COMPOSITIONS CONTAINING AMBIENT-TEMPERATURE STABLE, INACTIVATED BUT THERAPEUTICALLY ACTIVE BIOPHARMACEUTICALS & METHODS FOR FORMULATION THEREOF

Abstract

The disclosure concerns compositions containing inactivated but therapeutically active biopharmaceuticals, and methods for formulation thereof. Biopharmaceuticals are encapsulated and immobilized in dry amorphous carbohydrate-glass and irradiated for inactivation while in the dry state. The resulting compositions provide ambient-temperature stable, therapeutically active but inactivated biopharmaceuticals for use in vaccines and other applications.

Claims

1. A method for formulation of a thermostable inactivated but potent (TIBP) vaccine, comprising: preserving one or more vaccines in a dry carbohydrate-glass, said preserving including: vacuum drying the vaccines for at least 6 hours at 40 C., or a higher temperature, to yield thermostable vaccines; the thermostable vaccines being modified for long-term stability at all ambient temperatures between 20 C. to +37 C.; wherein said thermostable vaccines are configured for long-term stability for storage and distribution without a need for refrigeration of the preserved vaccines; and subsequent to preserving the vaccines, inactivating the thermostable vaccines by irradiating said dry carbohydrate-glass containing the thermostable vaccines using a permeated ionizing radiation dose above 12.5kGa.

2. The method of claim 1, wherein said preserving further comprises preservation by vaporization (PBV), said PBV comprising: providing said one or more vaccines in a carbohydrate aqueous solution or a hydrogel to form a vaccine composition; partially freezing said vaccine composition to form a two-phase slush state thereof, wherein said two phase slush comprises a mixture of ice crystals and aqueous amorphous liquid; performing primary drying of the slush by vaporizing water under vacuum and heat application, where said vaporizing comprising simultaneous boiling of the liquid, sublimation of ice, and evaporation of water molecules from a surface of the amorphous phase; and continuing said vaporization to transform said slush into a glassy dry amorphous foam stable under said vacuum, wherein said vaccines are immobilized in the glassy carbohydrate metrics of the foam; performing secondary drying of the foam under vacuum by desorption of water from the foam to increase the glass transition temperature of the foam, where at least a part of the secondary drying is performed above 40 C.

3. The method of claim 1, wherein said foam drying is executed using preservation by foam formation (PFF), said PFF comprising: providing said one or more vaccines in a carbohydrate aqueous solution to form a vaccine composition; boiling the vaccine composition under vacuum without freezing to transform the composition into a stable glassy foam under said vacuum, wherein said vaccines are immobilized in the glassy carbohydrate metrics of the foam Performing secondary drying of the foam under vacuum by desorption of water from the foam to increase the glass transition temperature of the foam, where at least a part of the secondary drying is performed above 40 C.

4. The method of claim 1, wherein said ionizing radiation is one of: alpha, beta, or gamma radiation; electron beam radiation; proton radiation; neutron radiation; or x-ray radiation.

5. The method of claim 1, wherein said TIBP vaccine comprises a multicomponent vaccine including: one or a plurality of: viruses; bacterium; archaeon; fungi; protista; proteins, or other antigens, or a combination thereof.

6. The method of claim 2, further comprising: mixing a suspension containing one or more of: viruses; bacterium; archaeon; fungi; protista; proteins, and antigens prior to said drying; and subsequent to mixing, immobilizing the resulting mixture in the carbohydrate glass by said vacuum drying.

7. The method of claim 2, wherein said method further comprises: mixing powders prepared by milling of different dry inactivated thermostable vaccines.

8. The method of claim 7, wherein said method further comprises mixing the dry powders of protein antigens and attenuated bacterial vaccine, followed by inactivation by irradiation of the powders in the dry state.

9. The method of claim 7, wherein said method further comprises mixing the dry powders of protein antigens and attenuated viral vaccine producing the antigen prior to inactivation by irradiation of the powders in the dry state.

10. The method of claim 7, wherein said method further comprises mixing dry powders of anthrax rPA antigens and attenuated anthrax vegetative bacterial vaccine producing the antigens, followed by inactivation by irradiation of the powders in the dry state.

