Link:  Pharm/Biotech Resources

Title:  Oral vaccinia formulation

United States Patent:  6,960,345

Issued:  November 1, 2005

Inventors:  Moyer; Mary Pat (San Antonio, TX)

Assignee:  Incell Corporation, LLC (San Antonio, TX)

Appl. No.:  379572

Filed:  March 6, 2003

This invention relates to methods and systems for generating a safe and effective oral smallpox vaccine for humans using a genetically defective strain of vaccinia virus to confer immunity following oral delivery of the vaccine. This invention is one that expands on current use of vaccinia virus propagation developed for gene therapy applications, and pharmaceuticals and nutraceuticals packaging and formulation technologies. The vaccine invention can be delivered as a live virus with the ability to express viral proteins but unable to achieve complete, lytic virus replication, or it may be derived from such a virus, contain additional immunogens, or be delivered as viral antigens. Furthermore, the invention establishes innovative methods for formulation and packaging and for preclinical testing of the vaccine invention for safety, efficacy and potency with the use of human intestinal and other test cells and diagnostic test systems and kits.

SUMMARY OF THE INVENTION

The oral vaccine system and methods described herein use a live, defective vaccinia virus or a viral antigen preparation of such a virus, that can confer anti-smallpox immunity in the recipient. The invention encompasses the combined methods by which the virus is grown using in vitro cell culture methods (e.g., the methods of growing the I-MVA in the baby hamster kidney cell line, BHK-21, followed by steps to purify the virus, and quantitate the dosage), the characterization bioassays for its safety and efficacy prior to clinical use by oral delivery as per the immunization protocol, and the methods and components used for formulation. Cells used for vaccine preparation are derived from INCELL's reference Master Cell Bank (MCB) and Working Cell Bank (WCB: n>200 vials) stocks. The MVA virus is propagated, for example, on BHK-21 cells that are cultured to high culture density on microcarrier beads in plastic cell culture bags.

The vaccinia virus used for the vaccine can derive from the I-MVA strain or other defective vaccinia virus (DVV) strain incapable of generating infectious virus in a complete lytic cycle in human cells, but able to replicate in an animal host cell which is permissive for the virus.

Safety, efficacy and potency components of the invention include in vitro and immunoassays to evaluate the potential safety and potency using surrogate endpoint assays, such as infection of human intestinal cells, or other defined alimentary tract epithelial cells, and cell mediated immune (CMI) responses of cells from anti-vaccinia immunized individuals. The CMI responses can include bioassays for cytokines, cytotoxicity or other in vitro methods that reflect what would occur in vivo.

The vaccine might be packaged in various forms, including packaging in a liquid, gel, or solid form that may be a tablet or gelcap or a component of a food carrier material, such as a pudding or yogurt. In particular the live vaccine would require packaging in a form that would allow delivery into the human alimentary tract as whole virions that could be taken up in at the first part of the alimentary tract, i.e., the oral cavity, or at other sites, such as the intestine.

Various embodiments of the invention disclosed herein employ vaccinia virus, which was developed for gene therapy applications and has been used as a vector to deliver genes, (e.g., tumor or microbial antigen genes), to the host as a live vaccine and carrying information intended to confer immunity on the host by expression of the delivered gene.

The principle use of the oral vaccine system will be to protect against potential poxvirus infection, including smallpox, but incorporation of other genes into the vaccinia virus vector is envisaged, such that multi-valent vaccine(s) against a variety of potential bio-agents, potential pathogens, or products of pathogens (e.g., toxins) can be prepared for oral delivery as disclosed herein. These can be packaged as separate packages or may be in the same vector. They may be in a single package or multiple packages, as another use of the invention.

DETAILED DESCRIPTION OF THE INVENTION

A. Manufacturing Methods and Materials

1. Facilities and Standards

All cells, virus and reagents are handled according to cGMP (current Good Manufacturing Practice) standards. The manufacturing facility (anteroom, class 10,000 room, class 100 hoods) staff will use SOPs that meet FDA testing, validation, and QA/QC manufacturing standards. These measures are taken to accelerate the process from discovery to product.

