WO1997013531A1 - Solid, orally administrable viral vaccines and methods of preparation - Google Patents

Abstract

Compositions and excipients useful for vaccine formulations and delivery are disclosed. Materials and methods are provided for the efficacious immunization of patients against certain viruses, including rotaviruses, thereby conferring immune protection against subsequent viral challenge. The figure shows a graphical representation of the effect of various carriers on relative rotavirus infectivity retention post-drying.

Description

97/13531 PC17US96/16278

SOLID, ORALLY ADMINISTRABLE VIRAL VACCINES AND METHODS OF PREPARATION

Field of the Invention

This invention relates to vaccine formulations to prevent against viral infections. More specifically, the invention provides methodology for the formulation of a solid, oral delivery system for live rotavirus vaccines. The methodology disclosed herein may be applied to both living and attenuated viruses other than rotavirus.

Background of the Invention

In the U.S., rotavirus infections result in approximately 70,000 hospitalizations and 150 deaths of children less than five years of age annually. Infection with rotavirus causes severe, acute diarrhea in young children, and may be responsible for 1-2 million deaths per year worldwide. (Bishop, R.F., Vaccine 11 :247-253 1993; Snyder et al . , Bull. WHO 60:605, 1982) . Thus, an effective rotavirus virus vaccine would significantly reduce global childhood mortality (Bishop, supra) . Infection and reinfection with rotavirus are also common in adults (Marrie, T. , et al . , Arch. Inter. Med 142 :313- 316, 1982) and, although the symptoms are usually subclinical (Kapikian and Chanock, in Virology, B.F. Fields eds.. Raven Press, NY Pgs. 863-906, 1985), they contribute to significant loss of productivity in developed countries. Infection occurs after ingestion of viral particles. The rotaviruses colonize absorptive epithelial cells of the villi of the small intestine. Subsequent multiplication destroys the normal structure of the villi. The absorptive epithelial cells are replaced by glandular epithelial cells resulting in acute, copious watery diarrhea. This mechanism of infection renders the rotaviruses particularly susceptible to oral vaccination. Recently, progress has been made in the development of a live rotavirus vaccine. Problems remain however in the effective administration of vaccine, particularly in the development of suitable oral vaccine delivery system. Although the infectivity of rotavirus is relatively stable under various test conditions (Estes, M.K., et al., J. Virol. 31:879-88, 1981), the virus is acid- sensitive. Rotavirus begins to lose infectivity at pH 3.5 and the outer capsid of human rotavirus collapses below pH 3.0 (Estes, M.K., et al . , J. Gen. Virol. 43 :430- 409, 1979) . This acid-sensitivity affects the efficacy of orally-administered vaccines, as the live viruses can be inactivated in the acid environment of the stomach

(Kapikian, A. Z., et al. , Develop. Biol. Standardization .53_.:209, 1983) . Gastric acid can be neutralized by the administration of buffers before or concurrently with vaccine administration (Halsey, N.A. , et al. , J. Infec. Dis. 158 :1261-1267, 1988), but this procedure is time consuming, expensive, and poorly tolerated by infants (Edelman, R., Pediat. Infec. Dis. J. 6_:704-710, 1987) . One approach to overcoming this problem is to encapsulate the live rotavirus with enteric polymers which are stable at acid pH and protect the virus from inactivation, but which dissolve at intestinal pH and release the infectious rotavirus in the gut. However, insofar as it is known, successful incorporation of a live rotavirus vaccine into an enteric delivery system with subsequent recovery of significant levels of rotavirus infectivity has not previously been demonstrated. The carrier formulation described herein sustains viral infectivity and is suitable for further vaccine processing into a variety of delivery systems, including simple resuspension for liquid delivery.

A limitation on the effective use of the above- described vaccines is loss of infectivity of the virus during lyophilization and the measures required to protect the acid-sensitive vaccines from degradation in the stomach. In current practice, due to the lack of effective alternatives, one log loss of infectivity is said to be tolerated by manufacturers at the preliminary lyophilization step. Current vaccines are prepared with bicarbonate or citrate and resuspended in water before oral administration. Identification of an oral delivery system that performs better than, or at least equivalent to, the current norm with its concomitant loss of infectivity would be highly beneficial. The object of this invention is to provide such a vaccine for oral delivery that is superior to that which is currently available.

SUMMARY

The present invention provides a viral vaccine formulation suitable for processing into a variety of final dosage systems for oral administration to humans and animals, either adult or infant, including a therapeutic form of the vaccine and a carrier comprising cellulose and modified cellulose selected from the group consisting of cellulose esters, microcrystalline cellulose, carboxymethyl cellulose and mixtures thereof; a sugar selected from the group consisting of sucrose, fructose, glucose, mannose or any sugar suitable for use in pharmaceutical compositions; starch; and optionally gelatin.

Vaccine formulations retaining high levels of infectivity have been prepared using a carrier comprising 10-50 wt % of a cellulose component, 30-70 wt % of the sugar component, 10-50 wt % of starch and 10-30 wt % of gelatin based on the total weight of the carrier material. Particularly good results have been obtained with a live rotavirus vaccine including as the carrier substantially equal parts of cellulose acetate, starch, sucrose. Gelatin may beneficially be included in the carrier formulation for certain applications. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graphical representation of the relative effect on rotavirus infectivity retention produced by incorporation into various delivery systems. (1) PLG Microspheres; (2) Buchi spray drying; (3) Alginate microcapsules; (4) Cellulose granules.

Figure 2 is a graphical representation of the effect of various carriers on relative rotavirus infectivity retention post-drying. (1) Control solution rotavirus; (2) Rotavirus dried with sucrose; (3) Rotavirus dried with gelatin; (4) Rotavirus dried with lactose; (5) Rotavirus dried with a carrier blend of cellulose, sucrose, starch, gelatin at a 30:30:30:10 ratio.

Figure 3 is a set (A-F) of micrographs of the outer surface of granules (rotavirus dried with the carrier blend) either uncoated or coated with a polymer film coating. A) Surface of entire uncoated granule;

B) Photograph showing a close-up of the surface of the granule shown in 3A; C) Surface of an entire granule after applying primer coat of Opadry which is a hydroxymethylpropylcellulose (HPMC) ; D) Micrograph showing a close-up of the coated granule shown in 3C; E) Micrograph of the surface of a granule coated with both HPMC and Eudragit L30D. Eudragit L30D is methacrylic acid and methyl methacrylate copolymer; F) Micrograph showing a close-up of the coated granule shown in 3E.

Figure 4 is a micrograph of a cross section of the outer surface of a tablet formulated in accordance with this invention and uniformly coated with a polymer film. A) The viral core layer; B) The Opadry layer;

C) The Eudragit layer

DETAILED DESCRIPTION OF THE INVENTION

Effective delivery of rotavirus vaccine depends on the protection of the live rotavirus from degradation in the stomach while allowing release in the small intestine.

Conventional oral delivery systems that protect against degradation of therapeutic agents in the stomach are typically prepared by incorporation the agent into a soluble carrier matrix and coating the matrix with a protective polymer layer that is resistant to stomach acid, but soluble at neutral or alkaline pH. However, the conditions under which these coating processes are performed often result in degradation of sensitive biological materials such as viruses. Therefore a comparative evaluation of various delivery systems and certain processing conditions used therein was performed to develope an effective oral delivery system that is suitable for live, rotavirus vaccines. A porcine rotavirus was used for this evaluation. During the course of this evaluation a carrier blend was identified which enables the incorporation into tablets or granules of virus which retains infectivity. The inventor based the choice of ingredients in the present formulation in part, on known uses of cellulose acetate as a filler, sucrose as a binder, and starch as a disintegrant, and, also prior indications that certain carriers may stabilize viral preparations. Thus, gelatin, which is also a binder was added. Surprisingly, a carrier blend of cellulose acetate, starch, sucrose, and gelatin (30:30:30:10) stabilized the live porcine rotavirus and produced outstanding results with only minimal loss of infectivity. The same carrier blend stabilized human rotavirus, but a modified carrier blend containing cellulose, starch, and sucrose (33:33:33) without gelatin performed even better.

