WO2010006326A2

WO2010006326A2 - Methods and compositions for spore-based vaccines

Info

Publication number: WO2010006326A2
Application number: PCT/US2009/050356
Authority: WO
Inventors: John E. Herrmann; Boris R. Belitsky; Abraham L. Sonenshein; Saul Tzipori
Original assignee: Tufts University
Priority date: 2008-07-11
Filing date: 2009-07-13
Publication date: 2010-01-14
Also published as: WO2010006326A3

Abstract

Methods for immunizing a subject to an infectious agent are provided, using vegetative cytoplasmic expression of an antigen encoding an infectious agent or spore surface display of the antigen, and contacting the subject with a composition including a spore or a vegetative cell or both with or without an adjuvant. Also included are a thermally- stable vaccine composition using the method described above and a kit for its use.

Description

Methods and compositions for spore-based vaccines

Related Applications This invention claims the benefit of U.S. provisional application serial number

61/134,700 filed July U₅ 2008, inventors Hermann et al., and which is hereby incorporated herein in its entirety by reference.

Technical Field Methods for immunizing a subject to an infectious agent using vegetative cytoplasmic expression of an antigen of an infectious agent and a thermally-stable vaccine composition arc provided.

Background Bacillus subtilis is a Gram-positive, catalase-positive bacterium commonly found in soil. Members of the genus Bacillus have the ability to form tough, protective endospores, a characteristic which allows the spores of the organism to tolerate extreme environmental conditions, to be heat resistant, and to quantitatively survive lengthy exposure to a wide range of temperatures including freezing and boiling, without loss of viability. B. subtilis has a long safety record as a food component and as a probiotic, e.g., used in microbial feed supplements to improve intestinal microbial balance by competitively excluding pathogens both in animals and humans. Other Bacillus species are well-known biological insecticidal agents, e.g., Bacillus thuringiensis (Dipel^®) is used to combat gypsy moths without harm to other wildlife. Bacillus natio includes food-grade strains mainly used for the fermentation of soybeans, which fermentation process eventually results in a cheap and nutritious food that is rich in amino acids. In fact the term "natto" refers to a Japanese soybean fermented product "Natto", which is a widely used commercial product.

Bacilli have been studied extensively by researchers and as a result this family includes species with well-characterized genetic and physiological systems. B. subtilis has become a model organism for Gram-positive bacteria, and numerous studies have been published involving manipulation of its genetic structure and regulation of expression of its proteins. There remains a need for vaccines that are easily produced in large quantities and at low cost to prevent and control emerging viral epidemic and epizootic diseases. Vaccines based on bacterial production systems that can be stabilized for use in tropical areas and under other conditions to minimize loss of activity in areas having minimal storage capabilities are available.

Summary

An aspect of the invention provides a method of immunizing a subject to an infectious agent, the method including steps of: sporulating a vegetative host bacterial cell which contains an isolated nucleotide sequence encoding an antigen of the infectious agent, such that the nucleotide sequence is operably linked to a promoter for cytoplasmic vegetative expression of the antigen, such that the vegetative cells and spores are associated with the antigen and, contacting the subject with a composition including the vegetative cells and the spores, such that the antigen immunizes the subject to the infectious agent. Another aspect of the invention provides a method of immunizing a subject to an infectious agent, the method including steps of: sporulating a vegetative host bacterial cell which contains an isolated nucleotide sequence encoding an antigen of the infectious agent, such that the nucleotide sequence is operably linked to a promoter for expression of the antigen as a fusion to a spore coat protein, such that spores arc associated with the antigen and, contacting the subject with a composition including the spores, such that the antigen immunizes the subject to the infectious agent.

For example, the infectious agent is viral or bacterial. In an embodiment of the method, the infectious agent is at least one bacterium selected from the group including Bacillus anthracis, Clostridium ietani, Corynβbacterium diphtheriae, Bordelella pertussis, Mycobacterium tuberculosis, Salmonella iyphimurium, Staphylococcus aureus,

Streptococcus pneumoniae, Treponema pallidum, Neisseria gonorrhoeae, and the like. In alternative embodiment, the infectious agent is at least one virus selected from the group consisting of human immunodeficiency virus (HIV), influenza, polio, herpes, smallpox, measles, mumps, rubella, rotavirus, chicken pox, rabies, West Nile virus, eastern equine encephalitis, norovirus, and the like. An antigen in related embodiments includes a rotovirus antigen or a Clostridium tetani antigen.

Another aspect of the invention provides a method of immunizing a subject to an infectious agent, the method including steps of: sporulating a vegetative host bacterial cell which contains an isolated nucleotide sequence encoding an antigen of the infectious agent, such that the nucleotide sequence is operably linked to a promoter and coding sequence for a spore coat protein, such that the spores display the antigen on their surfaces, and contacting the subject with a composition including the spores, such that the antigen immunizes the subject to the infectious agent.

Another aspect of the invention provides a method of immunizing a subject to an infectious agent, the method including steps of: cultivating a vegetative host bacterial cell which contains an isolated nucleotide sequence encoding an antigen of the infectious agent, such that the nucleotide sequence is operably linked to a promoter and coding sequence for cytoplasmic vegetative expression of the antigen, such that the vegetative cells are associated with the antigen and, contacting the subject with a composition including the vegetative cells, such that the antigen immunizes the subject to the infectious agent.

The method in various embodiments further includes prior to sporulatmg, obtaining the isolated nucleotide sequence encoding the rotavirus antigen from a rotavirus strain that is bovine or murine. For example, the rotavirus antigen is a viral virion protein. The viral virion protein, for example, is selected from the group consisting of VP2, VP4, VP6, VP7, NSP4, and a portion or a derivative thereof.

In other embodiments the method includes, prior to sporulatmg, obtaining the isolated nucleotide sequence encoding an antigen from a bacterial pathogen. For example, the antigen is a toxin protein or a polypeptide component of a toxin. The toxin protein, for example, is selected from the group consisting of tetanus toxin, diphtheria toxin, pertussis toxin, Staphylococcus aureus toxins, anthrax toxin, Salmonella typhimuriurn toxins, Streptococcus pneumoniae toxins, Treponema pallidum toxins, Neisserria gonorrhoeae toxins, and a portion or a derivative thereof. In general, the subject is a vertebrate animal. For example, the vertebrate animal is selected from the group consisting of an agricultural animal, a high value zoo animal, a research animal, a human, and a wild animal found in a dense human environment.

In related embodiments, the method includes contacting the subject by administering the composition by a route selected from the group consisting of intravenous, intramuscular, intraperitoneal, intradermal, mucosal, and subcutaneous routes. For example, contacting the subject is intranasal administration. For example, the intranasal administration further includes inhalation or nose drops. in general, an immunizing host bacterial cell is a Bacillus cell. For example, the Bacillus is Bacillus subtilis.

The composition used in certain embodiments of the method of the present invention further includes an adjuvant. For example, the adjuvant is selected from the group consisting of cholera toxin, a non-toxic variant of Escherichia coli labile toxin, and a portion or a derivative thereof.

In related embodiment, the method further includes observing resistance of the composition to a condition selected from at least one of the group of heat, drying, freezing, deleterious chemicals and radiation. For example, the method includes lyophilizing the composition prior to contacting. For example, resistance to heat involves observing resistance at 6O⁰C for one hour and 45⁰C for at least 30 days, such that a heat-treated composition maintains ability to generate full protective immunity.

An embodiment of the method includes measuring an antibody titer in serum of an infected subject, such that an increase in antibody for the antigen in comparison to a control serum is an indication of efficacy of the immunogenicity of the composition. In another embodiment, the method further involves measuring an amount of viral shedding in the subject having been afflicted by the infectious agent, such that a decrease in fecal virus as compared to that in a control also afflicted by the infectious agent and not contacted with the composition, is a measure of efficacy of the immunogenicity of the composition. An aspect of the invention provides a thermally-stable vaccine composition for immunizing a subject with an antigen from an infectious agent. For example, the composition includes at least one of vegetative cells and spores, i.e., vegetative cells or spores or both vegetative cells and spores from a Bacillus cell, such that the cell contains an isolated nucleotide sequence encoding the antigen, the nucleotide sequence being genetically engineered and having been integrated into the host bacterial chromosome or carried on a plasmid and provided with appropriate transcriptional and trans] ational regulatory sequences to express the antigen cytoplasmically as a soluble component during vegetative growth, and upon sporulation by the cell, the antigen is associated with the vegetative cells and the spores, and the composition including vegetative cells and spores is effective to immunize the subject.

An embodiment of the composition includes the antigen as a viral protein or a portion or a derivative thereof. For example, the viral protein is a viral virion protein. For example, the viral virion protein is selected from at least one of the group consisting of VP2, VP4, VP6, VP7, NSP4, and a portion or a derivative thereof. The composition Bacillus in certain embodiments is Bacillus sublilis.