11. The method of claim 7, wherein said method further comprises mixing the dry powders of anthrax rPA antigens and attenuated Sterne strain anthrax bacterial vaccine, followed by inactivation by irradiation of the powders in the dry state.

12. The method of claim 7, wherein said thermostable multicomponent anthrax vaccine was prepared by mixing dry powders of a Rabies antigen and attenuated rabies viral (ERA) vaccine inactivated by irradiation in the dry state.

13. The method of claim 1, wherein said method further comprises mixing powders made by milling different dry thermostable therapeutic products sterilized by irradiation.

14. The method of claim 5, wherein said multicomponent vaccine comprises coagulation factors, enzymes, hormones, cytokines, growth factors, peptides, or a combination thereof.

15. The method of claim 5, wherein said multicomponent vaccine comprises enzymes, hormones, cytokines, growth factors, peptides or nucleic acids.

16. A method for formulation of a thermostable composition comprising one or more inactivated but therapeutically active (IBTA) biopharmaceuticals, the method comprising: preserving the one or more biopharmaceuticals in a dry state, said preserving comprising: providing said one or more biopharmaceuticals in one of an aqueous solution or a hydrogel to form a first composition; partially freezing said first composition to form a two-phase state thereof, wherein said first composition comprises an amount of ice and an amount of liquid water in said two-phase state; vaporizing said first composition, said vaporizing comprising simultaneously applying vacuum and heat, wherein water is removed from said first composition through simultaneous boiling of the liquid, sublimation of ice, and evaporation of water molecules from a surface of the liquid; and continuing said vaporization to transform said first composition into a dry foam, wherein said dry foam forms an amorphous carbohydrate-glass matrix with said one or more biopharmaceuticals encapsulated and immobilized therein; following said preserving, irradiating said one or more biopharmaceuticals that are encapsulated and immobilized in the dry state to yield IBTA biopharmaceuticals, wherein said irradiating comprises: subjecting said amorphous carbohydrate-glass matrix containing the encapsulated and immobilized biopharmaceuticals to an electron beam; and forming said IBTA biopharmaceuticals into said composition, wherein said forming said IBTA biopharmaceuticals into said composition comprises at least one of: reconstituting said amorphous carbohydrate-glass matrix containing the IBTA biopharmaceuticals in an aqueous solution, combining said amorphous carbohydrate-glass matrix containing the IBTA biopharmaceuticals with a dry compound to form a therapeutic mixture, or micronizing said amorphous carbohydrate-glass matrix containing the IBTA biopharmaceuticals to form a therapeutic powder.

17. A method for formulation of a therapeutic composition comprising inactivated but therapeutically active (IBTA) biopharmaceuticals, the method comprising: immobilizing one or more biopharmaceuticals in a dry amorphous carbohydrate-glass matrix; irradiating said biopharmaceuticals immobilized in the dry amorphous carbohydrate-glass matrix to inactivate the biopharmaceuticals yielding immobilized IBTA biopharmaceuticals; and forming said immobilized IBTA biopharmaceuticals into the therapeutic composition.

18. The method of claim 17, wherein said immobilizing one or more biopharmaceuticals in a dry amorphous carbohydrate-glass matrix comprises: providing said biopharmaceuticals in one of an aqueous solution or a hydrogel to form a first composition; partially freezing said first composition to form a two-phase state thereof, wherein said first composition comprises an amount of ice and an amount of liquid water in said two-phase state; vaporizing said first composition, said vaporizing comprising simultaneously applying vacuum and heat, wherein water is removed from said first composition through simultaneous boiling of the liquid, sublimation of ice, and evaporation of water molecules from a surface of the liquid; and continuing said vaporization to transform said first composition into a dry foam, wherein said dry foam forms said carbohydrate-glass matrix.

19. The method of claim 18, wherein said hydrogel comprises calcium alginate.

20. The method of claim 17, wherein said immobilizing one or more biopharmaceuticals in a dry amorphous carbohydrate-glass matrix comprises: using a preservation by foam formulation technique.