By contrast, incorporation of live rotavirus into poly DL-lactide-co-glycolide (PLG) microspheres (a delivery system known to be useful when delivering vaccines to intestinal Peyer's patches) , alginate microcapsules, as well as the application to the surface of spray-coated non-pareil seeds resulted in complete or substantial loss of infectivity.

In addition to the delivery system per se, potentially critical points in vaccine formulation processes that were evaluated included exposure of the virus to solvent and drying. Porcine rotavirus was found to be relatively stable in some commonly used organic solvents, including ether, diethylcarbonate and methylene chloride (1 log reduction in infectivity as determined by plaque assay) , but was not stable in others, such as ethyl acetate or acetone. However, no infectivity was retained by rotavirus preparations that were microencapsulated in PLG microcapsules prepared by evaporation of the co-mixed virus and the copolymer from methyl chloride. Furthermore, no infectivity was retained by rotavirus incorporated directly in the enteric polymers cellulose acetate phthalate, or Eudragit

L30D by the Buchi spray drying process. As the rotavirus was known to be stable to the solvents used in these tests, the results implicated the drying process as the potential point of loss of infectivity. Incorporation of the rotavirus into granules, a process in which the rotavirus. solution was dried rapidly in the presence of the carriers cellulose, starch, sucrose, and gelatin, and then milled into granules, resulted in retention of substantial infectivity as previously noted. It was found, however, that this formulation is susceptible to acid degradation of the virus and that enteric coating with Eudragit L30D alone was not sufficient to prevent this loss due to the extensive porosity of these granules (Fig 3) . A review of the literature revealed two alternative delivery systems, tableting and spray coating on to non¬ pareil sugar beads, that are commonly used for the adminstration of therapeutic agents. During these initial studies it became apparent that the nature of carrier used to stabilize the rotavirus vaccine during drying was critical. Comparative analyses indicated that cellulose and sugars gave superior results during the drying process and stabilized the virus during subsequent manipulations in the preparation of the delivery system. However, a significant loss of infectivity was observed during the process of spray coating on the non-pareil sugar beads using either cellulose or lactose as the carrier.

In contrast, tableting with cellulose as the carrier was found to preserve the infectivity of the rotavirus with only one log loss in infectivity after the entire process of tableting, coating with HPMC and subsequent coating with Eudragit. The tableted rotavirus vaccine was found to be stable for up to sixty days at 4oc, and for four weeks at room temperature and thus would be suitable for clinical use. By altering the size of the tablets generated, particulate tablet vaccines suitable for administration to infants can be generated. Generally, the tablet size should be less than 0.5 mm, with tablet size in the range of 0.10 to 0.25 mm being preferred. To demonstrate that this methodology can be applied to other viruses, vaccine studies were carried out using live transmissible gastroenteritis virus (TGEV) , a lipid envelope containing corona virus that infects pigs. The oral administration of live, attenuated TGEV in enterically-coated tablets to piglets indicated that this delivery system protected live viruses as evidenced by seroconversion of the pigs and the infection of the contact control animals that were housed with inoculated animals. As mentioned previously, the compositions of the invention may be prepared in various forms for administration, including tablets or granules. Granules may be suspended in a suitable carrier medium. As used herein "pharmaceutically acceptable carrier medium" includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical Sciences. Fifteenth Edition, E.W. Martin (Mack Publishing Co., Easton, PA, 1975) . Except insofar as any conventional carrier medium is incompatible with the anti-viral compounds of the invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component (s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention.

The compounds of the present invention may be used to vaccinate patients against certain viruses. Thus, the expression "immunologically effective amount" as used herein, refers to a nontoxic but sufficient amount of the compositions of the invention to elicit an immune response thereby protecting the patient against subsequent viral infections. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the particular viral vaccine and the like. As used herein, the term patient may refer to an animal or a human being.

The following examples describe in further detail the preparation of rotavirus vaccines suitable for oral delivery. The examples are intended to illustrate and not limit the scope of the invention.

Examples Materials and Methods

Preparation of Rotavirus Vaccine Antigen. The porcine rotavirus used in these studies,

Gottfried Strain GP46, was a gift from Dr. L.J. Saif (Ohio State University, Wooster, OH) . Pancreatin was purchased from Gibco BRL, Grand Island, NY. Lactose, sucrose, starch, microcrystalline cellulose (Avicel) and gelatin were purchased from Foremost Ingredients, Barboo, WI; Sigma Chemical Co., St. Louis, MO Colorcon, West Point, PA; FMC Corporation, Philadelphia, PA and Geo A. Hormel and Co., Austin, MN; respectively. Tableting carriers, acdisol, stearic acid and talc were purchased from FMC, Mallinckrodt Specialty Chemical Co., St. Louis, MO and Luzenac America Inc., Englewood CO. The enteric coatings Eudragit L30D, Opadry and cellulose acetate phthalate were purchased from Rohm Pharma, Maiden, MA; Colorcon; and Eastman Chemical Co., Kingsport, TN; respectively. Eudragit L30D is an aqueous dispersion of an anionic copolymer based on methacrylic acid and acrylic acid ethyl ester. Opadry is a water-soluble hydroxylpropylmethylcellulose based polymer. Sodium alginate (Keltone HV) was obtained from the Kelco Division of Merck & Co., Inc., San Diego, CA and non¬ pareil seeds from Paulur Corporation, Robbinsville, NJ. The Coomassie Blue Binding Assay kit was purchased from Pierce, Rockford, IL.

Rotavirus, strain GP46, was grown on MA 104 fetal rhesus monkey kidney cells. Prior to inoculation, the cells were maintained in Dulbecco's modified medium (DMEM) supplemented with 10% bovine calf serum and antibiotics. Confluent monolayers of these cells in roller bottles were infected at a multiplicity of infection (M.O.I.) of 0.01 - 0.02. After absorption of the virus to the cells for 1 hour at 37oC, DMEM supplemented with antibiotics and containing 30 μg/ml of pancreatin was added. The culture supernatant containing cell debris and virus was harvested 2-3 days post- infection and clarified by centrifugation at 3,700 x g_av for twenty minutes at 4oC. The virus was pelleted from the clarified solution by centrifugation at 130,000 x g_av for 1.5 hour at 4©c. The virus was then pelleted by resuspending the virus pellet in media and centrifugation on a 30% glycerol - phosphate buffered saline (PBS) cushion at 200,000 x g_av for 4.5 hours at 97/13531 PCMJ 96/1627

- 10 - 4oC. These pellets were resuspended in PBS - 1% bovine serum albumin (BSA) and were stored at -80oC.

Preparation of Dried Rotavirus Vaccine with Carriers

As described above a formulation of cellulose acetate, sucrose, starch and gelatine in a ratio of 30:30:30:10 was selected for trial and its ability to stabilize the live, rotavirus vaccine was tested. The carriers were ground in a Waring blender. The solution of live rotavirus was then added and the ingredients mixed well until a wet mass formed. This was dried in a desiccator at 4°C under vacuum. Upon completion of drying to a level of 5% H₂0, the dried carrier blend was ground with a small mill to form a dry powder. These processing steps were accomplished in approximately 1 hour and, except for the drying step, were performed at room temperature. The blends were then assayed for infectivity.