In a related embodiment, the composition further includes an adjuvant. For example, the adjuvant is at least one of cholera toxin, a non-toxic variant of Escherichia coli labile toxin, and a portion or a derivative thereof. The composition includes the isolated nucleotide sequence encoding the antigen from a strain that is bovine or murine. Components of the composition in certain embodiments are treated with heat to remove substantially all water. For example, components are treated by at least one of centrifugation under vacuum, lyophilization, spray drying and the like. The present invention aiso features a vaccination kit that includes a unit dose of the composition according to any of the above, a container, and instructions for use. For example, the instructions include storage at a room temperature from about 4"C to about 45⁰C and the like.

Brief Description of the Drawings

Fig. 1 is a line graph showing serum antibody titer to rotavirus VP6 observed following intranasal immunization of mice with B. subtihs control spores (squares) or B. subiilis spores associated with bovine (triangles) or murine (circles) VP6 antigen as a function of time (days after the first immunization). B. subtilis spores were administered with cholera toxin (open symbols) or without cholera toxin (closed symbols) as an adjuvant.

The data show that animals administered VP6 antigen produced antibody, and that antibody titer was improved by use of the adjuvant.

Fig. 2 is a line graph showing amount of rotavirus in feces as a function of time (days) after rotavirus challenge of mice previously immunized with each of: B. subtilis control spores (squares) and B. subtilis spores associated with either VP6 antigen from rotavirus strains of bovine origin (triangles) or with rotavirus of murine origin (circles). The B. subtilis spores were administered with cholera toxin (ct; open symbols) or without cholera toxin (closed symbols). The disease model was epizootic diarrhea of infant mice (EDIM). The data show that fecal viral content was reduced in EDΪM mice previously inoculated with murine VP6 associated spores or with bovine VP6 associated spores, compared to control spores, and that adjuvant further decreased the fecal viral content. Fig. 3 is a line graph showing serum antibody titer specific for rotavirus VP6 observed after mice were immunized intranasally with B, subtilis control spores (squares) or B. sublilis spores associated with bovine derived VP6 (circles, diamonds) or murine derived VP6 (triangles) as a function of time (days after first immunization), B. subtilis spores were administered with an adjuvant prepared from non-toxic Escherichia coli LT (R192G having a mutation of arginine to glycine at residue 192) at 5 μg/dose or 10 μg/dose. The data show that an amount of 5 μg/dose or 10 μg/dose of LT (192G) was effective as an adjuvant. No serum antibody titer was observed in animals administered the control spores, even in the presence of the adjuvant.

Fig. 4 is a line graph showing amount of rotavirus in feces as a function of time (days) after rotavirus challenge of mice immunized with B. subtilis control spores (squares) or B. subtilis spores associated with VP6 antigen of rotavirus strains of bovine origin (circles, diamonds) or murine origin (triangles). Spores were administered with an adjuvant prepared from non-toxic Escherichia coli LT (R 192G) at 5 μg/dose or 10 μg/dose. The data show that the mice immunized with B. subtilis spores that were associated with bovine or murine VP6 recovered more quickly from the rotavirus infection than control mice, and that feces produced by the VP6-immunized mice contained far fewer virus particles than feces from the mice administered control spores.

Fig. 5 is a listing of the nucleotide sequence (SEQ ID No: 1) of the modified Vspac promoter for expression of antigens constitutively at a high level.

Fig. 6 is a set of photographs showing expression of TTFC in recombinant B. subtilis. Colonies grown of solid medium were labeled with rabbit anti-TT antibody followed by anti-rabbit IgG-FITC conjugate of TTFC-expressing strain BB2646 but not control BB2643 strain, and expression by colonies was observed by presence of fluorescein stain. Fig. 6 shows Coomassie blue stained 4 - 12% SDS-PAGE (left panel), and TTFC- specific Western blot (right panel) profiles of fractionated cell extracts from BB2643 (control) and BB2646 (TTFC-expressing). Arrows indicate TTFC at the predicted molecular weight of 5 O KDa. Fig. 7 is a bar graph showing serum anti-TTFC antibody titers after oral immunization of BALB/c mice with each of strains BB2646 carrying TetC, and strain BB2643, a negative control.

Fig. 8 is a bar graph showing serum anti-TTFC antibody titers after intranasal immunization of BALB/c mice with B. subtilis vegetative cells expressing TetC cytoplasmically or after intra-muscular (i.m.) immunization with a conventional DTaP vaccine (positive control). For immunization with B. subtilis, mice were inoculated intranasally with 1 x 10⁸ cells in a volume of 20 μl on days 0, 14, and 28 or on days 0, 2, 14, 16, 28, and 30. For DTaP vaccination, mice were injected i.m, with 50 μl of DTaP vaccine as provided by the manufacturer. Arrows indicate mice that died after challenge.

Fig. 9 is a set of line graphs and a bar graph showing dose response of immune response generated by spore preparations of strain BB2646 and protection against lethal tetanus toxin challenge in BALB/c mice. Each immunized mouse was tested for immune response by intraperitoneal injection with an amount of tetanus toxin equivalent to twice the 100% lethal dose (LD ιoo) of tetanus toxin and was examined for symptoms at the time indicated on the abcissa. It was observed that more than 10⁹ spores were required for effective immunization. Fig. 9 panel A is a line graph showing average serum anti-TetC antibody titers in animals administered with 10⁹ spores in control (closed squares), TTFC- associated spores at concentrations of: 10⁷ (open squares) , 10⁸ (closed triangles) and 10⁹ (open triangles). The arrows indicate the time points of immunization. Fig. 9 panel B is a bar graph showing individual serum anti-TetC anibody titers in four groups of animals immunized will] control and TTFC-associated spores at three concentrations 10⁷, 10⁸ and 10 . Fig. 9 panel C is a line graph showing mouse survival in four groups of animals challenged with 10⁹ spores in control (closed squares), TTFC-associated spores at concentrations of: 10⁷ (open squares) , 10⁸ (closed triangles) and 10⁹ (open triangles). It was observed that mouse survival rate was 100% after immunization with ¹⁰⁹ TTFC associated spores.

Fig. 10 is a line graph and a bar graph showing effect of incubation at 37⁰C on immunogenicity of BB2646 spores. It was observed that immunogcnicity was stable at 4°C after lyophilization, but not in liquid suspension after storing for 5 weeks at 37⁰C. Fig. 10 panel A is a line graph showing anti-TetC antibody titers in animals immunized with control spores (closed squares), TTFC-associated spores: not treated (open squares), treated with 37⁰C for 5 weeks (dosed triangles) and lyophilized (open triangles). The arrows indicate the dates of immunization. Fig. 10 panel B is a bar graph showing individual serum anti-TetC antibody titers after immunization of four groups of animals with spores stored at various conditions compared to animals immunized with control spores.

Fig. 1 1 is a set of graphs showing role of spore germination in immunogenicity of BB2646 spores. Fig. 11 panel A is a line graph showing average serum anti-TetC antibody titers in animals immunized with control spores (closed squares), TTFC-associated germinating spores (open triangles) and TTFC-associated germination deficient spores (open circles). Fig. 1 1 panel B is a bar graph showing individual serum anti-TetC antibody titers with germinating and germination deficient spores compared to control spores. Fig. 11 panel C is a line graph showing survival rate in challenged animals immunized with control spores (closed squares), TTFC-associated germinating spores (open triangles) and TTFC- associated germination deficient spores (open circles). It was observed that spore germination was not required for immunization.

Fig. 12 is a line graph and a bar graph showing antibody endpoint titers (panel A) and survival rate (panel B) of mice following intranasal immunization with vegetative cells of strain BB2646 expressing TetC in cytoplasm, and controls. Survival in mice receiving 10 (open squares) spores and mice receiving intramuscular (IM) injection of DTaP- associated spores was 100%, compared Io lower survival levels in mice receiving 10⁷ (open triangles) and control (closed diamonds) animals.

Fig.13 is a bar graph and a line graph showing a relationship between immunogenicity and geπnination of BB2646 spores and outgrowth of vegetative cells. Fig. 13 panel A shows individual serum anti-TetC antibody titers in mice after three rounds of inoculation with TTFC-associated spores before or 1-3 hrs after suspension in growth medium. Fig. 13 panel B shows mouse survival levels following immunization with 10⁹ untreated dormant (bright) (closed triangles), 10⁹ dormant (bright) spores heated to 8O⁰C for 10 min before inoculation (open triangles), 10⁹ germinated (dark) spores after incubation for Ih in growth medium (closed circle), 10⁹ germinated (dark) spores incubated for 3h in growth medium (closed squares) and 10 germinated (dark) spores incubated for 3h in growth medium (open squares). Highest titers and greatest survival was observed in mice inoculated with lθ' spores incubated for 3h in growth medium. It was observed that the population of cells in this population had substantially converted to vegetative cells.