21. The method of claim 17, wherein said irradiating said biopharmaceuticals immobilized in the amorphous carbohydrate-glass matrix comprises: subjecting said biopharmaceuticals immobilized in the amorphous carbohydrate-glass matrix to electron beam irradiation.

22. The method of claim 17, wherein said irradiating comprises any of: electron beam, Gamma, X-ray, proton, neutron, UV irradiation, or a combination thereof.

23. The method of claim 17, wherein said irradiating comprises a radiation dose of at least 12 kGy.

24. The method of claim 17, wherein said forming said immobilized IBTA biopharmaceuticals into the therapeutic composition comprises at least one of: reconstituting said amorphous carbohydrate-glass matrix containing immobilized IBTA biopharmaceuticals in an aqueous solution, combining said amorphous carbohydrate-glass matrix containing immobilized IBTA biopharmaceuticals with a dry compound to form a therapeutic mixture, or micronizing said amorphous carbohydrate-glass matrix containing immobilized IBTA biopharmaceuticals to form a therapeutic powder.

25. The method of claim 17, wherein said biopharmaceuticals comprises one or more: viruses; bacterium; archaeon; fungi; protista; proteins, antigens, or a combination thereof.

26. The method of claim 25, wherein said biopharmaceuticals comprises a combination of bacteria, viruses, and proteins.

27. The method of claim 26, wherein said combination of bacteria, viruses, and proteins is combined prior to immobilizing in the sugar-glass.

28. The method of claim 26, wherein said combination of bacteria, viruses, and proteins is combined after immobilizing in the sugar-glass, wherein said combination comprises, with three distinct amorphous carbohydrate-glass materials; each of the three materials containing one of the bacteria, viruses, and proteins; milling each of said three materials, and combining powders resulting therefrom.

29. The method of claim 17, wherein said biopharmaceuticals comprises: killed but metabolically active (KBMA) cellular biopharmaceuticals.

30. A therapeutic composition, comprising: an amount of inactivated but therapeutically active (IBTA) biopharmaceuticals immobilized in a dry amorphous carbohydrate-glass, said biopharmaceuticals being inactivated by irradiation while in the dry state; wherein said composition is ambient-temperature stable.

31. The composition of claim 30, comprising: a first IBTA biopharmaceutical powder comprising an amount of first inactivated but therapeutically active biopharmaceuticals immobilized in a first carbohydrate-glass and irradiated in a dry state; a second IBTA biopharmaceutical powder comprising an amount of second inactivated but therapeutically active biopharmaceuticals immobilized in a second carbohydrate-glass and irradiated in a dry state; each of the first and second inactivated but therapeutically active biopharmaceuticals being independently selected from the group consisting of: viruses; bacteria; archaea; fungi; protista; or a combination thereof; each of said first and second IBTA biopharmaceutical powders being combined in a mixture.

32. The composition of claim 30, comprising: an IBTA biopharmaceutical powder comprising micronized carbohydrate-glass particles; each of said particles forming an encapsulation of two or more inactivated but therapeutically active biopharmaceuticals immobilized therein.

33. The composition of claim 30, wherein said therapeutically active biopharmaceuticals comprises one or more: viruses; bacterium; archaeon; fungi; protista; proteins, antigens, or a combination thereof.

Description

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0066] For purposes of this invention, the terms foam drying and vacuum foam drying are herein to describe various drying techniques for obtaining preserved materials, including preservation by foam formulation (PFF) as described in U.S. Pat. No. 5,766,520; and preservation by vaporization (PBV) as described in WO 2005/117962; the contents of each of which are hereby incorporated by reference.

[0067] The term preservation by vaporization (PBV) describes a current state of the art foam drying technique for preserving sensitive biological material in an amorphous sugar-glass by simultaneous sublimation, evaporation and boiling of water from within a partially frozen slush-state, as is further described in WO 2005/117962.

[0068] The terms carbohydrate-glass or sugar-glass are used herein to describe an amorphous solid carbohydrate matrix including one or more carbohydrates, generally sugars. The matrix may further include amino acids, salts, surfactants and polymers that were dissolved in preservation solutions prior to drying.