Comparison of the effects of carriers on the drying process

As infectivity may be lost on drying of the rotavirus during granulation and prior to tableting, the effect on the drying process of varying ingredients of the carrier blend was evaluated. The formulations included lactose alone, sucrose alone, gelatin alone, or the carrier blend of cellulose, starch, sucrose, and gelatin at a ratio of 30:30:30:10. The ingredients were suspended in distilled H₂0, and mixed with rotavirus strain Gottfried GP46 (1.9 x IO⁷ pfu) until a wet mass was formed. The wet mass was dried in a desiccator at 4oc under vacuum until a level of 5% water weight was reached, then ground with a small mill to form a fine, dry powder. The viral activity retained after processing was determined by plaque assay. Application to the surface of non-pareil seeds and enteric coating

As an alternative to granules and tablets, which involve incorporating the live rotavirus inside the core material of the delivery system, the rotavirus was applied to the surface of non-pareil sugar seeds. The dried live rotavirus strain Gottfried GP46 at a concentration 10⁷ - IO⁹ pfu was suspended in 100 ml of an aqueous HPMC film-forming polymer that contained 1 - 2% sucrose. This was applied to the surface of 200 g non- pareil seeds by the Wurster spray coating method administered in a STREA-1 laboratory unit as previously described. Subsequently, a sub-coating of Opadry was applied to an approximate weight gain of 6-8%, followed by an enteric coating of Eudragit L30D that further increased the weight of the seeds by 20%. Seeds from the three processing steps including the uncoated seeds, seeds coated with Opadry, and seeds coated with both Opadry and Eudragit L30D, were stored at 4oC under vacuum with desiccant. The seeds were evaluated for in vi tro stability by plaque assay and disintegration analysis, as described below for the granules under the heading "Preparation of Granules for Oral Delivery".

Plague Assay of Rotavirus Titer

Confluent monolayers of MA 104 cells in six-well plates were washed twice with PBS and inoculated with 0.2 ml of a serial 10-fold dilution of the sample in PBS. After an absorption period of 1 hour at 37oC with tilting every 15 minutes, unabsorbed virus was removed and the cells overlayed with 4 ml per well of 0.9% agar-DMEM containing 30 μg/ml pancreatin. After incubation for 5 days at 37oC, 3 ml of a second overlay medium containing agar-DMEM and 0.03% neutral red was added and the plaques were counted on the next two days. The results are given as plaque forming units (pfu) . Scanning Electron Microscopy (SEM)

Microspheres/granules were mounted on a specimen disc and coated with a 20 angstrom layer of palladium gold. The coating was carried out using the electron microscope-500 sputter coater. After coating, the samples were examined and photographed using an ISI-SX40 SEM.

Microencapsulation in PLG Microspheres by Solvent Extraction

Rotavirus-containing microspheres were prepared by Southern Research Institute, Birmingham, Alabama. Three milligrams of purified rotavirus strain SB-IA containing approximately IO¹² pfu of infectious particles was microencapsulated in biodegradable and biocompatible polymers of PLG by a modification of an emulsion- based methylene chloride solvent evaporation procedure as described by Cowsar, D.R., et al . , Meth. Enz . 112 :101- 116, 1985. The surface morphology was evaluated by electron microscopy and a smooth surface of continuous polymeric coating was confirmed. The vaccine content (core loading) was estimated by dissolving a sample of the microspheres in methylene chloride, extracting the rotavirus, determining the amount of protein and calculating the percent antigen by weight. The core loading of 0.55% was satisfactory. The size of the microspheres was determined using a Malvern light scattering device and was found to range from 1 to 10 μm.

A sample of the microspheres was dissolved in methylene chloride, the virus extracted twice with PBS, and the infectivity measured by plaque assay.

Microencapsulation in Cellulose Acetate Phthalate (CAP) Microspheres bv Spray Drying with the Buchi 190 Mini- Spray Dryer

CAP has been used widely as an enteric coating polymer for pharmaceutical tablets or granules. CAP dissolves at approximately pH 5.5, and thus it withstands prolonged contact with acid contents of the stomach, but dissolves and releases drugs readily in the small intestine.

The rotavirus strain SB-IA preparation containing approximately IO⁸ pfu of infectious particles were resuspended in 100 μl of 1.5 M sucrose containing 10 mM poly-L-lysine and mixed with 40 ml of an aqueous solution of CAP (25 mg/ml, pH 6.5) . Microcapsules were produced by atomization of the CAP and vaccine emulsion through a Buchi 190 mini-spray dryer (Brinkmann, Westbury, NY) .

About 50% of the material was recovered as microcapsules of 1 to 5 μm in diameter, as determined by SEM.

The infectivity of the microencapsulated rotavirus was determined by in vi tro analysis. Aliquots containing fifty mg of microcapsules were made and placed into 2 tubes. One sample was treated with 0.1 N HCl and 37oc for 30 minutes. The microcapsules did not dissolve in the acidic solution and appeared to be intact. The microcapsules were then pelleted by low speed centrifugation and dissolved in 0.5 ml of simulated intestinal fluid, pH 6.8 - 7.5 (USP XXI) . The other 50 mg of microcapsules were dissolved in 0.5 ml of simulated intestinal fluid without pretreatment with acid. Both samples were then examined for virus infectivity by plaque assay.

Incorporation into Alginate Microcapsules bv the Process of Ionic Gelation

The alginate microcapsules containing live rotavirus strain SB-IA vaccine formed by chelation of the sodium alginate with calcium ions. Rotavirus at a concentration of 1 x 10^a pfu was mixed with 3 ml of sodium alginate solution (1.2% w/v in normal saline) . This suspension was then dripped slowly through a 19 gauge needle into a solution of calcium chloride (1.5% w/v in distilled water) while stirring at 500 rpm. The microcapsules containing rotavirus were collected by sieving, rinsed three times with a normal saline solution, and then dried at 4oc under vacuum.

Half of the alginate microcapsules containing rotavirus were incubated at 37oc in simulated gastric fluid, pH 1.2 (USP XXI) for 30 minutes. They were pelleted and then suspended in phosphate buffered saline (PBS) , pH 7.7. The other half of the alginate microcapsules were not exposed to gastric fluid, but were resuspended in PBS, pH 7.7 until dissolved. The rotavirus was released from the alginate microcapsules, which dissolved completely at the higher pH. The rotavirus infectivity of both samples was then determined by plaque assay. The infectivity of the live rotavirus was reduced by 2 log after exposure to gastric fluid, but reduced by only 0.73 log when exposed to PBS alone.

Preparation of Granules for Oral Delivery

Granules were prepared by forming a carrier blend of cellulose, starch, sucrose and gelatin at a ratio of 30:30:30:10: in a Waring blender. One batch of granules was prepared by adding a solution of rotavirus strain SB- IA at a concentration of 4.51 x IO⁷ pfu per 300 mg of carrier blend, and a second batch prepared by adding a solution of SB-IA at a concentration of 3.91 x IO⁴ pfu per 20 mg of carrier blend. The wet mass was dried under vacuum at 4°C and then ground with a small mill to form granules. These were passed through a series of sieves (300 μm - 3 mm, USA standard testing sieves) , and granules of 1-3 mm were collected and stored at 4oc under vacuum with desiccant . One batch of granules containing 4.51 x 10⁷ pfu rotavirus strain SB-1A/300 mg of carrier blend was enterically coated with Eudragit L30D, using a Wurster spray coating method in a fluid bed laboratory unit, STREA-1 (Aeromatic Inc., Columbia, MD) . Eudragit dissolves above pH 5.5 by forming salts with alkalis, 97/13531 PC17US96/16278

- 15 - thus affording coatings that are resistant in gastric media, but soluble in the small intestine. Sufficient coating was applied to increase the weight of the granules by 25% (w/w) . Although a weight gain of 12-18% is considered to be protective by manufacturer's standards, the extensive porosity of the granules required a heavier coating to adequately seal the pores.

A second batch of granules containing 3.91 x IO⁴ pfu rotavirus strain SB-1A/20 mg carrier blend was enterically coated with Eudragit L30D as described above except that granules received a protective coating of

Opadry prior to the application of the Eudragit. It has previously been determined that enteric polymers can inactivate certain ingredients such as viruses, proteins, and peptides. The protective coating, Opadry, is a water-soluble hydroxypropylmethlycellulose-based polymer. The Opadry was applied to the granules by the Wurster spray coating method in a fluid bed laboratory unit, STREA-1, with a weight increase of the granules of approximately 8-10%. A further weight gain of 25% occurred on coating with Eudragit.