Fig. 14 panel A is a line graph showing serum anti-TetC antibody titers after intranasal immunization of BALB/c mice with dried, heated B. subtilis vegetative cells expressing TTFC cytoplasmically (open circles) or control (closed squares). The dried vegetative cells were treated at 6O⁰C for 1 hr and resuspendcd in sterile H₂O before immunization. Mice were inoculated intranasally in a volume of 20 μi per dose on days 0, 14, and 28. Serum titer in mice immunized with TTFC was five orders of magnitude greater than in control mice.

Fig.14 panel B is a line graph showing protection against lethal tetanus toxin challenge in BALB/c mice after intranasal immunization with dried, heated B. subtilis vegetative cells expressing TTFC cytoplasmically (open circles) or control (closed squares). Each mouse was injected intraperitoneally with a dose of two LD_JQ_O amount of tetanus toxin and was examined for symptoms during the time period indicated. Data show survival of immunized mice

Fig. 15 is a set of line graphs showing serum anti-TetC antibody titers and survival rates in mice after intranasal immunization of BALB/c mice with dried, heated B. suhtilϊs spores displaying TTFC on the spore surface, compared to control spores. Fig. 15 panel A is a line graph showing serum aiiti-TetC antibody titers in animals immunized with TTFC- displaying spores that were either dried and heated to 6O⁰C for 60 min (open diamonds) or untreated (open circles) in comparison with control spores that were dried and heated (open squares). Fig. 15 panel B is a line graph showing survival rate in animals inoculated with TTFC-displaying spores that were either dried and heated 6O⁰C for 60 min (open diamonds) or untreated (open circles) in comparison to control spores that were dried and heated (open squares). It was observed that heating spores in the dry state did not diminish the immune response in mice. Fig. 16 is a line graph showing development of antibody response in groups of mice inoculated with vegetative cells of strain BB3059, which contains three copies of the Vspac- tetC construct: freshly grown, unheated BB3059 vegetative cells (open squares), 4 x 10⁸ lyophilized BB3059 cells incubated at 45⁰C for 30 days (open circles), 4 x 10⁷ lyophilized BB3059 cells at 45^υC for 30 days (gray filled circles), 4 x 10⁶ lyophilized BB3O59 cells incubated at 45⁰C (black filled circles) compared to unhealed, freshly grown vegetative cells of the control strain BB2643 (closed squares) and cells immunized IP with DTaP vaccine (open diamonds). Long-term heat stability of lyophilized cells of strain BB3059 incubated at 45°C for month was observed.

Fig, 17 a set of line graphs showing antibody development in mice treated with spores incubated at 45°C for month (panel B) compared to control (panel A), demonstrating long-term heat stability of strain BB3184, which contains three copies of the cotC-tetC construct Fig. 17 panel A shows the immune response in animals immunized with CotC- TetC spores in H₂O (open, squares), 10 lyophilized spores (open circles), 10 lyophilized spores (gray filled circles), 10⁷ lyophilized spores compared to spores of the control strain BB2643 (closed squares). Fig. 17 panel B shows immune response in animals treated with CotC-TetC spores lyophilized and stored at 4°C (open squares), 10⁹ spores lyophilized and incubated at 45⁰C for 1 month (open circles), 10⁸ spores lyophilized and incubated at 45⁰C for 1 month (gray filled circles) in comparison with spores of the control strain BB2643 stored in H₂O at 4⁰C (closed squares).

Fig. 18 is a scatter graph showing IgGl :G2a ratio observed in serum after intranasal immunization with B. subtilis BB3059vegetative cells, and control i.m. injection of DTaP.

Fig. 19 is a bar graph showing IgA levels in scrum and feces of mice following intranasal immunization with B. subtilis spores associated with bovine VP6 antigen from construct BB2666 in the presence of mLT adjuvant. The data show that immunized animals produced higher levels of IgA in feces than in serum.

Fig. 20 is a bar graph showing IgA levels following intranasal immunization of mice with B. subtilis spores (light gray bars) or B. subtilis vegetative cells (dark gray bars) associated with bovine VP6 antigen from construct BB2666. Spores and vegetative cells were administered with mLT adjuvant. The data show that animals administered spores associated VP6 produced higher levels of IgA than animals administered with vegetative cells.

Fig. 21 is a set of drawings showing a schematic growth curve of B. subtilis vegetative cells and initiation of sporulation after onset of growing phase. Fig. 21 panel A shows antigen display on surface of the spore. Fig. 21 panel B shows antigen displayed on the vegetative cell surface. Fig 21 panel C shows antigen displayed in the vegetative cytoplasm.

Fig. 22 is a set of drawings showing construction of genetic fusions for antigen display. Fig. 22 panel A shows organization of a recombinant fusion of an exemplary gene CotC with TetC coding gene. Fig. 22 panel B shows organization of an exemplary gene for expressing antigen during vegetative growth.

Detailed Description

Infectious diseases remain a public health problem, in spite of the progress in antibiotic and anti-viral chemotherapeulic agents. A class of viral diseases referred to as emerging diseases and exemplified by SARS and avian and/or swine influenza, have been causally associated with increased contact between wild animals that migrate, such as ducks and geese, with intensely farmed agricultural animals such as pigs, and from rapid global travel.

Spore-forming microorganisms offer the possibilities of new classes of vectors for administering one or more antigens of an infectious virus, in order to immunize human or animal subjects. Great variety in choice of types of host cells enables the designer of the vaccine to use a cell genotype that results in a single round of immunization, for example in human subjects, by using chromosomal markers that allow growth only under highly restricted conditions, or by using a cell genotype chosen to allow transmission from subject to subject, such as in a bird population, Most important, because bacterial and fungal spores remain viable under a very wide range of ambient environmental conditions, a spore-based vaccine offers the possibility of storage at room temperatures rather than under refrigeration or freezing. See Acheson et al., U.S. patent number 5,800,821, issued September 1, 1998, and incorporated herein by reference in its entirety.

Spores of bacterial genera within the group of streptomycetes are sufficiently heat resistant to survive extreme fluctuations of room temperature, for example substantial quantitative survival for at least a few minutes at 50 ⁰C. Strains of the yeast Saccharoinyces, a fungus that produces ascospores, are resistant to several minutes of heat at 60⁰C. See, Put ct al., 1982, J. Appl. Bact. 52: 235-243. Similarly, spores of non-toxic strains of fungi, such as PenicilHum strains that are well known edible components of cheese (Roquefort, gorgonzola, etc.) and produce spores may be used. Heat resistance for 10 minutes at 50 "C was observed with spores from a variety of species of the fungus Aspergillus (Pitt et al., 1970, Appl Microbiol. 20(5): 682-686). Genetics and recombinant techniques for many strains and species of both streptomycetes and fungi are well developed. See Hopwood et al., Streptomyces, 1985, publ. John limes Press; Kieser et al., Practical Streptomyces Genetics, 2008, publ, John lnnes Press. However these spores are not so resistant to extreme conditions as are the spores of

Bacillus strains (which survive quantitatively even at such extreme conditions as boiling, and have been recovered as viable colony forming units from insects preserved for millions of years in amber; Cano et al., 1995, Science 268: 1060-1064). The methods herein are suitable for use with spores of bacteria or fungi capable of withstanding ambient conditions of storage at room temperature.

The use of B. subtilis as a vehicle for vaccine antigen delivery is a promising new approach to mucosal immunization (Due et al., 2003, Infect. Immun. 71 : 2810-2818; Oggioni et al., 2003, Vaccine 21 Suppl. 2: S96-101). The primary model used to date has been the spore form of B. subtilis displaying tetanus toxin antigen on its surface. An advantage of using spores as vectors is that the spores are highly resistant to environmental stresses such as extremes of heat, pH, desiccation, freezing and thawing, and radiation (Nicholson et al., 2000, Microbiol. MoL Biol. Rev. 64(3): 548-557). Heterologous antigens displayed on the spore surface as a fusion product with spore coat proteins have been shown to elicit protective immune responses to tetanus toxin when spores displaying tetanus toxin fragment C (TTFC) were given either orally or intranasally (Due et al,, 2003, Infect. Immun. 71: 2810-2818). For oral immunization, several rounds of high doses of spores (> 10^!0) were necessary and the long-term immunogenic stability of these preparations has not been rigorously tested. Moreover, the exposure of the antigen to proteases in the Gi tract may reduce the availability of immunogenic protein to the GI immune system (Due et al. 2003). Orally administered spores of B. subtilis survive passage through the gastrointestinal tract of mice and may germinate in the intestines to yield replicative vegetative cells; the intestinal tract becomes briefly colonized (Spinosa et al., 2000, Res. Microbiol. 151 : 361- 368; Casula et al., 2002, Appl. Environ. Microbiol. 68: 2344-2352). If spores were designed to generate antigen only after germination in the intestinal tract, such a spore-based vaccine would address the issues of antigen degradation during storage and during passage through the GI tract and would potentially be stable indefinitely. The B. subtilis spore-based vaccine induces a serum antibody response to Yersinia pseudotuberculosis invasin by spores engineered to display invasin on the vegetative cell surface after germination and outgrowth (Acheson ct al., U.S. patent number 5,800,821, issued September 1 , 1998). Oral inoculation with B. subtilis spores engineered to express TTFC after germination in the vegetative cell cytoplasm was shown to induce protective antibodies (Uyen et al., 2007, Vaccine 25(2): 356-365). It is not known how well engineered strains of B. subtilis colonize the intestine of humans or if there would be interference of colonization from other intestinal mico flora. Because of the lack of immune responses found by some to live bacterial vectors given orally, another approach to mucosal immunization is the intranasal route. Attenuated Salmonella typhi expressing TTFC elicited protective immunity to tetanus toxin after the vaccine was administered intranasal Iy, but not orally (Galen et al., 1997, Vaccine 15(6-7): 700-708). Use of attenuated pathogenic bacteria as vectors has the general disadvantage that sufficient attenuation of virulence is required to assure safety. For this reason, bacteria that are generally regarded as safe are preferable. For instance, Lactobacillus plant arum expressing TTFC was shown to protect against tetanus toxin challenge after intranasal administration (Grangette et al., 2001 , Infect. Inimun. 69(3): 1547- 1553). Like lactobacilli, B. subtilis is also generally regarded as safe, and is neither pathogenic nor toxigenic to humans, animals, or plants (Sonenshein et al 1993). B. subtilis has been extensively studied a model gram-positive bacterium, and is advantageous for genetic manipulation. Stable genetically engineered constructs can be integrated into the bacterial chromosome, making it a good candidate for vaccine preparation.