[0069] In accordance with aspects of the invention, it is a primary objective to provide vaccines and other biopharmaceutical products with improved therapeutic activity, and to enhance methods for formulating such products.

[0070] In a general embodiment, a method for formulation of a therapeutic composition containing inactivated but therapeutically active (IBTA) biopharmaceuticals comprises: (i) preserving biopharmaceuticals in a dry state; (ii) irradiating the biopharmaceuticals in the dry state to yield IBTA biopharmaceuticals; and (iii) using the IBTA biopharmaceuticals to form the therapeutic composition.

[0071] Preserving the biopharmaceuticals in the dry state can comprise any foam drying technique, including preservation by foam formulation, or preservation by vaporization as referenced above. In each of these techniques, the result is an amount of biopharmaceuticals being stabilized within a dry amorphous carbohydrate-glass matrix.

[0072] Although not preferred, it is conceivable that a similar result can be achieved using lyophilization of a biopharmaceuticals suspension; however, at the risk of harming sensitive biomaterial structures of the biopharmaceuticals and thereby reducing therapeutic effectiveness. Other techniques can be similarly implemented for preserving the biopharmaceuticals.

[0073] Once stabilized in the dry amorphous carbohydrate-glass matrix, the encapsulated and immobilized biopharmaceuticals are subjected to irradiation. The irradiation ideally comprises electron beam (EB) irradiation, but may alternatively include: Gamma, X-ray, proton, neutron, UV irradiation, or a combination thereof. Although radiation dose can be varied, the radiation dose should be at least 12 kGy in certain embodiments.

[0074] Once preserved, and subsequently irradiated, the resulting inactivated but therapeutically active (IBTA) biopharmaceuticals can be further formed into a composition by any of: (i) reconstituting the encapsulated and immobilized IBTA biopharmaceuticals in an aqueous solution, (ii) combining the encapsulated and immobilized IBTA biopharmaceuticals with a dry compound to form a therapeutic mixture, or (iii) micronizing the encapsulated and immobilized IBTA biopharmaceuticals to form a therapeutic powder.

[0075] In an embodiment, an amount of first therapeutically active biopharmaceuticals is preserved using a foam drying technique, the first preserved biopharmaceuticals are further inactivated by irradiation in the dry foam state, and subsequently micronized to yield a first preserved biopharmaceutical powder. An amount of second therapeutically active biopharmaceuticals is preserved using a foam drying technique, the second preserved biopharmaceuticals are inactivated by irradiation in the dry foam state, and subsequently micronized to yield a second preserved biopharmaceutical powder. Each of the first and second biopharmaceutical powders are then combined to yield a therapeutic composition, in this case a mixture of distinct preserved and inactivated biopharmaceuticals. Note that the foam drying technique can be any foam drying technique, and is independent for producing each of the first and second powders.

[0076] In another embodiment, an amount of first therapeutically active biopharmaceuticals is mixed with an amount of second therapeutically active biopharmaceuticals. The first and second biopharmaceuticals are collectively preserved using a foam drying technique, and further irradiated when in the dry state. The resulting foam is micronized to yield a powder containing inactivated but therapeutically active (IBTA) biopharmaceuticals for biopharmaceutical applications.

[0077] Experiments have shown that with biopharmaceuticals immobilized in carbohydrate-glass, electron beam irradiation can be used to reduce the viability of dry preserved bacteria more than one million times with minimal loss of metabolic activity, indicating that the internal bacterial proteins and enzymes are structurally and functionally intact after EB irradiation. In contrast, UV irradiation of cells in the liquid state can result in the formation of highly reactive radicals that can degrade proteins. An important benefit of using EB irradiation to produce vaccines is that radiation in the dry state will damage nucleic acids and not protein antigens on the bacterial surface. EB irradiation of dried biologicals is a simple and inexpensive procedure that does not require addition of psoralen.

[0078] In the embodiments herein, it is preferred to first make the biopharmaceuticals ambient-temperature stable by encapsulating in a carbohydrate-glass matrix, and then attenuate them by EB irradiation. This approach enables one to evaluate survival of dry preserved bacteria prior to irradiation, and assess protein integrity after irradiation by measuring metabolic activity instead of performing expensive animal immunogenicity studies.