Uncoated granules, granules coated with Eudragit L30D alone, granules coated with Opadry alone and granules coated with both Opadry and Eudragit L30D, were stored at 4°C under vacuum with desiccant. The viral infectivity was determined by plaque assay.

Disintegration analysis was used to determine the effectiveness of the film coatings in protecting the live rotavirus vaccine from exposure to gastric fluid. The granules were weighed prior to and after exposure to simulated gastric fluid (USP XXI; pH 1.2) at 37oC for 1 hour, and the percent gastric uptake determined. The granules then were exposed to simulated intestinal fluid (USP; pH 6.8 - 7.5) at 37°C, and the time required for complete disintegration determined. Preparation of Tablets

Rotavirus Gottfried strain GP46 was prepared for tableting using a drying procedure as described for the preparation of the granules except that 1) the rotavirus was dried with the carrier blend of cellulose, starch, sucrose and gelatin at a concentration of 9.7 x 10^s pfu/lOOmg of carrier blend, or 2) the rotavirus was dried with lactose at a concentration of 6.7 x 10⁴ pfu/ 20 mg lactose.

Tablets formed from preparation 1 were composed of the following ingredients (% dry weight) : lactose filler (74.70 %) , acdisol disintegrant (3 %) , stearic acid lubricant (1.5 %) , talc as a processing aid (1.0 %) , and the dried rotavirus preparation with carriers (19.70 %) . Tablets formed from preparation 2 were composed as follows: lactose (54.10 %) , acdisol (3 %) , stearic acid (1.50 %) , talc (1.0%) , and the dried rotavirus preparation with carriers (40.40 %) . The dry ingredients from each preparation were mixed well to form a dry blend, incorporated into a 3 mm dye and pressed at 550 lb pressure to produce 50 mg, 3 mm tablets.

The tablets were enterically coated with Opadry and Eudragit L30D as described for the coating of the granules. The subcoating of Opadry was applied until the weight of the tablets increased by 6-8%. Subsequently, an enteric coating of Eudragit L30D was applied until the weight of the tablets was further increased by 20-25%. Tablets from the three processing steps including uncoated tablets, tablets coated with Opadry, and tablets coated with both Opadry and Eudragit L30D, were stored at 4oc under vacuum with desiccant. The tablets were evaluated for in vi tro stability by plaque assay and disintegration testing as described for the granules. 97/13531 PC17US96/16278

- 17 -

Example I

Initial experiments were performed to compare the infectivity of rotavirus after drying in the presence and absence of the chosen carrier blend. The results are shown in Table I .

TABLE I INFECTIVITY LOST AFTER DRYING WITH ORIGINAL CARRIER FORMULATION

Expt Formulation of Carriers input Recovery Rotavirus

Cellulose Sucrose Starch Gelatine (pfu x IO⁷) Infectivity Log Acetate Bloom 175 (pfu x IO⁷) Loss

1 30 30 30 10 1.9 1.0 0.29

2 No excipients, solution of rotavirus 1.9 x IO⁷ pfu in 100 μl 1.1 0.24

Surprisingly, the chosen carrier blend of cellulose acetate, starch, sucrose and gelatine (bloom 175) at a ratio of 30:30:30:10 (w/w) resulted in essentially complete retention of the rotavirus vaccine infectivity after drying compared to a control solution of the virus as determined by plaque assay. This preparation was readily soluble in PBS and this carrier blend would facilitate subsequent granulation and tableting procedures.

Example II

To determine if any one commonly used carrier of the classes used in the original formulation was capable of maintaining the infectivity of the live rotavirus vaccine during the drying process, the rotavirus vaccine was mixed with individual carriers. These carriers included cellulose acetate, starch, sucrose or gelatin bloom 175 or compounds with similar properties including microcrystalline cellulose, fructose, glucose, mannose, or gelatin blooms 150, 175, 200 or 300. The live, rotavirus vaccine was prepared, blended with the respective carrier, and the blend was then dried and assayed for infectivity as described in the above materials and methods. In this experiment, some of the formulations were not readily soluble in PBS and required incubation at 37oC for 10 minutes to dissolve. The formulations blended with gelatin blooms 200 and 300 were relatively insoluble and required incubation at 37o for 20 and 30 minutes respectively.

TABLED

INFECTIVITY LOST IN DRYING

WITH INDIVIDUAL CARRIERS

EXP. FORMULATION Input Recovery tt OF Rotavirus

CARRIERS (pfu x 10*)

Infectivity Log (pfu x 10*) loss

EXPERIMENTAL:

3 Cellulose acetate 1.0 0.00 6.00

4 Microcrystalline cellulose 1.0 0.00 6.00

5 Starch 1.0 0.01 4.00

6 Sucrose 1.0 1.06 1.97

7 Fructose 1.0 1.50 1.82

8 Glucose 1.0 1.30 1.88

9 Mannose 1.0 0.95 2.02

10 Gelatin 150 bloom 1.0 1.00 2.00

11 Gelatin 175 bloom 1.0 0.35 2.45

12 Gelatin 200 bloom 1.0 0.45 2.34

13 Gelatin 300 bloom 1.0 0.14 2.85 No single carrier was sufficient to retain the activity of the live rotavirus vaccine. The dried preparations of rotavirus in sucrose, fructose, mannose or glucose, or gelatin (blooms 150, 175, 200 or 300) alone showed a loss of infectivity of approximately two logs. The dried preparations of rotavirus in cellulose alone, either as cellulose acetate or microcrystalline cellulose, or in starch alone showed almost complete loss of infectivity.

Example III As individual carriers were not able to stabilize the live, rotavirus vaccine during drying, an experiment was conducted to determine if any two carriers in combination could stabilize this vaccine. The live, rotavirus vaccine was prepared, blended with the carriers, and then dried as described above in materials and methods. These processing steps were accomplished in approximately 1 hour 40 minutes and, except for the drying step, were performed at room temperature. In this experiment, some of the formulations were not readily soluble in PBS and required incubation at 40oC for 10 minutes. The blends were then assayed for infectivity as described in materials and methods.

TABLE ID

INFECTΓVTΓY LOST IN DRYING WΠΉ

COMBINAΗONS OF TWO

CARRIERS

Expt. Formulation of Carriers Input Recovery tt Rotavirus

Cellulose Sucrose Starch Gelatin (pfu x 10*) Infectivity Log Acetate Bloom 175 (pfu x 10*) loss

14 50 0 50 0 1.0 0.00 6.00

15 50 50 0 0 1.0 0.04 3.40

16 50 0 0 50 1.0 0.20 2.70

17 0 50 50 0 1.0 1.19 1.92

18 0 0 50 50 1.0 N.D. N.D.

19 0 50 0 50 1.0 1.24 1.91

Combinations of sucrose and starch, and sucrose and gelatin resulted in improved retention of rotavirus infectivity, but all of these resulted in a greater than 1 log loss (Table II) . In addition, routine use of such high levels of gelatin presents difficulties in solubilizing the formulation and in fabrication of final dosage units. The formulation of sucrose and gelatine would present difficulties in subsequent granulation and tableting procedures, and a combination of sucrose and starch is probably not suitable for subsequent granulation.

Example IV

To determine if a combination of any three of the carriers stabilized the rotavirus, the carriers were blended and mixed with free rotavirus as described above in materials and methods. These processing steps were accomplished in approximately 1 hour 50 minutes and, except for the drying process, were performed at room temperature. In this experiment some of the formulations were not readily soluble in PBS and required incubation at 40°C for 10 minutes. The blends were then assayed for infectivity as described above in materials and methods .