Bacterial genera such as Bacillus and others that produce spores, and fungal species are within the scope of embodiments of the methods and compositions herein, if they satisfy criteria of suitability for engineering vaccines, viz., production of stable spores, and non- toxicity to animals of spores and vegetative cells. For example, cells of non-toxic streptomycete strains such as Streptomyces lividans, S. coelicolor, and S. reticuli may be engineered by conventional genetic techniques to express cytopiasmicaliy an antigen encoded by the genome of an infectious agent, such that the antigen is synthesized in soluble form, during vegetative growth of the cells. The antigen while made as a soluble material becomes associated with spores during the sporulation process. The spores are prepared by conventional techniques into a vaccine compositon, and when administered to a subject results in an immune response capable of protecting the subject from infection by the infectious agent that is the source of the antigen. The term, "antigen" as used herein and in the claims refers to a protein or a portion of a protein, isolated from nature or synthesized, or expressed in and purified from a recombinant cell, or a peptide, or a derivatized version thereof containing one or a few additional amino acids, including sequences of amino acids that are of biological origin, or are not found in nature. The antigen is a peptide of sufficient length to provoke an immune response in an animal having an immune system, generally at least about 4 to 7 amino acids in length.

The term "derivative" as used herein and in the claims may be a protein, peptide, or chemically related form of that protein having an additional substituent on an amino acid, for example, N -carboxy anhydride group, a γ-benzyl group, an e,N-trifluoroacetyl group, or a halide group attached to an atom of the amino acid of a protein.

The term, "immune response" means any natural function of an immune system, and includes without limitation, any cellular function or organ function characteristic of ability to distinguish a component of self and non-self by an organism, for example, ability to bind to an MHC class 1 or class II protein, or ability to bind to a cell having such proteins, ability to activate killer T cells, or to elicit production of antibodies of any type are all within the definition.

Immunizations have been well-known in the medical arts since discovery of cowpox vaccination over two hundred years ago, and include injection by a variety of routes including subcutaneous, and oral delivery, e.g., of Sabin polio vaccine. Discovery herein of efficiency of a spore-based vaccine delivered by an intranasal route is an important and surprising result.

The term, "associated" as used herein and in the claims refers to the physical relationship of an antigen to a spore, or a vegetative cell or portion thereof. During sporulation, the antigen is produced during vegetative phase of cell growth and is physically associated with the spore. The antigen may be displayed on the spore's surface, or may be entrained within the spore coat, and the ability of the spores or vegetative cells or portions thereof to elicit an immune response does not depend on any particular physical location of the antigen within the spore or cell, nor any mechanism of packaging, in contrast to embodiments of the methods and compositions herein, in which the antigen is associated with spores and is not necessarily covalently bound to any particular spore component, Acheson et al., issued September 1, 1998, U.S. patent number 5,800,821 shows a vaccine using spores engineered by genetically manipulating spore-forming bacterial cells to contain a certain DNA sequence encoding an antigen, producing spores produced as a genetic fusion to spore surface proteins during the sporulation phase in the cells.

The term, "adjuvant" as used herein and in the claims refers to any compound which when administered together with an antigen nonspecifically enhances the immune response to that antigen. Adjuvants may be insoluble and undegradable substances (e.g., inorganic gels such as aluminum hydroxide), or watcr-in-oil emulsions such as incomplete Freund's adjuvant. Generally, adjuvants retard the destruction of antigen and allow the persistence of low but effective levels of antigen in the tissues and also nonspecifically activate the lymphoid system by provoking an inflammatory response. One of the most effective adjuvants is Freund's complete adjuvant having mycobacteria suspended in a water-in-oil emulsion, however the intense inflammatory response it provokes precludes its clinical use. Examples herein show enhanced immune responses with several adjuvants including cholera toxin and a non-toxic Escherichia coli heat-labile enterotoxin variant, which are administered with spores.

Both cholera toxin produced by Vibrio cholerae and Escherichia coli heat labile enterotoxin have been widely used as adjuvants in experimental systems, and each induces significant antibody responses to adjuvants and are also potent mucosal adjuvants for co- administrated, unrelated antigens, especially when give by the oral route. Cholera toxin functions as an adjuvant by inducing antigen specific CD4⁺ T cells to secrete interleukin 4 (IL-4), IL-S, 1L-6, and IL- 10. Heat labile enterotoxin produced by some enterotoxigenic strains of Escherichia coli functions as an adjuvant by inducing ThI and Th2 cytokine responses. Both cholera toxin produced by Vibrio cholerae and heat labile enterotoxin produced by Escherichia coli are multi-subunit macromolecules composed of two structurally, functionally, and immunologically separate A and B sub units (Yamamoto et al., 1997, Proc. Natl. Acad. Sd. U S A 94(10): 5267-5272).

The term "thermally-stable" as used herein and in the claims relates to an enhanced persistence of an active substance or pharmaceutical product as a function of time under the influence of a variety of environmental factors, primarily temperature, and is also affected by other conditions such as humidity and light in comparison with a control preparation that is not thermally stable. In making this determination, product-related factors also influence the stability, e.g., the chemical and physical properties of the active substance and the pharmaceutical excipients, the dosage form and its composition, the manufacturing process, the nature of the container-closure system, and the properties of the packaging materials. Also, the stability of excipients that may contain or form reactive degradation products are considered.

Stress testing of the active substance is used to identify the potential degradation products, and to establish degradation pathways and stability of the molecule. The nature of the stress testing depends on the individual active substance and the type of pharmaceutical product involved,

In general, an active substance is evaluated under storage conditions (with appropriate tolerances) that test thermal stability and, if applicable, sensitivity to moisture. The storage conditions and the lengths of time period for study are appropriate to consider storage, shipment, and subsequent use appropriate to the climatic zone or zones in which the active substance is likely intended to be stored. Long-term testing extends for a period about a month, or, about 3 months, about 6 months, or about 12 months. Testing includes a number of product batches in conditions of packaging and temperatures that are representative of the product's intended use. Data from an "accelerated" storage condition, if appropriate, are obtained to test the product at conditions beyond those intended, i.e., excessively high or low temperatures compared to potential actual ambient conditions. An accelerated storage condition includes tests of conditions that mimic handling issues such as prolonged exposure to excess moisture and variable volume delivery of a composition including the active substance. Calculations of the data obtained under accelerated conditions are then used to extrapolate the presumed stability of the product in normal environments and conditions, although these calculations are an estimate of stability of the product under normal conditions. Data obtained from accelerated storage conditions are combined with other data including long- term testing described above to determine the stability of the product.