[0079] Compositions containing ambient-temperature stable, inactivated but therapeutically active (IBTA) biopharmaceuticals present a unique opportunity for safe and efficacious treatment options.

[0080] It is important to note that primary irradiation in the liquid state produces harmful free radicals that unpredictably damage the biological components. Accordingly, the methods described herein incorporate irradiation in the dry state, after stabilization, which effectively limits production of free radicals, and targets only the nucleic acids required for replication while maintaining the metabolism-associated structures intact.

[0081] In contrast, methods which irradiate first and preserve second will fail to retain cellular metabolism. Because they are liquid-irradiated first, all the cells that are subsequently stabilized are preserved in the damaged state with much of their metabolic activity already lost.

[0082] Thus, when inactivated properly, appropriate organisms will retain therapeutic potency and be a safer alternative to their live counterparts.

[0083] In one embodiment, the step of encapsulating the therapeutically active biopharmaceuticals in a dry amorphous carbohydrate-glass matrix is achieved using the technique known as Preservation by Vaporization (PBV). PBV generally includes: (i) providing the biopharmaceuticals in one of an aqueous solution or a hydrogel to form a first composition; (ii) partially freezing the first composition to form a two-phase state thereof, wherein the first composition comprises an amount of ice and an amount of liquid water in the two-phase state; (iii) vaporizing the first composition, the vaporizing comprising simultaneously applying vacuum and heat, wherein water is removed from the first composition through simultaneous boiling of the liquid, sublimation of ice, and evaporation of water molecules from a surface of the liquid; and continuing the vaporization to transform the first composition into a dry foam, wherein the dry foam forms the carbohydrate-glass matrix that encapsulates the biopharmaceuticals.

[0084] Where a hydrogel is incorporated into the first composition, the hydrogel can comprise calcium alginate.

[0085] PBV is a current state of the art because the process yields high thermostability without significant disturbance of activity. For this reason, PBV is preferred, however, other foam drying techniques can be similarly implemented to encapsulate the biopharmaceuticals in a dry amorphous carbohydrate-glass matrix, for example, by using the technique known as preservation by foam formulation (PFF) as referenced above.

[0086] FIG. 5 illustrates a method for producing thermostable inactivated but therapeutically active biopharmaceuticals.

EXAMPLES

Example 1

Preparation of Inactivated but Therapeutically active (IBTA) Viral Lave Attenuated Vaccine

[0087] Fixed rabies virus (RV), Evelyn-Rokitnicki-Abelseth (ERA) strain, was attenuated using a reverse genetics system. The recovered virus was sequenced and had only the desired change (R333E). The resulting virus, referred to as ERAg333, was grown.

[0088] Supernatant was mixed (1:2) with 30% sucrose and 15% methylglucoside in phosphate buffer (pH=7.0). 0.5 ml of mixture was distributed into crimp vials and dried using Genesis and Virtis Ultra freeze-dryers that were modified to allow better vacuum pressure control. After two hours of processing, the solid material formed a stable dry foam. Secondary drying was performed under vacuum at 35 C. and 45 C. for 20-24 hours. RV preservation by vaporization (PBV) in crimp vials at 22 C. with desiccant was electron beam-irradiated at various doses. Virus titers were measured as described below except in 96-well plates on four consecutive days post infection.

[0089] Commercially available RV vaccine RabAvert was purchased and reconstituted according to the manufacturer's instructions.

[0090] RV PBV in crimp vials was placed at 22 C. with desiccant, in a dry incubator at 37 C., in mineral oil bath at 80 C. and 90 C. for viability, or in a water bath at 80 C. for electrochemiluminescent (ECL) assays. Vials were removed at different time points and reconstituted with 0.4 ml phosphate buffered saline (PBS) (0.01M, pH 7.4). Virus titers were measured by serially diluting vaccine with BHK-21 cells in an 8-well chamber slide as described previously. The mean focus forming units (ffu)/ml and standard deviation were calculated from at least three statistical replicates.