TABLEIV

INFECTIVITYLOSTINDRYING WITH COMBINATION OFTHREE CARRIERS

Expt. Formulation of Carriers Input Recovery tt Rotavirus

Cellulose Sucrose Starch Gelatin (pfu x 10«) Infectivity Log Acetate Bloom 175 (pfu x 10*) loss

Experimental:

20 25 50 25 0 1.0 1.23 1.91

21 15 70 15 0 1.0 1.50 1.82

22 10 80 10 0 1.0 0.59 2.23

23 25 0 25 50 1.0 0.73 2.14

24 30 0 30 40 1.0 0.87 2.06

25 40 0 40 20 1.0 1.02 1.99

26 50 25 0 25 1.0 2.43 1.61

27 40 35 0 25 1.0 2.08 1.68

28 30 50 0 20 1.0 2.18 1.66

29 0 25 50 25 1.0 2.20 1.66

30 0 40 40 20 1.0 1.38 1.86

31 0 50 30 20 1.0 0.91 2.04

The results indicated that various combinations of three of the carriers were capable of improving the retention of rotavirus infectivity. In particular, combinations of cellulose acetate, sucrose, and gelatin, and sucrose, starch and gelatine resulted in good retention of infectivity. However, none of the blends resulted in log losses lower than 1. These results indicated that the ratio of the formulation components was critical to retention of infectivity.

Example V

To determine if the formulation could be optimized, various ratios of the carriers were used in the formulation. All four carriers were represented in the formulations. The live, rotavirus vaccine was prepared, blended with the carriers, and the blend dried as described above in materials and methods.

TABLEV

INFECTΓVΠΎ LOST IN DRYING WΠΉ VARYING PROPORTIONS OF CARRIERS

Expt. Formulation of Carriers Input Recovery tt Rotavirus

Cellulose Sucrose Starch Gelatin (pfu x 10«) Infectivity Log Acetate Bloom 175 (pfu x 10*) loss

Experimental:

32 40 30 20 10 1.0 1.76 1.75

33 50 30 10 10 1.0 0.16 2.79

34 40 40 10 10 1.0 2.26 1.64

35 35 50 5 10 1.0 0.93 2.03

36 50 35 5 10 1.0 0.33 2.48

37 60 20 10 10 1.0 1.79 1.75

38 30 50 10 10 1.0 3.28 1.48

39 30 40 20 10 1.0 3.83 1.41

40 20 60 10 10 1.0 4.00 1.39

41 10 70 10 10 1.0 0.60 2.22 TABLE VI

FURTHER VARIATIONS ON THE PROPORTIONS OF CARRIERS

Expt. Formulation of Carriers Input Recovery tt Rotavirus

Cellulose Sucrose Starch Gelatin (pfu x 10«) Infectivity Log Acetate Bloom 175 (pfu x 10*) loss

Experimental:

42 25 25 25 25 1.0 9.6 1.02

43 70 10 10 10 1.0 3.2 1.49

44 50 30 10 10 1.0 21.0 0.68

45 30 50 10 10 1.0 36.0 0.44

46 10 70 10 10 1.0 46.0 0.33

47 20 20 50 10 1.0 27.0 0.57

48 30 30 10 30 1.0 51.0 0.29

49 20 20 30 30 1.0 5.6 1.25

50 10 10 10 70 1.0 5.5 1.26

51 10 10 30 50 1.0 5.3 1.28

52 10 10 50 30 1.0 1.3 1.89

53 10 10 70 10 1.0 3.1 1.51

54 30 30 30 10 1.0 14.0 0.85

These results indicate that the formulation for the stabilization of rotavirus during drying can be optimized by routine experimentation. In this example, the best formulation was found to be cellulose acetate, sucrose, starch and gelatin (bloom 175) in a ratio of 30:30:10:30 (Table VI) . Other formulations also resulted in good retention, including 50:30:10:10, 30:50:10:10,

10:70:10:10. However, these combinations of carriers were difficult to solubilize requiring incubation at 37 - 40oc and would likely present problems on subsequent tableting and granulation. In contrast, the formulation of cellulose acetate, sucrose, starch and gelatine in a ratio of 30:30:30:10, showed a good yield of infectivity, was readily soluble, and would facilitate subsequent tableting or granulation. Optimally, the various components can be added in slightly different ratios, for example, cellulose acetate can be added in the range of 10-50, sucrose in the range of 30-70, starch in the range of 10-50, and gelatin bloom 175 in the range of 10-30.

Example VI Preparation of Dried Human Rotavirus Vaccine with Carriers■ An experiment was conducted using human rotavirus to determine if the carrier formulation preserved the activity of human rotavirus and to determine if it was possible to further improve the results from the combination of any three carriers used previously in Example IV. Fewer ingredients would be advantageous in subsequent process development of the formulation into a product. A formulation of cellulose acetate, sucrose, and starch in a ratio of 33:33:33 was chosen because it is readily soluble, and would facilitate subsequent tableting or granulation. The formulation of cellulose acetate, sucrose, starch, and gelatin in a ratio of 30:30:30:10 was used as a basis of comparison because it showed good retention of infectivity in Experiments I and V. Also, two other formulations were tried, which are identical to the previous ones used in this experiment, except for the carrier cellulose acetate, which was replaced with microcrystalline cellulose, because this preparation of cellulose has demonstrated improved granulation and tableting over other celluloses.

Human Rotavirus

Human rotavirus (HRV strain D x BRV strain UK, Reassortant clone 41-1-1 FRHL-2) was supplied to us by Dr. Kapikian (NIH) . This virus was grown on MA 104 fetal rhesus monkey kidney cells. Prior to inoculation, these cells were maintained in Dulbecco's Modified Eagle medium (DMEM) supplemented with 10% bovine calf serum and antibiotics. Confluent monolayers of these cells in roller bottles were infected at a multiplicity of infection (M.O.I.) of 0.01. After absorption of the virus to the cells for 1 hour at 37oC, DMEM supplemented with antibiotics and containing 0.5 μg/ml trypsin was added. The culture supernatant containing cell debris and virus was harvested 48 hours post-infection and fresh medium without trypsin was added for another 24 hours. The harvested media were pooled and clarified by centrifugation at 3,700 x g tor 20 minutes at 4©c. The virus was then purified by resuspending the virus pellet in medium and centrifugation on a 30% glycerol-phosphate buffered saline (PBS) cushion at 200,000 x g_av for 4.5 hours at 4oc. These pellets were resuspended in PBS supplemented with 1% bovine serum albumin (BSA) , divalent cations, Ca⁺⁺ and Mg⁺⁺ and stored at -80°c. The live rotavirus vaccine was prepared, blended with the carriers and the blend dried as described in materials and methods. These processing steps were accomplished in approximately 1 hour and, except for the drying step, were performed at room temperature.

TABLE VH

INFECTΓVΠΎ LOST IN DRYING OF HUMAN ROTAVIRUS WITH COMBINATION OF THREE CARRIERS

The substitution of microcrystalline cellulose for cellulose acetate resulted in greater loss of infectivity. Surprisingly, the chosen carrier blend of cellulose acetate, starch and sucrose at a ratio of 33:33:33 was the best formulation with the most retention of human rotavirus infectivity. This formulation is also readily soluble and would facilitate subsequent tableting or granulation.

Example VII

Comparison of delivery systems

Microencapsulation of live rotavirus indicated that incorporation of live rotavirus into PLG microspheres with solvent removal by either extraction or evaporation, completely destroyed all viral infectivity (Fig. 1) . Furthermore, microencapsulation of rotavirus in CAP polymer particles by the process of atomization in a Brinkmann Buchi 190 mini-spray dryer, completely destroyed rotavirus infectivity. Incorporation of live rotavirus into alginate microcapsules resulted in significant loss of infectivity of approximately 2 log after exposure of microcapsules to gastric fluid, but reduced infectivity by only 0.73 log when exposed to PBS alone. The application of rotavirus to the surface of non-pareil seeds with subsequent coating also resulted in a 2-3 log loss (Table VIII) . In contrast, preparation of granules in an carrier blend of starch, sucrose, gelatin and cellulose reduced infectivity by only 0.29 log, and tableting with an carrier blend of starch, sucrose, gelatin and cellulose reduced infectivity by only 0.77 log (Fig. 1 and Table IX) .