2. The BHK-21 Clone 13 (BHK21-CL13} Cell Line

The BHK-21 CL 13 cell line (ATCC #CCL-10) is used as the permissive cells to propagate the stock virus. The rationale for choosing this line is that it is permissive for DVV such as MVA and is easily grown in culture. An example showing BHK-21 cells growing in culture is shown in FIG. 1. Cells are maintained as recommended by the ATCC in a modified Minimal Essential Medium with Earle's Salts [EMEM], 0.1 mM non-essential amino acids, and 1 mM sodium pyruvate (e.g., GIBCO or other vendor) with 10% v/v fetal bovine serum (FBS; e.g., Hyclone or other vendor), or another suitable growth medium. They are subcultured using 0.25% trypsin, 0.03% EDTA (GIBCO) at subcultivation ratios of 1:2 to 1:10. Cells used for vaccine preparation are derived from INCELL's reference Master Cell Bank (MCB) and Working Cell Bank (WCB; n≧200 vials) stocks. The banked cells have been checked for sterility by standard microbial growth and mycoplasma PCR assays of the MCB and WCB and characterized by DNA fingerprinting to assure identity.

3. Virus Propagation and Analyses

The INCELL propagated strain of MVA (ATCC #VR01508), designated I-MVA, has been routinely propagated by standard methods and titered by the preferred method of immunoplaque assay as detailed below. Other quantitative methods that have been used include either end point dilution in BHK-21 cells to obtain a 50% tissue culture infectious dose (TCID50/ml) or IU (infectious units), as detailed by Dresden et al. [15]. For in vitro and in vivo assays, virus has been purified by ultracentrifugation through a 36% sucrose cushion using standard virus purification methods. The BHK-21 cells are grown in culture (also termed "in vitro") and infected at 0.1 FFU per cell to generate large lots of virus harvested at 72+/-2 hours post infection (p.i). The BHK-21 cell cultures can be monolayers in various types of bioreactor or scale-up cultures, including culture flasks, stacked systems, culture microcarrier beads, or other appropriate substrates to culture the cells. The resultant virus stocks can be concentrated or purified by ultracentrifugation, ultrafiltration, or other standard methods, then titered on BHK-21 cells and stored for packaging. Part of the stock is aliquoted for use in the bioassays and for QA testing. All lot information is entered into the master database and inventory management system which were developed as part of the invention and its use.

For immunoplaque assays, BHK-21 cells are seeded at a density of 4.5×10⁴ cells per well of a 24-well plate in growth medium (EMEM, 10% FBS, plus additives, as described above). After overnight attachment, when the cells are 80-90% confluent, they are infected with I-MVA by mixing the virus with EMEM prepared as per the growth medium but with 2% rather than 10% FBS (=EMEM:2 infectivity medium). Test I-MVA source materials are generally diluted in 10^-3 to 10^-7 for cell culture derived, unconcentrated supernatants, and 10^-6 to 10^-10 for gradient centrifugation purified or otherwise concentrated (e.g., ultrafiltered) virus. Dilutions are made in EMEM:2 infectivity medium. Cells that receive no virus but are otherwise incubated with EMEM:2 infectivity medium and treated the same are used as negative controls. Reference virus stocks that are known to produce 100-200 plaques per well are included in the assays as positive controls to assure performance of the assay. Cultures are gently swirled to assure even virus distribution then incubated for 24 hr at 37° C., in a 5% CO₂, 95% air environment. At the end of the incubation period, the medium is removed from the wells, the cells are fixed with 0.5 ml 1:1 acetone:methanol for 5 min, the fixative is removed and 1 ml CMF-PBS is added to each well. The rinse solution is removed and anti-vaccinia virus primary antibodies (e.g., rabbit, sheep, human or other source) and the biotin or other chromagen-labeled secondary antibodies used at an effective dilution (e.g., 1:500 to 1:1000) to easily visualize the immunoplaques. For most studies, 1:500 dilutions of each of the primary (rabbit anti-vaccinia; Accurate Chemical or INCELL-prepared) and secondary (HRP anti-rabbit IgG; SIGMA or other vendor) antibodies were used. An example showing BHK-21 cells and the appearance of plaques in the immunoplaque assay is shown in FIG. 2.

B. Antiviral Antibodies and Applications

1. Intramuscular Depot Immunization with TiterMax Gold

For rabbit immunizations, 10⁸ FFU in 0.5 ml PBS were combined with 0.5 ml TiterMax Gold using a double hub emulsification needle (push antigen into TiterMax first, aqueous into oil phase) for mixing. The emulsion was injected into 4 sites (0.2 ml each) over both shoulders and both hind quadriceps. For sheep immunizations, 2×10⁸ FFU in 1 ml PBS were combined with 1 ml TiterMax Gold as above and inject 0.4 ml twice into each hind quadriceps. Animals were bled periodically to test antibody production. Good antibody titers are present within 4-6 weeks and remain high for several months.