[0091] The Meso Scale Discovery platform was used to perform RV antigen capture ECL assays as described previously. RV glycoprotein (G) monoclonal antibody (MAb) 62-80-6 was used at 1 g/ml for capture and 0.5 g/ml for detection.

[0092] Approved animal use protocols were established with CDC IACUC. Blood was collected as described previously from female, 4-week-old, CD-1 mice assigned to groups of 10, and the geometric mean titer (GMT) of RV neutralizing antibodies (rVNA) in international units (IU)/ml was determined using a rapid fluorescent focus inhibition test (RFFIT) or a modified RV neutralization test for small volumes. Live attenuated RV PBV vaccine, placebo, and inactivated RV PBV, stored for 36 days at 22 C. in the dark with desiccant, were reconstituted with 0.4 ml of sterile PBS (0.01M, pH 7.4) without calcium or magnesium. Reconstituted vaccine and RV ERAg333 from frozen stock was subsequently diluted using the same PBS. On day 0, mice were vaccinated intramuscularly (IM) in the right leg as described previously. Back titrations of dilutions used to vaccinate mice were completed as described above. For inactivated vaccines, the BCA Protein Assay was used according to manufacturer's instructions to determine total protein concentration. Blood was collected again from all mice on day 14 and 30, and rVNA GMT was determined. On day 30 all mice were challenged IM in the left leg with 50 l of canine RV 3374R. Animals were monitored and euthanized when showing signs of rabies as described previously. The brain stem was collected from euthanized animals and subjected to the direct fluorescent antibody (DFA) test for rabies. The experiment was terminated 30 days after the last death in the placebo group. Probability values were calculated using chi-square test with a 95% confidence interval.

[0093] Results

[0094] The starting titer of RV ERAg333 before PBV was 8.3 log 10 ffu/ml. After PBV, about 0.2 log 10 of viable virus was lost resulting in 8.110.12 log 10 ffu/ml. For inactivated vaccines, electron beam-irradiation at all tested doses damaged RV and resulted in lower virus titers; no viable virus was recovered in samples treated with the highest dose of 12 kGy (Table 1). The complete inactivation of rabies virus after treatment with 12 kGy was confirmed in three blind cell passages.

TABLE-US-00001 TABLE 1 Inactivation of RV PBV by electron beam Dose Rabies virus titer (log.sub.10 ffu/ml) (kGy) 24 hrs 48 hrs 72 hrs 96 hrs 0 8.16 0.1 TNC.sup.a TNC TNC 2.9 6.03 0.04 TNC TNC TNC 4.2 4.82 0.24 TNC TNC TNC 6 4.32 0.25 TNC TNC TNC 12 BLD.sup.b BLD BLD BLD .sup.aToo numerous to count (TNC) .sup.bBelow level of detection (BLD); no virus detected

[0095] RV PBV was stored at 22 C. with desiccant for 1, 2, 3, 15, or 23 months. After a 0.5 log 10 drop in the first two months, the vaccine was stable up to the end of the experiment, when viability only decreased approximately 0.6 log 10 (Table 2). RV PBV was incubated at 37 C. for 1, 2, 15, or 23 months. After 2 months, viability dropped <1 log 10 and after 15 months dropped 1.5 log 10. RV PBV was placed at 80 C. or 90 C. After 3 hours at 80 C., viability was essentially the same, and only 1 log 10 of viable virus was lost after 16 hours. Incubation at 90 C. was significantly more damaging, and RV PBV lost >1 log 10 of activity after 1 hour at 90 C.

