The invention provides attenuated vaccinia virus vaccines that can be used in methods to prevent or treat small pox in patients, as well as methods of obtaining such vaccines.

BACKGROUND OF THE INVENTION

This invention relates to methods and compositions for use in vaccination against smallpox.

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.sup.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, Ga., 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.RTM. 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.RTM. 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.RTM.. Preferably, the vaccinia virus is produced in substantially the same or greater amounts as Dryvax.RTM. when inoculated into cell cultures, and/or has substantially the same digestion pattern as Dryvax.RTM. 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. 19, 2001 with the American Type Culture Collection, P.O. Box 1549, Manassas, Va. 20108, U.S.A.; 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.times.10.sup.4 to 1.times.10.sup.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.RTM. 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.RTM. or vaccinia virus strain ACAM1000. 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 modern 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.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides clonal strains of attenuated vaccinia viruses that can be used in vaccination methods against smallpox (i.e., variola virus). As is described further below, the attenuated vaccinia strains of the invention are obtained by isolating vaccinia clones from cell cultures in which Dryvax.RTM. has been propagated. The invention also provides methods of using vaccines that include these vaccinia viruses in the prevention of smallpox, as well as methods of obtaining such clonal strains of vaccinia viruses.

The vaccines of the invention are derived from, and have similar characteristics to, Dryvax.RTM. (New York City Board of Health strain, Wyeth Laboratories), which is currently licensed by the U.S. Food and Drug Administration (FDA) and consists of a mixed population of vaccinia viruses generated in calf skin. The vaccines must have acceptably attenuated virulence for humans who are vaccinated with them. An acceptable level of attenuation can be, for example, a level that is similar to (e.g., does not differ in a statistically significant manner from) that observed with Dryvax.RTM., and can be determined using any of the in vitro or in vivo tests described below. A property of vaccinia virus is its neurotropism, or ability to replicate in cells of the central nervous system, causing inflammation (i.e., encephalitis). Preferably, the vaccines of the invention are not more neurotropic than Dryvax.RTM. and do not cause postvaccinal encephalitis in treated patients.

The vaccines and methods of the present invention are described further below.

Indications for Use

The principal indication for use of the vaccines of the invention is in the prevention of smallpox in populations exposed or potentially exposed to smallpox after an act of bioterrorism or biowarfare. Efficacy of the vaccines of the invention advantageously is high (>95%), and the vaccines protect against both person--person spread of the virus and primary exposure to high-dose aerosol exposure to biological weapons. Given this principal indication, the vaccines of the invention can be used, for example, to create a new national stockpile of smallpox (vaccinia) vaccine, and manufacture can be continued annually to maintain a continuous stock of in-date vaccine for an extended period of time.

The vaccines are not intended for routine use, except in laboratory workers who are exposed to vaccinia, cowpox, monkey pox, variola, or other members of the Orthopoxvirus genus. Otherwise, the vaccines are to be released under emergency conditions, as determined by the national security and public health authorities. Under the circumstances of such an emergency, the risks of adverse events associated with vaccinia would be outweighed by the potential benefits of protecting individuals against smallpox and society against spread of the disease. It is recognized that emergency use of the vaccines may be difficult to control, that infants, who are at higher risk of postvaccinal encephalitis, will receive the vaccines, and that precautions and contraindications for use in persons with underlying conditions (e.g., history of eczema, pregnancy, and immunosuppression) may be ignored. For these reasons, it is important that the cell culture-derived vaccines of the invention are not more virulent than the currently licensed product.

Depending upon events that cannot be accurately predicted, there may be a decision to undertake pre-exposure prophylaxis of certain groups, including military personnel, civilian medical personnel, and so-called `first responders.` The inherent safety profile of the vaccines in these groups, while of great importance, is enhanced by deliberate application of the product and avoidance of use in individuals with risk factors for adverse events. Under these circumstances, the principal risks are autoinoculation, ocular vaccinia, and accidental infection, all of which are self-limited adverse events. There is a small risk of accidental infection of others with underlying risk factors.