TABLE VIII

RETENTION OF INFECTIVITY AFTER SPRAYING ROTAVIRUS TO THE SURFACE OF SUGAR BEADS

Batch Input Recovered Loss number pfu/ g beads pfu/ g beads log

1 8.5 x 10⁴ 3.5 x IO² 2.4

2 2.7 x IO⁶ 2.4 x 10³ 3.0

3 4.0 x 10⁷ 7.5 x 10³ 2.7

Beads of each batch were coated with HPMC, and additionally batch #2 was coated with Eudragit.

TABLE IX

EFFECT OF TABLETING AND COATING ON ROTAVIRUS INFECTIVITY

pfu/dose pfu/tablet pfu/tablet pfu/tablet (no coating) (HPMC (HPMC and coating) Eudragit coating)

RV dried 6.7 x 10* 7.1 X 10* 3.8 X IO³ 1.0 X 10³ with lactose log loss 2.45 2.43 3.70 4.28

RV dried 9.7 x 10« 3.2 x 10« 4.4 x 106 6.4 x IO⁶ with carrier blend log loss 0.29 0.77 0.63 1.46*

* The log loss of infectivity is slightly higher in this experiment due to the sensitivity of the virus to procedures used to remove the Eudragit prior to plaque assay. Effect of formulation of the carrier blend

To determine the appropriate carriers to be used in forming granules, the live rotavirus was dried on several different substrates (Fig. 2) . Drying of the live rotavirus in solution at a dose of 1.9 x IO⁷ pfu, on sucrose alone, gelatin alone, or lactose alone resulted in a loss of infectivity of between 1.39 and 2.45 logs. In contrast, drying on the carrier blend containing cellulose, sucrose, gelatin and starch resulted in preservation of infectivity with a log loss of 0.29.

Effect of coating processes on recovery of rotavirus infectivity from granules.

Enteric coating of the granules with Eudragit L30D resulted in a reduction of infectivity of 1.61 log (Table X) . There was an additional 2.39 log loss of infectivity after exposure of the coated granules to gastric fluid, for a total cumulative loss of 4.0 log. There was an approximate weight gain of 8-10% on disintegration testing.

TABLE X

RECOVERY OF ROTAVIRUS INFECTIVITY AFTER INCORPORATION INTO GRANULES

PreDaration of Granules Recovered Log loss original rotavirus input = (pfu/300 mg)

4.51 x 10⁷/300mg cellulose blend

Uncoated granules 2.3 x 10⁷ 0.30

Granules coated with Eudracrit

Treated with simulated intestinal fluid 1.1 x IO⁶ 1.61

Treated with simulated gastric fluid 4.5 x 10³ 4.00

Consequently, a second batch of granules was coated with HPMC, Opadry primer, prior to enteric coating (Table XI) . In contrast to the uncoated granules, which exhibited a 0.30 log reduction in infectivity, coating with HPMC resulted in a loss of infectivity of 0.78 logs, and the subsequent enteric coating with Eudragit reduced the infectivity by 1.19 log. After treatment of the enterically coated granules with USP XXI, pH 1.2 simulated gastric fluid, there was an additional loss of infectivity of 0.10 log. Thus, the total losses incurred during the coating processes and gastric exposure was 1.29 log. In this batch of granules, there was an approximate weight gain of 4-6% on disintegration testing.

Table XI

EFFECT OF COATING PROCESSES ON RECOVERY OF ROTAVIRUS INFECTIVITY FROM GRANULES

All granules were treated with simulated intestinal fluid and titer of the viruses released in solμtion was measured. Original rotavirus input was 3.91 x 10⁴/20 mg cellulose blend.

As losses in rotavirus infectivity were observed after exposure of the enterically coated granules to the USP XXI, pH 1.2 simulated gastric fluid, the granules were examined by SEM to determine the effectiveness of the coatings in sealing the pores and crevices on the surfaces of the granules (Fig. 3) . The HPMC and Eudragit enteric coatings smoothed the surfaces of the granules and rounded out the rough edges, but did not adequately seal the pores. (Figure 3B, D and F) Pores and crevices were apparent in the SEM analyses of the whole granules, despite the application of a 50-80% weight gain of the Eudragit L30D. Effect of incorporation of live rotavirus into tablets and enteric coating.

Preparation of tablets after drying of the live rotavirus vaccine onto lactose resulted in a significant loss of infectivity at each step of processing with a total accumulative loss of infectivity of 4.28 log (Table IX) . In contrast, initial drying of the live rotavirus vaccine onto an carrier blend of cellulose, sucrose, starch and gelatin, followed by tableting and coating steps, resulted in a significant improvement in the retention of infectivity with a cumulative log loss of 1.46. Only 0.29 log loss of infectivity was observed after drying of the rotavirus on the carrier blend, compared to 2.45 log loss when the rotavirus vaccine was dried on lactose (Fig. 2 and Table IX) . As minimal losses in infectivity were observed after all tableting processes of the dried rotavirus and carrier blend, the tablets were cross-sectioned and examined by SEM. The SEM microscopy revealed a very uniform coating of Opadry and Eudragit L30D around the surface of the tablet providing a more effective seal and substantially complete protection from gastric fluid (Fig. 4) . No weight gain was observed in either preparation on disintegration testing.

Using current techniques, rotavirus infectivity is not retained during incorporation into commonly used delivery systems. Although prior work indicated that live rotavirus is stable in some organic solvents such as methylene chloride, diethylcarbonate, and ether, the process of microencapsulation in PLG copolymer almost completely ablates the infectivity. This loss of infectivity could be associated with the mechanical action, possible increases in temperature, prolonged exposure to organic solvent or the lyophilization steps involved in the microencapsulation process. We and others have demonstrated success using PLG to encapsulate viruses, proteins, and peptides with excellent results (Moldoveanu, Z., et al. , J. Infec. Dis. 167 :84-90. 1993), but this procedure is apparently not suitable for the preparation of a live rotavirus vaccine (Fig. 1) .

Spray drying is commonly used to encapsulate active ingredients, but also involves temperature changes, mechanical action and drying steps. Although, no organic solvent was used in this case, this process also resulted in almost complete loss of infectivity (Fig. 1) .

The alginate microcapsule delivery system with a liquid core and semipermeable membrane is an acceptable environment for live cells, however, it has not been determined that alginate is an acceptable environment for a live rotavirus vaccine. A satisfactory level of rotavirus infectivity was maintained in alginate microcapsules that had not been exposed to gastric acid, but after exposure to gastric acid, rotavirus infectivity was reduced by 2 log (Fig. 1) . The porosity of the alginate microspheres produces a delivery system that cannot be effective for oral administration of a live rotavirus vaccine, unless these pores can be sealed completely.