2. Oral Immunization Formulae and Methods

A variety of oral immunization formulae can be used for immunization. Oral immunization is done by preparing a formula in which the virus remains viable (as determined by infectivity of released virus from the orally delivered paste and separate components of the paste formulae listed below) and is captured in nanoparticles and micelles as part of the protective formulation that includes aqueous and oil-based components, as well as suspending agents and carriers that protect the virus from degradation and allow it to be absorbed from the oral cavity and the intestine.

As an example of the formulation used for the the studies shown in the Figures, virus is prepared (at 10⁸ per rabbit or 2×10⁸ per sheep) by mixing virus in a solution of Hetastarch (hydroxyethyl starch, clinical grade; 6% w/v; Baxter), 40% (v/v) mannitol [UPS grade higher; SIGMA or other vendor], 0.15% (v/v) AAFA™ (nutritional supplement grade fish oil; INCELL 5% (v/v) glycerol (UPS grade; SIGMA or other vendor), 0.5% (w/v) gelatin (SIGMA) at a volume that will achieve a final concentration of 5×10⁴ to 2×10⁸ infectious FFU, depending on the effective or test dose expected (e.g., 10⁶ to 10⁸ for humans, depending on immunization status). In the animal studies, doses were at 10⁸ per rabbit and 2×10⁸ per sheep. Gel-sol virus carrier (GSVC) excipient components were prepared as an equal mixture (1:1:1; Avicel® CE-15(microcrystalline cellulose and guar gum), Avicel® 591 (water-dispersible microcrystalline cellulose containing sodium carboxymethylcellulose (NaCMC) and Ac-Di-Sol® (internally-crosslinked, water insoluble sodium carboxymethylcellulose (NaCMC)) [source of all components: FMC Products]) which was slowly added (with vortexing) to a final concentration of 10% (w/v).

Taste-testing (humans and animals) revealed that the formulae was palatable as a slightly sweet paste-gel type of formulae that caused no aftertaste and which could be subsequently dried (e.g., for tableting) and still maintain infectious virus as measured by infection of dissolved materials on BHK-21 cells after they had been dried and stored for various time periods, supporting long-term storage as a tablet or paste-gel material that maintains biological activity.

C. Bioassays and Biochemical Methods for Safety, Efficacy and Potency

A variety of bioassays and biochemical analyses are done to evaluate the vaccine. These include: (a) human cell line nonpermissiveness with expression of vaccine antigens (a safety test); (b) viral antigen expression and production compared to previous lots and reference standards (i.e., potency); and (c) activation of humoral and cell-mediated immunity (e.g., potency and efficacy) in infected animals. These are imperative types of assays to evaluate each virus lot and the overall potential variability between lots of virus.

1. Safety and Potency Bioassays: I-MVA Infection of Human Cells in vitro

INCELL has the only long-term continuous cell lines derived from human intestine (HI). As part of the pre-clinical testing, the HI cells were be grown in M3:10™ medium (INCELL) as monolayer cultures using standard methods so that they maintained functional cell and organ-specific markers that make them useful in vitro surrogates for orally administered products, including vaccines or drugs. Master and Working Cell Banks of these cells were banked in the INCELL repository prior to initiating these studies.

As part of the evaluation of I-MVA lots of oral vaccine, the HI test cell line(s) lines were seeded into culture vessels in M3:10™ (INCELL) growth medium, allowed to attach, then infected with test lots of virus essentially as described above for the FFU immunoplaque assays or as detailed elsewhere (15) for alternate cell infectivity studies. For each set, parallel cultures of uninfected and infected permissive BHK-21 cell controls, and dilutions of prepared reference virus, were tested to validate the bioactivity of the virus stocks.

An example of the study showing comparative infectivity of I-MVA for human intestinal and other human cells compared to the permissive BHK-21 cells are shown in Table 1. The important vaccine safety-related conclusion from the results shown in this table is that the I-MVA strain used to prepare the vaccine does not grow in human cells but readily replicates in the permissive BHK-21 cells.

Claim 1 of 25 Claims

1. An oral vaccine comprising:

a replication-defective or deficient vaccinia virus, or a modified vaccinia virus strain that is unable to generate infectious virus in human cells but is able to replicate in an animal host cell which is permissive for the virus, in a formulation comprising hydroxyethyl starch, mannitol, nutritional supplement grade fish oil, glycerol, and gelatin;

wherein gel-sol virus carrier excipient components comprising a mixture of equal parts of microcrystalline cellulose and guar gum; water-dispersible microcrystalline cellulose containing sodium carboxymethylcellulose (NaCMC); and internally cross-linked, water-insoluble sodium carboxymethylcellulose (NaCMC) is added to the formulation to form a paste or gel for oral delivery.

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