U.S. patent number 6,187,319 (Herrmann et al., issued February 13, 2001) describes a method of producing an immune response in an animal to a rotavirus antigen, by administering an isolated rotavirus VP6 polypeptide of a different strain of rotavirus to produce an effective immune response. The VP6 polypeptide was delivered directly, or by using a DNA plasmid or a virus to express the polypeptide in the recipient, with transcriptional and translational regulatory sequences encoded by the plasmid or virus. U.S. patent number 6,165,993 (Herrmann et al, issued December 26, 2000) describes a method of eliciting an immune response or protective immunity with a vaccine having DNA encoding an antigen (e.g., capsid proteins or polypeptides of a rotavirus such as VP4, VP6 and VP7), the antigen encoded by a nucleotide sequence in a plasmid vector. A feature provided by the present invention herein is a method of immunizing a subject to an infectious agent, the method including steps of: sporulating a vegetative host bacterial cell which contains an isolated nucleotide sequence encoding an antigen of the infectious agent, such that the nucleotide sequence is operably linked to a promoter for cytoplasmic vegetative expression of the antigen, such that the spores are associated with the antigen and, contacting the subject with a composition including the spores, such that the antigen immunizes the subject to the infectious agent. In general, the infectious agent is viral or bacterial. For example, the infectious agent is at least one bacterium selected from the group of consisting of Bacillus anthracis, Clostridium letani, Cυrynehacterium diphtheriae, Bordetella pertussis, Mycobacterium tuberculosis, Salmonella typhimurium, Staphylococcus aureus, Streptococcus pneumoniae, Treponema pallidum, Neisseria gonorrhoeae, and the like. For example, the infectious agent is at least one virus selected from the group consisting of human immunodeficiency virus (HlV), influenza, polio, herpes, smallpox, measles, mumps, rubella, rotavirus, chicken pox, rabies, West Nile virus, eastern equine encephalitis, norovirus, and the like. An exemplary antigen is a rotavirus antigen.

The method in related embodiments further includes prior to sporulating, obtaining the isolated nucleotide sequence encoding the rotavirus antigen from a rotavirus strain that is bovine or murine. For example, the rotavirus antigen is a viral virion protein, for example, the viral virion protein is selected from at least one of the group consisting of VP2, VP4, VP6, VP7, NSP4, and a portion or a derivative thereof. In general, the subject is a vertebrate animal. For example, the vertebrate animal is from at least one of the group of an agricultural animal, a high value zoo animal, a research animal, a human, and a wild animal found in a densely populated human environment such as a wild bird. The method in related embodiments further includes contacting the subject by administering the composition by a route selected from at least one of intravenous, intramuscular, intraperitoneal, intradermal, mucosal, and subcutaneous routes. For example, contacting the subject is by intranasal administration. For example, the intranasal administration includes inhalation or nose drops. Inhalation methods include use of a nebulizer or an atomizer, and include a measured dose.

In general, the vegetative host bacterial cell is a Bacillus cell. For example, the Bacillus is Bacillus subtilis, although other species of bacilli and other spore-forming organisms are also within the scope of the methods herein.

The composition used in the method herein includes in related embodiments an adjuvant, for example, the adjuvant is selected from at least one of the group consisting of cholera toxin, a non-toxic variant of Escherichia coli labile toxin, and a portion or a derivative thereof.

The method according to related embodiments further involves observing resistance of the composition to at least one condition selected from the group of heat, drying, freezing, deleterious chemicals and radiation.

An embodiment of the method further involves measuring an antibody titer in serum of an infected subject, such that increase in antibody for the antigen in comparison to a control serum is an indication of efficacy of the immunogenicity of the composition. Suitable control sera include pro immune serum from the subject, or serum from a different subject receiving a different antigen. Still another feature of the invention provided herein is measuring an amount of viral shedding in the subject afflicted by the infectious agent, such that a decrease in fecal virus compared to that in a control subject also afflicted by the infectious agent and not contacted with the composition, is a measure of efficacy of the immunogenicity of the composition. A featured embodiment of the invention provided herein is a therm ally- stable vaccine composition for immunizing a subject with an antigen from an infectious agent, the composition including spores from a Bacillus cell that contains an isolated nucleotide sequence encoding the antigen, the nucleotide sequence being genetically engineered and having been integrated into the host bacterial chromosome or carried on a plasmid and provided with appropriate transcriptional and translalional regulatory sequences, such that the cell expresses the antigen cytoplasmically as a soluble component during vegetative growth, and upon sporulation by the cell, the antigen is associated with the spores, and the composition comprising the spores is effective Io immunize the subject. For example, the antigen is a viral protein or a portion or a derivative thereof. For example, the viral protein is a viral virion protein. For example, the viral virion protein is selected from at least one of the group consisting of VP2, VP4, VP6, VP7, NSP4, and a portion or a derivative thereof. An exemplary, Bacillus is Bacillus subtilis. The composition in related embodiments includes an adjuvant. For example, the adjuvant is selected from at least one of the group consisting of cholera toxin, a non-toxic variant of Escherichia cυli labile toxin, and a portion or a derivative thereof.

In related embodiment of the composition, the isolated nucleotide sequence encoding the antigen is from a strain that is bovine or murine. The invention herein also features a vaccination kit that includes a unit dose of the composition according to any of the above embodiments, a container, and instructions for use. In related embodiments, the instructions include storage at a room temperature of from about 4⁰C to about 45°C and the like (calculation of 45⁰C is that this temperature is the same as l l3°F). Pharmaceutical compositions

An aspect of the present invention provides pharmaceutical compositions, wherein these compositions comprise spores associated with an antigen from an infectious agent, and optionally further include an adjuvant, and optionally further include a pharmaceutically acceptable carrier. In certain embodiments, these compositions optionally further comprise one or more additional therapeutic agents. In certain embodiments, the additional therapeutic agent or agents are selected from the group consisting of growth factors, anti-inflammatory agents, vasopressor agents, collagenase inhibitors, topical steroids, matrix metalloproteinase inhibitors, ascorbates, angiotensin II, angiotensin III, calreticulin, tetracyclines, flbronectin, collagen, thrombospondin, transforming growth factors (TGF), keratinocyte growth factor (KGF), fibroblast growth factor (FGF), insulin-like growth factors (IGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF), neu differentiation factor (NDF), hcpatocyte growth factor (FIGF), and hyaluronic acid. As used herein, the term "pharmaceutically acceptable carrier" 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 Ed. by Gennaro, Mack Publishing, Easton, PA, 1995 discloses various earners used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Carriers are selected to prolong dwell time for example following inhalation or other form of intranasal administration, or other route of administration.

Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as glucose, and sucrose; starches such as com starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurale; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other nontoxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

In yet another aspect, according to the methods of treatment of the present invention, the immunization is promoted by contacting the animal with a pharmaceutical composition, as described herein. Thus, the invention provides methods for immunization comprising administering a therapeutically effective amount of a pharmaceutical composition comprising active agents that include a spore preparation having an associated antigen from an infectious agent, to a subject in need thereof, in such amounts and for such time as is necessary to achieve the desired result. It will be appreciated that this encompasses administering an inventive vaccine as described herein, as a preventive or therapeutic measure to promote immunity to the infectious agent, to minimize complications associated with the slow development of immunity (especially in compromised patients such as those who are nutritionally challenged, or at risk patients such as the elderly or infants). In certain embodiments of the present invention a "therapeutically effective amount" of the pharmaceutical composition is that amount effective for promoting appearance of antibodies in serum specific for the chosen antigen, or disappearance of disease symptoms, such as amount of virus in feces or in bodily fluids or in other secreted products. The compositions, according to the method of the present invention, may be administered using any amount and any route of administration effective for generating an antibody response. Thus, the expression "amount effective for promoting immunity", as used herein, refers to a sufficient amount of composition to result in antibody production or remediation of a disease symptom. The exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active agent(s) or to maintain the desired effect. Additional factors which may be taken into account include the severity of the disease state, e.g., exposure to infectious agent in the past or potential future exposure, or exposure to a seasonal allergen; age, weight and gender of the patient; diet, time and frequency of administration; drug combinations; reaction sensitivities; and tolerance/response to therapy. Long acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular composition.

The active agents of the invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. The expression "dosage unit form" as used herein refers to a physically discrete unit of active agent appropriate for one dose to be administered to the patient to be treated. It will be understood, however, that the total daily usage of the compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. For any active agent, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of active agent which ameliorates at least one symptom or condition. Therapeutic efficacy and toxicity of active agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose is therapeutically effective in 50% of the population) and LD50 (the dose is lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The therapeutic dose shown in examples herein is at least about 10 , about 3 x 10 , about 10 , or at least about 3 x 10 spores/dose/animal. As bacterial spores are readily produced and arc inexpensively engineered and designed and stored, greater doses for large animals are economically feasible. For an animal several orders of magnitude larger than experimental animals used in examples herein, the dose is easily adjusted, for example, to about 3 x 10¹⁰, 3 x 10¹¹, to 3 x 10^l2 or about 3 x 10°, for animals such as humans and small agricultural animals. However doses of about 3 x 10¹⁴, 3 x 10¹⁵, or even about 3 x 10^!6, or about 3 x lO¹⁷, for example for a high value zoo animal or agricultural animal such as an elephant, are within the scope of the invention. For preventive immunizations, or periodic treatment, or treatment of a small wild animal, smaller doses such as less than about 3 x 10⁹, or less than about 3 x 10 , or even less than about 3 x 10 per dose, are within the scope of the invention. Administration of pharmaceutical compositions

After formulation with an appropriate pharmaceutically acceptable carrier in a desired dosage, the pharmaceutical compositions of this invention can be administered to humans and other mammals topically (as by powders, ointments, or drops), orally, rectally, parenterally, intracistemally, intravaginally, intraperiloneally, bucally, ocularly, or nasally, depending on the severity and nature of the infectious agent being treated.