TABLE-US-00002 TABLE 2 Viability of RV after PBV and storage at different temperatures Rabies virus titer (log.sub.10 ffu/ml) Temp. Initial 1 hrs 2 hrs 3 hrs 16 hrs 1 M 2 M 3 M 15 M 23 M 22 C. 7.91 0.05 ND.sup.a ND ND ND 7.51 0.07 7.40 0.09 7.39 0.12 7.42 0.04 7.33 0.05 37 C. 7.56 0.14 ND ND ND ND 7.13 0.17 6.99 0.01 ND 6.1 0.09 5.58 0.22 80 C. 7.51 0.07 7.56 0.09 7.48 7.46 0.05 6.53 0.05 ND ND ND ND ND 0.15 90 C. 7.51 0.07 6.07 0.09 ND ND ND ND ND ND ND ND .sup.aNot determined (ND)

[0096] MAb 62-80-6 was used for capture and detection of RV G in an antigen capture assay and counts g-1 ml-1 were estimated from the best fit linear regression. In agreement with the measured virus titers, live attenuated RV PBV had the same counts g-1 ml-1 as the original ERAg333 virus (Table 3).

TABLE-US-00003 TABLE 3 Antigenic G content of different RV vaccines measured by antigen capture assay using the 62-80-6 RV G MAb Storage Conditions Temper- ECL Counts Antigen Time ature g.sup.1 ml.sup.1a ERAg333.sup.b 20 M 80 C. 2200 Live attenuated RV PBV 20 M 22 C. 2200 Commercial vaccine 25 M 4 C. 1400 3 hrs 80 C. 980 Inactivated RV PBV 20 M 22 C. 1300 3 hrs 80 C. 680 Native ERA G.sup.c 18 M 80 C. 9100 Denatured ERA G.sup.c 10 mins 98 C. 7 Placebo 20 M 22 C. 3 .sup.aEstimated from the best fit linear regression of means of at least four statistical replicates from at least two biological replicates of eight 5-fold serial dilutions .sup.bParent strain for both live attenuated and inactivated vaccines; generated by reverse genetics .sup.cPurified RV ERA glycoprotein

[0097] Inactivation of RV PBV by electron beam irradiation resulted in a decrease in antigen content but was similar to a commercial inactivated vaccine. When inactivated RV PBV was placed at 80 C. with high humidity for 3 hours, antigen decreased 48% while decreasing 30% in a commercial vaccine incubated under the same conditions.

[0098] Live attenuated and inactivated RV PBV was used to vaccinate mice IM. Both live and inactivated RV PBV effectively induced rVNA titers by day 14 (Table 4). Live vaccine induced rVNA titers similar to ERAg333 and commercial vaccine. On day 30 rVNA titers increased in groups that received live RV PBV surpassing ERAg333 and commercial vaccine. Inactivated RV PBV induced rVNA titers on day 30 similar to commercial vaccine on day 14.

[0099] The different dilutions of live attenuated RV PBV induced similar rVNA titers on day 14 and 30. Only the undiluted and 10-1 dilution of inactivated RV PBV vaccine induced rVNA titers by day 30. The decreased immunogenicity of the inactivated RV PBV is consistent with the in vitro antigen capture results.

[0100] On day 30 all mice were challenged with canine street RV IM in the hind leg. All animals that received commercial vaccine survived (Table 4, p<0.01 compared to placebo). All animals also survived in groups that received ERAg333 or live RV PBV, consistent with the observed rVNA responses. In groups that received inactivated RV PBV all animals survived except in the 10-2 group. In this group, 80% survived despite only 3 individuals having a measurable rVNA response. Survivorship in this group was significantly different compared to the placebo (p<0.05) but not compared to the commercial vaccine or other inactivated RV PBV groups. At the experimental endpoint, animals from each group were randomly selected for rabies diagnosis, and all were rabies DFA negative.