Non-pareil seeds, or sugar beads, were also included in this comparison. This technology which involves the loading of an active ingredient onto the surface of a seed is commonly used to deliver drugs, especially if a sustained or controlled release of the active ingredient is desired throughout the gut. Furthermore, the active ingredient is close to the surface and thus very quickly available if a burst release of the active ingredient is desired throughout the gut . It would seem that this type of delivery system could be appropriately modified for the delivery of most vaccines, proteins or peptides. However, our results indicate that this technology is not suitable for the delivery of live rotavirus vaccines (Table VIII) . Significant loss of rotavirus infectivity was observed after spraying onto the surface of non¬ pareil seeds and subsequent coating processes even though disintegration testing indicated that the enteric coating was successful. As non-pareil seeds are a sucrose formulation, and our results indicated that sucrose alone is not effective in maintaining rotavirus infectivity, these results are not surprising. Comparison of the delivery systems indicated that granules prepared from the carrier blend (Fig. 1 and 2) performed significantly better than the other delivery systems (Fig. l) . This suggested that stabilization of the rotavirus during the drying process is an important factor in maintaining rotavirus infectivity. Thus, it is possible that the polymers used for microencapsulating the rotavirus vaccine such as PLG, CAP, and alginate do not stabilize the live rotavirus vaccine during the drying process. Apparently a carrier blend of cellulose, starch, sucrose and gelatin is capable of maintaining rotavirus infectivity. However, when these carriers were used individually, the rotavirus infectivity was decreased significantly (Fig. 2) . Consequently, the carrier blend was chosen to form granules for further analysis. The effect of the enteric coating processes, such as application of HPMC and Eudragit L30D, on the recovery of rotavirus infectivity after exposure to simulated gastric fluid was determined (Tables X and XI) . The results show an improvement in the retention of rotavirus infectivity after exposure to simulated gastric fluid when the granules were coated with both HPMC and Eudragit L30D as opposed to coating with Eudragit L30D alone (Tables X and IX) . Apparently, a primer coat is needed prior to the application of the Eudragit L30D for protection of the live rotavirus. Some cells, proteins and peptides are very sensitive to this polymer and precoating with HPMC affords some protection. In addition, the crevices and pores of the granules were difficult to seal, even after application of a thick coat of polymer. The weight gain on disintegration testing indicated that a completed seal was not achieved even after pre-coating with HPMC. It is apparent (Fig. 3) that after completion of all the coating processes, there were still some pores and crevices present on the surface of the granules. Thus, it was extremely difficult to ensure the perfect seal required for complete protection from the gastric fluid. This problem will be addressed by using more current granulation technology to create small, agglomerated particles that are hard, round and smooth, in contrast to the more irregular, porous granules produced by the standard laboratory methods used in this study. The improved technology will provide a surface that can be more uniformly coated and provide a better seal with subsequent, improved protection from gastric fluid. Tablets are round, hard and smooth and provide a surface that can be coated uniformly (Table IX and Fig. 4) . The SEM of the tablet cross-section reveals a very uniform and even distribution of Opadry and Eudragit L30D around the surface of the tablet providing an effective seal. Tableting is also a practical and economical laboratory procedure that can be used to demonstrate the feasibility of preparing an effective rotavirus vaccine delivery system based on smaller, aggregated particles. Tablets, of themselves, are not considered an appropriate delivery system for infants and children, who would be the most important target group for this vaccine. Tableting resulted in improved retention of infectivity at every step in the processing with a total cumulative loss of 1.46 log (Table IX) . Disintegration testing and SEM photography indicated complete sealing on enteric coating (Fig. 4) . Furthermore, the actual loss of infectivity may be lower than that recorded in the analysis as the methods used to remove the Eudragit L30D to assay contents for remaining rotavirus infectivity contributed to the loss of rotavirus infectivity. This concept is supported by the comparative losses assayed after the HPMC and Eudragit L30D coating. As HPMC coating involves longer exposure to increased temperatures and mechanical processes than Eudragit coating, it is reasonable to assume that a greater loss of rotavirus infectivity would be observed during the HPMC coating than during the Eudragit L30D coating. Consequently, a more accurate assessment of loss of infectivity may be indicated by the loss subsequent to the HPMC coating step. Based on this assumption, the total loss of rotavirus infectivity after all four processing steps including 1) drying, 2) tableting, 3) HPMC coating, and 4) Eudragit L30D enteric coating was only 0.63 log. This is a significant improvement considering that other investigators routinely observe greater losses of rotavirus infectivity after the preliminary drying or lyophilization steps.

Example VIII

The procedures described above may be utilized to make vaccines based on other viruses. The following examples are provided to demonstrate the applicability of the above-described methodology in the synthesis of vaccines based on Transmissible Gastroenteritis Virus

Transmissible Gastroenteritis Virus Antigen

Two batches of live, attenuated TGEV (transmissible gastroenteritis virus) were prepared. Batch 1 was prepared from virus obtained from Dr. Richard Hesse from Schering-Plough as a harvest of TGEV that had been concentrated ten-fold. A volume of 890 ml was centrifuged at 4,000 rpm for 45 minutes. The pellet obtained was resuspended in 40 ml PBS and kept at -80° until further use. The virus was concentrated by ultracentrifugation at 45,000 rpm for 1 hour and the pellet resuspended in 9.3 ml PBS (5 x 10^e TCID₅₀/ml) . An aliquot of this preparation (2 ml) was further concentrated for tableting using a Centricon-100 concentrator. The filtration device was spun at 5,300 rpm in a Sorvall centrifuge for 3 hours. The TGEV was harvested from the filter in a volume of 200 μl . The filter was washed twice with 175 ul of dH₂0, bringing the total volume of TGEV solution of 550 μl. This virus preparation was used for wet massing and tableting.

Batch 2 was prepared from porcine coronavirus TGEV also obtained from Dr. Hesse (Schering-Plough) . The virus was grown on ST cells in 850 cm² roller bottles. Prior to inoculation with virus, the cells were maintained in Dulbecco's modified Eagle medium supplemented with 7% fetal bovine serum and antibiotics. A confluent monolayer of cells was infected at an M.O.I, ot 0.1-0.001. After absorption ot the virus to the cells for 1 hour at 37oC, DMEM supplemented with fetal bovine serum and antibiotics was added. The culture supernatant containing cell debris and virus was harvested 2-3 days post-infection from 184 roller bottles (13.8 liters) and clarified by centrifugation in a Sorvall centrifuge at 6,000 rpm for 20 min. The supernatant was harvested and the virus concentrated using an Amicon Spiral Ultracentrifuge Cartridge S1Y100 under pressure (20-30 p.s.i.) . The permeate containing the virus (750 ml) was then further concentrated by centrifugation using a Beckman TI 45 rotor at 45,000 rpm for 1 hour. The pellet was resuspended in 50 ml of PBS. At this stage of purification, the virus had been concentrated 276-fold and the infectious titer was 8.11 x 10^s TCID_S0/ml .

Preparation of Dried TGEV Antigen

Two lots of dried TGEV were prepared. The first lot incorporated 550 ul of Batch 1 TGEV prepared as described above. A formulation of cellulose acetate, sucrose, starch, and gelatin in a ratio of 30:30:30:10 was chosen. The carriers were ground and blended in a Waring blender, the TGEV solution was added to 500 mg of the carrier blend and mixed until a wet mass was formed. This was dried in a desiccator at 4° under vacuum. Upon completion of drying to a level of 5% H₂0, the dried TGEV carrier blend was ground with a small mill to form a dry powder. This powder was then incorporated into tablets.

The second lot of dried TGEV was also formulated with cellulose acetate, sucrose, starch, and gelatin in a ratio of 30:30:30:10 as described above except that 500 μl of Batch 2 of the purified virus was used. Thus, 4.055 x IO⁹ TCID₅₀/500 μl of virus was incorporated into the carrier blend.

Preparation of Tablets

The two batches ot dried TGEV antigen were separately incorporated into tablets. The same procedure was used in both cases. The tablets were composed of the following ingredients (% dry weight) : lactose filler (78%) , acdisol disintegrant (8%) , stearic acid lubricant (1.5%) , talc as a processing aid (1.0%) , and the dried rotavirus antigen (12.5%) . The dry ingredients from each preparation were mixed well to form a dry blend, incorporated into a 3 mm dye and pressed at 550 lb pressure to produce 40 mg, 3 mm tablets.

The tablets were coated with Opadry, which is an HPMC-based polymer. This was applied by the Wurster

Spray Coating method in a fluid bed laboratory-scale unit (Strea-1; Aeromatic Inc., Columbia, MD) such that the weight increase of the tablets was 4.4%. The tablets also were coated with the enteric coating Eudragit L30D using the Wurster spray coating method. Eudragit is an aqueous dispersion of an anionic copolymer based on methacrylic acid and acrylic acid ethyl ester. The polymer dissolves at approximately pH 5.5 by forming salts with alkalis, thus affording a coating that is insoluble in gastric media, but soluble in the small intestine. Sufficient coating was applied to increase the weight of the tablets by 10.3%. The viral infectivity of both batches of tablets was determined by plaque assay. The infectivity titer of the tablets in Batch 1 was 1.37 x IO⁵ TCID₅₀/tablet, and the infectivity of the tablets in Batch 2 was 1.6 x IO⁶ TCID₅₀/tablet .