In various embodiments of the invention herein, oral and intranasal inoculation using spores and vegetative cells of B. subtitis engineered to express TTFC are compared. It was observed that high titers of antibodies, sufficient for protection against a lethal dose of tetanus toxin, were produced in mice after intranasal administration of vegetative cells expressing cytoplasmic TTFC or spores displaying TTFC as a fusion protein to a spore coat protein. These vaccines proved to have a long shelf life at elevated temperatures when stored in the dry state. While intranasal administration was demonstrated to be surprisingly effective in examples herein, the antigen associated with spores and/or vegetative cells, preferably lyophilized, following vegetative cytoplasmic expression of the antigen prior to sporulation leads to a vaccine that is administered in any of a variety of routes. For example, it is envisioned that for agricultural animals, such as immunizing chicken or ducks for viral influenza, oral or intranasal administration would be highly suitable.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active agent(s), the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoatc, propylene glycol, 1,3-butylene glycol, dimetliylformamidc, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Dosage forms for topical or transdermal administration of an inventive pharmaceutical composition include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active agent is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. For example, ocular or cutaneous infections may be treated with aqueous drops, a mist, an emulsion, or a cream. Administration may be therapeutic or it may be prophylactic. Prophylactic formulations may be present or applied to the site of entry of potential disease organisms, such as, in the case of topical infectious organisms such as herpes virus, to wounds, such as contact lenses, contact lens cleaning and rinsing solutions, containers for contact lens storage or transport, devices for contact lens handling, eye drops, surgical irrigation solutions, ear drops, eye patches, and cosmetics for the eye area, including creams, lotions, mascara, eyeliner, and eyeshadow. The invention includes products which contain the compositions having the lyophilizcd vegetative cells or spores (e.g., gauze bandages or strips), and methods of making or using such devices or products. These devices may be coated with, impregnated with, bonded to or otherwise treated with a vaccine composition. For sites of disease entry that are primarily spread by droplet infection, such as rhinovirus and influenza, intranasal administration is suitable.

The ointments, pastes, creams, and gels may contain, in addition to an active agent of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonitcs, silicic acid, talc, zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the agents of this invention, excipients such as talc, silicic acid, aluminum hydroxide, calcium silicates, polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorotluorohydrocarbons.

Transdermal patches have the added advantage of providing controlled delivery of the active ingredients to the body. Such dosage forms can be made by suspending spores in the matrix applied to the patches, or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the antigenic peptide released from spores into the compound, for passage across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. The injectable formulations can be sterilized prior to addition of spores, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents io the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. In order to prolong the effect of an active agent, it is often desirable Io slow the absorption of the agent from subcutaneous or intramuscular injection. Delayed absorption of a parenterally administered active agent may be accomplished by dissolving or suspending the agent in an oil vehicle. Injectable depot forms are made by forming micro en capsule matrices of the agent in biodegradable polymers such as polylactide-polygiycolide. Depending upon the ratio of active agent to polymer and the nature of the particular polymer employed, the rate of active agent release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly( anhydrides). Depot injectable formulations are also prepared by entrapping the agent in liposomes or microemulsions which are compatible with body tissues.

Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the active agent(s) of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active agcnt(s).

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active agent is mixed with at least one inert, pharmaceutically acceptable cxcipient or earner such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylccllulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectanls such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonitc clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof.

Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active agent(s) may be admixed with at least one inert diluent such as sucrose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active agent(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Uses of pharmaceutical compositions

As discussed above and described in greater detail in the Examples, spores, spore preparations, vegetative cells, and vegetative cell preparations, e.g., by lyophilization, particularly bacterial spores, e.g., spores of a Bacillus, are used to prepare a heat resistant stable active vaccine by associating with an antigen from an infectious agent during sporulation. En general, it is believed that these vaccines will be clinically useful in immunizing subjects for resistance to infectious diseases. The present invention encompasses the treatment of a variety of infectious diseases arising from infection with bacteria, viruses, fungi, and parasites. The vaccines herein are particularly useful to treat compromised patients, particularly those anticipating therapy involving, for example, immunosuppression and complications associated with systemic treatment with steroids, radiation therapy, non-steroidal anti -inflammatory drugs (NSAlD), anti-neoplastic drugs and anti-mctabolitcs.

In addition, spores associated with antigens from allergy producing proteins are contemplated, such as dust mite proteases, cat and dog salivary proteases, and proteins found in pollens of allergens such as pollen of grass and trees such as, ragweed, timothy and mapte trees. The unprecedented stability of the vaccine compositions, and the rapid response as shown by appearance of serum antibodies following intranasal administration, indicates that the vaccines can be used by patients in a home setting, and can be supplied in suitable single dose or measured dose formats, to be used as needed, for example, seasonally.

A skilled person will recognize that many suitable variations of the methods may be substituted for or used in addition to those described above and in the claims. It should be understood that the implementation of other variations and modifications of the embodiments of the invention and its various aspects will be apparent to one skilled in the art, and that the invention is not limited by the specific embodiments described herein and in the claims. The present application mentions various patents, scientific articles, and other publications, each of which is hereby incorporated herein in its entirety by reference.

The invention having how been fully described, it is exemplified by the following examples and claims which are for illustrative purpose only and are not meant to be further limiting. EXAMPLES Example 1 : Genetic engineering of rotavirus VP6 and chromosomal insertion

VP6 is the inner capsid protein of rotavirus and has a molecular weight of approximately 45 kd, and is a known immunogen for use in vaccines to treat rotaviral infection. Fusions of the bovine and murine VP6 coding regions to a version of the Pspac promoter (Yansura et al., 1984, Proc. Natl. Acad. Sci. USA 81:439-443) were integrated at the sacA locus of the B. subtilis chromosome. The Pspac promoter was altered by introducing two site-directed mutations, C-12T and A+1G. These mutations increased the strength of the promoter. The VP6 coding regions were recovered from pCR.2.1-VP6 bovine (obtained from Dr. LJ. Saif, Ohio State University, Wooster, OH) and pBluescript- VP6 murine (GenBank accession no. U36474; obtained from Dr. H. B.Greenberg, Stanford University, Palo Alto, CA ) by PCR and were cloned initially in vector pBBl 378. The latter plasmid carries the B. subtilis veg promoter and a kanamycin resistance gene surrounded by the 5' and 3' ends of the sacA locus. An appropriately placed ribosome binding site was incorporated into VP6 constructs during the PCR step. The relevant fragments containing the ribosome binding site and the VP6 coding sequences were excised from the resulting plasmids by treatment with Pacl and Sacϊ and were cloned in similarly digested pBB1375. The latter plasmid carries the mutant Pspac promoter and a kanamycin resistance gene surrounded by the 5' and 3' ends of the sacA locus. When introduced into competent cells of B. subtilis strain BB2534 [MhyA AthyB trpC2 ΔsacA::(thyΛ^¥ cat)], the resulting transformants, BB2543 for bovine VP6 and BB2547 for murine VP6, arose by double crossover recombination at the sacA locus. B. subtilis strains BB2666 (containing bovine VP6) and BB2667 (containing murine VP6) are Thy¹ versions of the latter strains, respectively. The control strain, BB2643, had the same genetic organization as BB2666 and BB2667, except that the VP6 coding sequence was absent.

Example 2: Growth of bacteria and preparation of vegetative cells and spores

Bacillus subtilis type 168 strain was used to construct the recombinant strains expressing TTFC. E. coli strain JM107 was used for cloning experiments. Bacterial strains were routinely grown in Luria broth (LB) and plates containing solid LB medium were prepared with neomycin (5 μg/ml) for B. subtilis, or kanamycin (25 μg/ml) or ampicillin (100 μg/ml) for E. coli. For some experiments, B. subtilis cells were grown in a defined medium (TSS) supplemented with glucose (0.5 - 1%), ammonium chloride (0.2%) and sodium glutamate (0.2%).

B. subtilis strains grown overnight on L agar plates were used to inoculate 4-L cultures in DS medium (Fouet et al, 1990; J. Bacterid. 172: 835-844). After incubation with shaking (200 rpm) at 37⁰C for 48 hrs, the mixture of spores and non-sporulating bacteria was harvested by ccntrifugation, washed with sterile deionized water, treated witli egg white lysozyme (1 mg/ml) to kill non-sporulating cells, washed five additional times with sterile deionized water and stored at 4⁰C in sterile water. Spores were titered by direct counting using a Pelroff-Hauser chamber and by comparing colony- forming ability before and after heating a sample to 80⁰C for 10 min.

