prevent smallpox infection and disease.
Accordingly, in a first aspect, the invention provides a clonal strain of attenuated vaccinia virus that is isolated from cultured cells in which Dryvax® has been cultured and, when administered to a human in an amount effective to induce a protective or therapeutic immune response against variola virus in the human, is acceptably attenuated in the human.
The clonal strains can have, for example, substantially the same virulence and/or immunogenicity as Dryvax®. Preferably, the vaccinia virus is produced in substantially the same or greater amounts as Dryvax® when inoculated into cell cultures, and/or has substantially the same digestion pattern as Dryvax® when digested with a restriction endonuclease.
The clonal strain can also have, for example, substantially the same virulence and/or immunogenicity as vaccinia virus strain ACAM1000 (deposited as ATCC Deposit No. PTA-3321 on Apr. 20, 2001; see clone
2
, below) when tested in appropriate animal models or in humans. Preferably, such a vaccinia virus is produced in substantially the same or greater amounts as vaccinia virus strain ACAM1000 when inoculated into cell cultures, and/or has substantially the same digestion pattern as vaccinia virus strain ACAM1000 when digested with a restriction endonuclease. One example of a vaccinia virus that is included in the invention is ACAM1000 (ATCC Deposit No. PTA-3321).
In a second aspect, the invention provides a pharmaceutical composition including a clonal strain of vaccinia virus, as described above and elsewhere herein, and a pharmaceutically acceptable carrier or diluent.
In a third aspect, the invention provides a method of preventing or treating variola virus infection in a patient by administering such a pharmaceutical composition to the patient. The pharmaceutical composition can be administered to the patient by, for example, scarification, in an amount ranging from, for example, 1×10
4
to 1×10
6
plaque-forming units.
In a fourth aspect, the invention provides a method of obtaining a clonal strain of attenuated vaccinia virus for use as a vaccine. This method involves (i) propagating Dryvax® in a cell culture system, and (ii) isolating from the cell culture system a clonal strain of vaccinia virus that has substantially the same virulence, immunogenicity, growth characteristics in culture, or restriction endonuclease digestion pattern as Dryvax® or vaccinia virus strain ACAM 1000. The virulence of the vaccinia virus can be tested in this method by, for example, a rabbit skin test or a suckling mouse neurovirulence test. Growth characteristics in culture can be determined using, e.g., human diploid (MRC-5) cells. Preferably, the vaccinia virus identified using this method, when administered to a human in an amount effective to induce a protective or therapeutic immune response against variola virus in the human, is acceptably avirulent in the human.
The invention provides several advantages. For example, previously, smallpox vaccine was produced by inoculation of vaccinia virus into the skin of calves, followed by scraping of the skin of the calves to harvest live virus. The crude virus preparation obtained underwent minimal purification before use in vaccinating human recipients, leaving open the possibility of pathogen contamination. The vaccines of the present invention are produced in a cell culture system that is acceptable by modem standards for vaccine manufacture and is highly purified, thus eliminating this problem. An additional advantage of using cloned viruses, such as those of the present invention, is that the characteristics of such viruses are unlikely to change during propagation and vaccine manufacturing, as compared to mixed populations of viruses. Indeed, we have shown that a virus according to the invention maintains its phenotype under repeated passage and expansion in cell culture, is free from contaminants, and is capable of being produced in cell culture in amounts suitable for large-scale vaccine manufacture.
REFERENCES:
patent: 4567147 (1986-01-01), Ooi et al.
patent: 5656465 (1997-08-01), Panicali et al.
R. Jennings et al. “Virus Vaccines”. In: Virus Culture, a Practical Approach, ed. A.J. Cann, Oxford University Press, New York, 1999,
Monath Thomas P.
Weltzin Richard A.
Acambis Inc.
Clark & Elbing LLP
Mosher Mary E.
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