Preparation of Liquid Oral Polvmer and TGEV

The liquid oral polymer was prepared by mixing 3 g carboxymethylcellulose with 100 ml H₂0, and stirring overnight. Live virus at a concentration of IO⁷

TCID₅₀/0.5 ml H₂0 was thoroughly mixed with 5 ml of the liquid oral polymer and administered to the pigs orally, for the primary inoculation, as well as the booster immunization given at week 8.

Oral Immunization of Pigs

Young (8 week-old) piglets were immunized at week 0 and week 8 with varying doses, forms of immunogen, and delivery modes, as indicated. Antibody titers in the serum were determined at weeks 0, 3, and 8 by virus neutralization. Animals had been prescreened to select seronegative animals, but through an apparent screening error, all were seropositive as indicated by the virus neutralization assay before initial dosing, most likely due to maternal antibody. Titers fell in all animals at week 3 and by week 8, 14 of 21 animals were seronegative and the remaining animals had low titers. Therefore a second dose of TGEV was administered at week 8 and the virus neutralization assayed at week 11. By week 11, 4 of the 5 pigs that had been inoculated with enterically coated tablets containing live TGEV were seropositive with an average geometric mean titer of 12 and all three of the contact controls were seropositive with an average geometric mean titer of 18. This indicated that the group that had been administered tableted live virus had become infected and were shedding virus. It is interesting to note that those animals receiving live virus in the liquid oral polymer formulation were not able to infect the contact control pigs suggesting that this formulation inhibits viral shedding. The data describing this experiment are set forth below in Table XII, in which the asterisks (*) indicate contact controls.

TABLE Xn

ORAL INOCULATION OF PIGLETS WITH

LIVE ATTENUATED TRANSMISSIBLE

GASTROENTERΓΠS VIRUS VACCINE USING A VARIETY

OF SYNTHEΗC DELIVERY SYSTEMS

Rise in Titer

Delivery from Week 8 Seropositive Geometric

Group Dose Mode to Week 11 at Week 11 Mean Titer

B 10* Enteric tablets 3/5 4/5 12

BC* - Contact 2/3 3/3 18 controls

C IO⁷ Liquid Oral 5/5 5/5 15 Polymer

CC* - Contact 0/2 0/2 1 controls

F 10⁷ Intraduodenal 2/4 2/4 4 Intubation

FC* - Contact 0/2 0/2 1 controls

Example IX

The following example pertains to vaccine formulations that would be suitable for administration to human infants. Minute tablets or pellets can be synthesized by the above protocols with slight modifications. The tablets would then be beneficially resuspended in a suitable fluid and the vaccine would be administered orally. The need for concomitant buffer administration would be eliminated as the tablets would be coated as described in the previous examples . Thus the following methodology provides for a commercially superior, orally administered vaccine for the immunization of infants against rotavirus infection. Human Rotavirus Vaccine Antigen

Human Rotavirus vaccine (1.6 x IO¹⁰ TCID50) would be prepared exactly as in Example VI. The pellets would be resuspended in 800 ml H₂0. This solution would be used to prepare the dried human rotavirus antigen.

Preparation of the Dried Rotavirus Antigen

One lot of dried rotavirus would be prepared. The first lot would incorporate 800 ml of the rotavirus vaccine as prepared above. A formulation of cellulose acetate, sucrose, starch, and gelatin in a ratio of 30:30:30:10 would be chosen (800g) . The carrier would be ground and blended in a waring blender, the rotavirus solution would be added to 800g of the carrier blend and mixed until a wet mass forms. This would be dried in a desiccator at 4°C under vacuum. Upon completion of drying to a level of 5% H₂0, the dried rotavirus carrier blend would be ground with a mill to form a dry powder. This powder would then be incorporated into tiny granules and enterically coated.

Preparation of Granules from the Dried Rotavirus Antigen The granules would be formed in a Glatt GPCG-1 fluid bed coater/granulator apparatus with a rota- processing insert. The 800 g of dried rotavirus blend would be placed in the rota-processing unit and rotated at a high speed. Water would be sprayed with an atomizing gun while the powder is rotating in the Glatt processing unit. As the dried powder wets, agglomerates would begin to form. These should become larger as more water is applied. When the agglomerates reach an acceptable size (100-200μ) , the process would be stopped. The agglomerated particles should be round and hard because of the centrifugal process used to form them. Assuming that the particles are acceptable, a coating of Opadry and Eudragit L30D would be applied as described in Example VII. Opadry would be applied such that a weight increase of the granules would be 4-6%. Eudragit L30D would then be applied such that a weight increase would be 10-12%. Although enteric coatings have been exemplified above, other biologically acceptable coating materials may be employed, such as time release coating compositions

Pla ue Assay of Rotavirus Infectivity

After coating, the viral infectivity of the agglomerates would then be determined by plaque assay as described in materials and methods.

The foregoing experimental results indicate that successful incorporation of a live rotavirus vaccine into a delivery system is dependent on the stabilization of the rotavirus vaccine during the drying process (Fig. 1 and 2) . The use of the carrier blend of this invention produced outstanding results with only minimal losses of infectivity after the drying step (Fig. 1 and 2, Table 2) and is well-suited for further processing into a variety of final dosage forms. This blend could also be very effective in stabilizing other viruses, proteins and peptides and in the optimization of various delivery systems. Of the dosage forms assessed, tablets produced the best results, in part because the surface characteristics were conducive to the production of complete, uniform, and thus more effective, enteric coating (Fig. 4 and Table 2) . However, this dosage form must be optimized to produce compacted granules of sufficiently small size that can be successfully enterically coated and suspended in a formula suitable for immunizing infants.

Claims

What is claimed is:

1. A vaccine carrier composition in solid form suitable for oral delivery, comprising: a) a cellulosic substance, selected from the group consisting of cellulose, cellulose ester, microcrystalline cellulose, carboxymethyl cellulose and mixtures thereof; b) a sugar selected from the group consisting of sucrose, fructose, glucose, and mannose; and c) starch.

2. A vaccine carrier composition as claimed in claim 1, said composition further comprising gelatin.

3. A vaccine composition, comprising: a) an immunogenically effective amount of a virus; and b) a carrier comprising 10%-50% weight of a cellulosic substance, 30%-70% weight of a sugar, 10%-50% weight of starch and 10%-30% weight of gelatin, said percentages being based on the total weight of the carrier.

4. A vaccine composition as claimed in claim 3, wherein said virus is a live virus.

5. A vaccine composition as claimed in claim 3, wherein said virus is a rotavirus and said carrier consists essentially of a blend of 30 weight % of cellulose acetate, 30 weight % of sucrose, 30 weight % of starch, and 10 weight % of gelatin.

6. A vaccine composition as claimed in claim 3, wherein said virus is rotavirus and said carrier consists essentially of 33 weight % of cellulose acetate, 33 weight % of sucrose, and 33 weight % starch.

7. A vaccine composition as claimed in claim 3 in the form of a tablet.

8. A vaccine composition as claimed in claim 7, wherein said tablet is coated with an enteric coating.

9. A vaccine composition as claimed in claim 3, in a granular form.

10. A vaccine composition as claimed in claim 9, wherein said granules are coated with an enteric coating.

11. A method for immunizing a patient against viral infection comprising orally administering to said patient the vaccine composition of claim 3 in an amount effective to produce in said patient an immune response to said virus .

12. A method for preparing a viral vaccine for oral administration, comprising: a) adding to a carrier composition according to claim 1, a live, attenuated virus; b) drying said composition under conditions maintaining infectivity of said virus; and c) forming said dried composition into tablets.

13. A method as claimed in claim 12, wherein said composition is converted into tablet form.

14. A method as claimed in claim 12, wherein said composition is converted into granular form.

15. A method according to claim 12, further comprising coating wherein said tablets are coated with a biologically acceptable coating.