The following bacterial strains were used in the examples: control B. subtilis spores (not carrying genes encoding antigens for vaccines), and B. subtilis capable of displaying either bovine or murine-derived VP6 (e.g., bovine (Bo)VP6 or murine (Mu) VP6 as described herein).

Example 3: Administration and response testing

BALB/c female mice of about 4-6 weeks were immunized intranasally with spores according to a specific schedule (e.g., dose administered at 0, 14, and 28 days) as shown in the figures. Each animal was administered 3 x 10 spores per dose. Adjuvant was used with an antigen in examples herein to co-immunize animals to enhance the immune response. Cholera toxin (CT) produced by various strains of Vibrio cholcrae promotes Th2 cytokine responses, and improves efficacy of the immune response involving one or more of IgGl , IgE, and mucosal IgA antibodies. Escherichia coli LT (Rl 92G) is a mutated variant of a heat-labile enterotoxin produced by enterotoxigenic strains of R coli. The LT (Rl 92G) variant is non-toxic, and induces ThI and Th2 cytokine responses and improves efficacy of the immune response involving one or more of IgGl , lgG2a, IgG2b, and mucosal IgA antibodies. Adjuvant volumes of 20 μl were used for immunization; CT was administered at 10 μg/dose, and E. coli LT (Rl 92G) was administered at 5 μg/dose or 10 μg/dose. Methods to determine effectiveness of immunization of mice administered with VP6 spore preparations (bovine or murine-derived VP6 spores or a negative control) included: mice were sampled for titer of serum anti~VP6 antibody, which was measured using ELISA. Mice were challenged orally with the agent that causes epizootic diarrhea of infant mice (EDlM), rotavirus, and were monitored for the course of rotavirus infection by measuring appearance of vims VP6 antigen in feces.

Example 4: Immunization with bovine or murine-derived VP6 spore preparations in presence of CT caused increased serum anti-VP6 antibody titer

Animals were immunized with VPό spores (with or without CT) and were tested for appearance of anti-VP6 antibody in serum. The mice were immunized intranasally on days 0, 14, and 28 and were tested on days 14, 28 and 42.

Animals immunized with spore preparations associated with bovine-or murine VP6 (Fig. 1, triangles or circles) showed significant production in serum of anti-VP6 as measured by titer response at day 28 and day 42 compared to animals administered control VP6 spores. Murine-derived VP6 spore preparations showed a slightly greater ability to elicit an antibody titer response than bovine-derived VP6 at day 42, and bovine derived VP6 spores resulted in a slightly greater response at day 28. See Fig. 1. Animals immunized with spore preparations of BB2643, a B. subtilis strain that does not contain rotavirus-deiϊved sequences (with or without CT; squares) produced almost no serum anti~VP6 ELISA titer response at any of the tested dates.

Immunizing animals with bovine-or murinc-associated VP6 spore preparations with CT adjuvant increased the serum anti~VP6 titer response compared to immunizing with bovine or murine-derived VP6 spore preparations absent adjuvant, at each of day 28 and day 42. (Fig. 1, open) The relative increase for serum anti-VP6 titer response for bovine- associated VP6 spore preparations with CT compared to control spore preparations absent adjuvant was similar at day 28 and day 42.

Intranasal immunizations with bovine-or murine-associated VP6 spore preparations caused increased serum antibody titer compared to control spore preparations not displaying VP6. Also, use of an adjuvant, i.e., administering bovine-or murine-associated VP6 spore preparations with CT, increased the serum antibody titer even further (open circles, open triangles). Example 5: Intranasal immunization with bovine-or murine-associated VP6 spores with CT reduced amount of rotavirus in feces in EDIM rotavirus infection model

Animals immunized with VPό spore preparations were challenged orally with rotavirus, using the EDIM animal model of rotavirus infection, and mice were tested for infection by measuring viral antigen in feces by ELISA each day for seven days.

Immunizing animals with VP6-associated spore preparations with or without adjuvant CT resulted in almost complete suppression of the disease, Animals administered control spores showed massive viral production from days 2 to 6 (Fig. 2). Bovine-or murine- associated VP6 spore preparations with CT adjuvant resulted in substantially reduced viral presence in comparison to administration of control spore preparations (with or without CT) with the infection appearing only at day 2, or at reduced levels from days 2 to 6. Animals administered murine-associated VP6 spore preparations and CT adjuvant showed substantially no virus in feces on day 3 (less than 0.2 OD). In contrast, animals administered control spore preparations continued to produce massive amounts of virus through day 6. These data show that intranasal immunizations with VP6-associated spore preparations with CT caused reduced rotavirus infection after EDlM rotavirus challenge. This result is surprising because the VP6 antigen associated with the spores was expressed during vegetative growth of the cells, and as a cytoplasmically soluble product, rather than as a fusion to a sporulation protein during the sporulation phase. Animals immunized with bovine-or murine-associated VP6 spores showed effective immunization: data show increased serum anti-VP6 antibody titer as measured by ELISA and reduced fecal rotavirus antigen in rotavirus challenged mice (Fig. 1 and Fig. 2, respectively). The use of adjuvant CT with bovine-or murine-associated VP6 spore preparations further improved immune response in mice, as shown by the increased scrum antϊ-VPό ELISA titer (100-fold increase) and reduced rotavirus presence (3-fold to 10-fold) compared to control administered spore preparations in absence of adjuvant.

To test the effect of adjuvant alone, groups of animals were administered CT with control spore preparations, and the data show no appearance of serum anti-VP6 antibody, or reduction of rotavirus presence in EDlM animals (Figs. 1 and 2).

Example 6: Intranasal immunizations with bovine-or murine-associated VP6 spores with LT (R192G) show increased serum anti-VP6 titer response Animals were administered VP6~associated spore preparations with adjuvant LT (Rl 92G), and were tested for serum anti-VPό antibody titer. The animals were administered spore preparations intranasally on days 0, 14, and 28 and serum was obtained on days 14, 28 and 42. It was observed that animals immunized with bovine-associated VP6 spores with 5 μg/dose or 10 μg /dose LT (Rl 92G) produced a substantial titer of antibody (see Fig. 3). Bovine-associated VP6 spore preparations with adjuvant LT (R 192G) resulted in greater serum antibody titers to rotavirus VP6, than murine-associated VP6 spores absent adjuvant (Figs. 1 and 3). Both amounts of LT (R 192G) of 5 μg /dose or of 10 μg /dose with the bovine- or murine-associated VP6 spores was effective in increasing serum antibody response to rotavirus VP6.

Control spore preparations without antigen with 10 μg /dose LT (Rl 92G) produced no serum anti-VP6 ELISA titer response at all time periods, similar to the results observed with animals immunized with control spore preparations with 10 μg /dose CT in Fig. 1, as expected. These data shows that adjuvant enhances immune responses to spore preparations containing rotavirus VP 6

Example 7: Intranasal immunizations with bovine-or murine-associated VP6 spore preparations with LT (Rl 92G) show reduced rotavirus infection in an EDIM disease model Animals were immunized intranasally with VP6-associated spore preparations, challenged orally with EDIM rotavirus, and were tested for viral production in feces, by ELISA for presence of the VP6 antigen, for each of seven days.

Animals immunized with murine-associated VP6 spore preparations with 10 μg/dose LT (Rl 92G), bovine-associated VP6 with μg /dose LT (Rl 92G), or bovine-associated VP6 with 5 μg/dose LT (Rl 92G) showed substantially reduced disease symptoms (Fig. 4, open symbols). The animals administered bovine-or murine-associated VP6 spores preparations showed almost no viral antigen in feces nor did animals administered bovine VP6 spore- associated preparations with adjuvant LT (Rl 92G) at 5 μg/dose or 10 μg/dose (Fig.4).

These data show that immunizing intranasally with bovine or murine derived VPό spores preparations with LT (Rl 92G) substantially reduced rotavirus in feces.

Animals administered bovine-or murine-associated VP6 spores preparations with LT (192G) were effectively immunized, as demonstrated by the increased serum anti-VP6 ELISA titers and the reduced fecal rotavirus content in EDIM rotavirus challenged animals. B. subiilis spore preparations associated with a viral antigen were effective vaccines, generating protective immunity against rotavirus challenge in the EDIM animal disease model. Animals immunized with bovine-or murine-associated VP6 spores had significantly increased serum anti-VP6 response titers as compared to control spores, and an adjuvant such as CT or LT (R 192G) further increased the serum antibody response to rotavirus VP6.