Of course, should circumstances in the country or world change such that routine vaccination of additional members of the population (e.g., children), or even the entire population, is thought to be desirable, the vaccines of the present invention can be used for these purposes as well.

Modes and Amounts of Administration

The vaccines of the invention are prepared by propagation of a desired strain of vaccinia virus (e.g., strain ACAM1000; ATCC Deposit No. PTA-3321; see below) in a cell culture system, and purification of the cultured strain from the system using standard methods. For example, the strain can be cultured in diploid human lung fibroblast cells, such as MRC-5 cells, primary chick embryo fibroblast cells, or any other appropriate cell type, as can be determined by one of skill in this art. The culture can take place using any appropriate system such as, for example, the Nunc Cell Factory.RTM..

Purified virus can be lyophilized for later use or can be immediately prepared in a pharmaceutical solution. Numerous pharmaceutically acceptable solutions for use in vaccine preparation are well known in the art and can readily be adapted for use in the present invention by one of skill in this art. (See, e.g., Remington's Pharmaceutical Sciences (18.sup.th edition), ed. A. Gennaro, 1990, Mack Publishing Co., Easton, Pa.) However, the viruses can simply be diluted in a physiologically acceptable solution, such as sterile saline or sterile buffered saline, with or without an adjuvant or carrier. Optionally, the pharmaceutical solution can contain a component that provides viscosity (e.g., glycerol) and/or a component that has bactericidal properties (e.g., phenol). The vaccines can be stored at a concentration of 10.sup.7 10.sup.9 plaque-forming units (PFU)/ml, for example, 10.sup.8 PFU/ml.

The vaccines of the invention can be administered to patients by, for example, scarification, using standard methods. For example, a bifurcated needle can be used in such an approach. Alternatively, the vaccine can be administered using any other standard route that is found to be acceptable by one of skill in this art. For example, the vaccine can be administered by subcutaneous or intradermal injection, or by another parenteral route, such as by intramuscular injection. The amount of vaccine administered to an average-sized adult can be, for example, 1.times.10.sup.4 to 1.times.10.sup.6 plaque-forming units. As a specific example, 2.5.times.10.sup.5 plaque-forming units can be used.

Preferably, vaccination is carried out before any exposure to variola, but vaccination can also be carried out with patients who have been exposed to variola, preferably within a few days of the exposure. Vaccination can be carried out only once in a person's lifetime or can be repeated after a period of time, such as several years (e.g., 5 10 years), as determined to be appropriate by one of skill in this art.

Identification of Vaccine Candidates

The invention also includes methods of identifying vaccinia vaccine candidates. These candidates can be identified by isolating clonal strains from cell cultures inoculated with Dryvax.RTM., and characterizing these clones using any of the in vitro or in vivo methods described below. For example, a candidate vaccine strain can be compared with Dryvax.RTM. for plaque size, yield in cell culture (using, e.g., MRC-5 cells), rabbit cutaneous virulence, suckling mouse neurovirulence, monkey neurovirulence, or protection in a mouse challenge model. Preferred candidates are those with virulence that is similar to or less than that of Dryvax.RTM., which induce protective immunity that is similar to or greater than that of Dryvax.RTM., and also have growth characteristics that are similar to or greater than those of Dryvax.RTM..

Prior to the present invention, isolation of a clonal strain that has satisfactory characteristics of a vaccine candidate was unpredictable, because the long history of passage of vaccinia has resulted in the generation of multiple subpopulations of variants (i.e., a genetic swarm), with potentially different biological properties. It was also uncertain whether a single variant, isolated by plaque purification (i.e., biological cloning) would have the same phenotypic characteristics as the sum of the multiple variants in the original mixed virus population. In fact, prior to the present invention, it would have been surprising if this were the case.

 

Claim 1 0f 18 Claims

1. A clonal strain of attenuated vaccinia virus isolated from cell cultures in which ACAM1000 (ATCC Deposit No. PTA-3321) or a progeny strain thereof has been propagated.

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