TABLE-US-00004 TABLE 4 Immunogenicity and efficacy of rabies vaccine preserved by vaporization in mice Live Attenuated Commercial Vaccine ERAg333.sup.a Placebo Vaccine Inactivated Vaccine Group 1 2 3 4 5 6 7 8 9 Dilution 1-Oct 2-Oct 3-Oct 2-Oct None None None 1-Oct 2-Oct Titer 6.8.sup.b 5.7 4.4 7.9 NA.sup.c NA NA NA NA Load ND.sup.d ND ND ND 300.sup.e 620 350 34 2.3 GMT day 14.sup.f 0.26.sup.g 0.11 0.2 0.37 <0.05 0.23 0.07 <0.05 <0.05 SD day14.sup.h 0.50.sup.g 0.70 0.95 0.98 0.0097 0.56 0.11 0.24 0.022 GMT day 30.sup.f 1.60.sup.g 0.96 1.7 0.84 <0.05 0.58 0.27 0.13 <0.05 SD day30.sup.h 2.4.sup.g 15 2.9 1.9 0.015 1.9 1.4 0.58 0.14 Survival.sup.i 100%.sup.j 100% 100% 100% 22% 100% 100% 100% 80%.sup.k .sup.aParent strain for both live attenuated and inactivated vaccines; generated by reverse genetics .sup.bLog.sub.10 ffu in 0.1 ml dose .sup.cNot applicable (NA); cannot be determined for inactivated vaccines .sup.dNot determined (ND) .sup.eg of total protein in 0.1 ml dose .sup.fGeometric mean titer (GMT) of rabies virus neutralizing antibodies .sup.gIU/ml .sup.hStandard deviation (SD) of rabies virus neutralizing antibody titers .sup.iGroup size = 10 except placebo n = 9 .sup.jp < 0.01 compared to placebo .sup.kp < 0.05 compared to placebo

[0101] Discussion

[0102] RV ERAg333 was successfully formulated into stable, dry foam using PBV technology. Live attenuated RV PBV was stable for 23 months at 22 C. and 2 months at 37 C. Stability decreased as temperature increased, yet RV PBV remained stable for at least 3 hours at 80 C.

[0103] An antigen capture assay was used to compare the antigen content of different vaccines. Since the ERAg333 virus was used for RV PBV preparation, MAb 62-80-6 which binds a linear epitope in the G was used for both antigen capture and detection. By using the same antibody for capture and detection, only trimeric G is detected. This was confirmed by low ECL counts for heat denatured purified RV G antigen. While the antigen capture assay is not a substitute for potency testing, it can be used to project if vaccines are immunogenic. Live attenuated and inactivated RV PBVs were both antigenic and immunogenic.

[0104] A single dose of live attenuated or inactivated RV PBV effectively induced rVNA and protected all mice from IM challenge with a canine RV. By day 30 the antibody response to live attenuated RV PBV surpassed commercial vaccine. Previous challenge experiments using the same RV, dose, and route found 100% mortality in unvaccinated mice. However, the IM challenge, while more closely modeling natural infection, introduces greater variability.

[0105] The advantages of PBV are that live attenuated RV can be stabilized and formulated into an oral vaccine suitable for use in domestic or wild animals. These results also support the use of PBV technology for other vaccines, e.g. RV-vectored ebola vaccine. Inactivated RV PBV if formulated into a potent vaccine and paired with a needle-less delivery system could be considered for human use in the future. Access to safe, potent vaccines is paramount for canine rabies elimination and prevention of rabies in humans.

Example 2

Preparation of Ambient-Temperature Stable, Inactivated but Therapeutically Active (IBTA) Probiotics and Other Bacteria

[0106] FIGS. 1-4 describe an example including formulation of ambient-temperature stable, inactivated and metabolically active vegetative form of Bacillus anthracis and Lactobacillus acidophilus bacteria.

[0107] B. anthracis Sterne strain bacteria were dry preserved using PBV (FIG. 1) and subsequently exposed to electron beam (EB) irradiation. Bacterial survival (CFU) and metabolic (reducing) activity of bacteria were measured using an MTT assay for two hours after the bacteria were reconstituted. A 12.5 kGy dose decreased survival of B. anthracis bacteria measured by colony forming units on agar more than one million times with only 0.5 log decrease in metabolic activity during the first hour after reconstitution (FIG. 2). In studies with other PBV-preserved L. acidophilus (see FIG. 3) we demonstrated that a 12.5 kGy EB irradiation dose decreased bacterial survival more than a million times with no detectable loss in metabolic activity during the first 1.5 hours after reconstitution (see FIG. 4). These results suggest that EB processing did not damage integrity of bacterial membranes, integrity of the bacterial intermembrane proteins and integrity of intracellular reducing enzymes. Thus EB radiation damages mostly nucleic acids.