Example 8: Recombinant strains expressing TTFC

To construct a recombinant strain that expresses TTFC during vegetative growth stage, vector pBB1375 for expression of cloned DNA under the control of a highly active version of the semi-synthetic spac promoter was constructed by site-directed mutagenesis. Plasmid pBB1375 was derived from pSac-Kan (Middleton et al., 2004, Plasmid 51 :238-245) by deleting the BseRII fragment (resulting in pBB1364) and then introducing V spac between the BgIIl and Xbal sites. The version of the spac promoter in pBB1427 has two singie-micleotide mutations (SEQ 3D No: 1) in conformance with the consensus sequences for promoters recognized by the sigma-A form of B. subtilis RNA polymerase (Fig. 5). The ribosomc binding site (RBS) and ATG initiation codon of the B. subtilis gsiB gene were inserted between the spac promoter and tetC The tetC sequence from positions 2855 to 4237 of the tetanus toxin gene of Clostridium tetani (GenBank no. X04436) were amplified and fused to the ATG initiation codon and the ribosomal binding site of the B, subtilis gsiB gene and cloned in parent plasmid pBB1375 to create pBB1427.

Competent cells of B. subtilis strain 168 were prepared by the two-step transformation method (Dubnau et al., 1994, Res. Microbiol. 145(5-6): 403-411). The plasmid pBB1427 was used to transform the competent cells (of genotype AthyA A lhyB sacA::[thyA cat]) to neomycin-resistance. Transformants arose by double-crossover recombination, resulting in the insertion of the Vspac-tetC construct within the sacA locus. A representative clone carrying genetic information for expression of TetC peptide cytoplasmic ally was named BB2646, A control strain, BB2643, carrying the Pspac promoter at the sacA locus without the appended tetC coding sequence was also prepared. This strain is a negative control that lacks genetic information encoding any antigen, i.e., carries an empty vector.

A strain displaying TTFC on. the surface of spores as a fusion protein with CotC, a spore coat protein, was constructed by introducing into pSac-Kan a 374-bp DNA fragment that includes the cotC promoter and coding sequence fused in-framc at its C -terminus with a 3»alanine-codon linker and the coding sequence of TTFC (residues 2581 to 4237 of the tetanus toxin gene). The resulting plasmid, pBBl 367, was introduced into the ΔthyA AthyB sacA::[thyA⁺ cat] B. sublilis recipient strain by transformation as described above, leading to integration at the sacA locus and resistance to neomycin. A resulting transformant was named BB2645.

Strains carrying three integrated copies of the Ϋspac-ieiC or cotC-teiC construct at different loci {sacA, thrC and amyE) in thci?. subiilis chromosome were constructed. These strains, BB3059 for Pspac-tetC and BB3184 for cotC-tetC, were constructed by methods analogous to those for the single copy integrants. The CotC fusion causes expression of the antigen on a spore coat protein. Expression of antigens on the surface of spores, or on the surface of vegetative cells or cytoplasmically in vegetative cells is illustrated in Fig, 21. Constructs are shown in Fig. 22.

Example 9: Preparation of vegetative cells and extraction of vegetative cell lysates Vegetative B. subtilis cells of strains BB2643 and BB2646 were prepared for use in immunization by growth at 37° C in LB to an absorbance at 600 nm (ODgoo) of 0.8-1.0. For vegetative cell lysates, cells were grown to OD_60O= 1-5 in LB medium or defined medium, the cell suspension was washed and lysed by sonication, and collected by high-speed centrifugation. Proteins concentrations were measured using the Pierce Protein Assay kit (Thermo Fisher Scientific, Rockford IL). For Western blotting, proteins were subjected to SDS-PAGE and blotted on nitrocellulose membranes. After successive incubation of the membrane with rabbit polyclonal anti-tetanus toxin antibody (1 :500) and goat anti -rabbit IgG conjugated with horseradish peroxidase (Pierce, 1 :500), the protein bands were visualized by chemilumincsccnce (Pierce), following the manufacturer's instructions (Fig. 6).

Example 1 1 : Immunofluorescence microscopy

B. subtilis strains (BB2643 and BB2646) grown in LB medium were fixed in situ as described previously (Harry et al., 1995, J. Bacterid. 177: 3386-3393). Cultures were vortexed to disrupt clumps of bacteria before fixation. A 0.25-ml volume of bacterial culture was mixed with concentrated fixative solution to give 2.4% (vol/vol) paraformaldehyde, 0.04% (vol/vol) glutaraldehyde, and 30 mM Na-PO4 buffer (pH 7.5) and the mixture was incubated for 10 min at room temperature (20-22° C) and then for 50 min on ice. The fixed bacteria were washed three times in PBS, pH 7.4, at room temperature and were resuspended in 100 μl of GTE (50 mM glucose, 20 mM Tris-HCl, pH 7.5, I O mM EDTA). A freshly prepared lysozyme solution in GTE was added to a final concentration of 2 mg/ml. Samples (10 μl ) were immediately distributed into wells of a multiwcll microscope slide (ICN Biochemicals; Aurora, OH) that had been treated with 0.1% (wt/vol) poly-L- lysine (Sigma). After 4 min, the liquid was aspirated from the wells, which were then allowed to dry completely. The slides were immersed in methanol at -20° C for 5 min and then at -20° C in acetone for 30 s and allowed to dry. Ten μl of blocking solution, 2% bovine serum albumin (BSA) in PBS (BSA-PBS) was added to each well, and slides were incubated for 15 min at room temperature and washed nine more times. Samples were incubated with polyclonal rabbit anti-tetanus toxin for 1 h at room temperature, washed three times, and were incubated with anti-rabbit immunoglobulin G (IgG) -fluorescein isothiocyanate (Southern Biotech; Birmingham, AL) for 1 h at room temperature. After three washings, the samples were observed and photographed with a Zeiss fluorescence microscope fitted with a Nikon DMX 1200 digital camera, and data were analyzed with Lucia GF software.

Example 11 : Immunization regimens

Groups of five 6- to 8-week old female BALB/c mice were inoculated via the intranasal route with vegetative cells of various B. suhiilis strains. B. siώtilis vegetative cells were cultured in LB broth for 4-6 hr until the culture reached an ODcoo of 0.8 to 1.0 at 600 nm. After harvesting, the cell pellets were resuspended in an equal volume of PBS. Spores were harvested after 48-72 hr of incubation with shaking in DS medium (Fouet et al., 1990).

The spores were washed repeatedly with sterile deionized water, treated with lysozyme (1 mg/ml) and washed again several times. Residual contamination by vegetative cells, as detected by phase contrast microscopy, was 1% or less. Spores were stored in deionized water at 4⁰C.

Mice were inoculated intranasally with 20 μl of cell or spore suspension per dose

(10⁷ to 10⁹ vegetative cells or 10⁸ to 10⁹ spores per dose; 10 μl per nare) on days 0, 2, 14, 16, 28, and 30 (6 inoculations) or 0, 14, and 28 (3 inoculations). For a positive control, mice were immunized intramuscularly (i.m.) with 50 μl of a commercial DTaP vaccine adsorbed

(triple vaccine for diphtheria, tetanus and pertussis; Tripedia®, Sanofϊ Pasteur inc., Swiftwater, PA, USA) on days O, 14, and 28 (3 inoculations). Blood samples from inoculated mice were acquired on days -1, 13, 27, and 41.

Example 12: Detection of TTFC-spgcjfic serum antibody responses Anti-TTFC antibody amount in serial three- fold dilutions of sera was measured by

ELISA. Absorbance values of pre-immune sera were used as reference blanks. Dilution curves were drawn for each serum sample and endpoint titers, representing the reciprocal value of the last dilution that gave an optical density =0.1 , were expressed as the means ±S.E. for animals submitted to the same vaccine regimen. Serum TTFC-specific IgG subclass responses were measured with same experimental procedure but using pcroxidase- conjugatcd rabbit anti-mouse IgGl and IgG2a. Titers lower than 100 (negative samples) were arbitrarily assigned as 33.

Example 13 : Tetanus toxin challenge Three weeks following the last immunization, mice were challenged intraperitoneal Iy with purified tetanus toxin (0.8 ng), determined previously to be an amount that is twice LDioo- Mice were observed for morbidity or mortality daily for 10 days.

Example 14: Recombinant TTFC expressed in B. subiilis vegetative cells A recombinant strain of B. subtilis was constructed to express the heavy chain C fragment of tetanus toxin (TTFC), corresponding to the 457 C-terminal amino acids of the 1315-residue tetanus holotoxiti, from a strong and constitutively active mutant version of the spac promoter. This construct was integrated at the sacA locus in strain BB2646. TTFC expression in BB2646 was confirmed by Western blotting and immunofluorescent (IF) staining (Fig. 6).

Example 15: Oral immunization with BB2646 spores

Ability of a spore preparations of strain BB2646 to generate a protective immune response after oral immunization of mice was tested. Mice immunized with the spore preparations showed very little increase in anti-TTFC serum antibody titer even after six inoculations with more than 10¹⁰ spores per inoculation, compared to the control strain BB2643 (Fig. 7). Constructs in which the TTFC-encoding sequence was fused to a vegetative cell wall protein (WapA) or a spore coat protein (CotC) were also tested. In neither of the latter cases was any significant increase in anti-TTFC titers in serum observed. Although some colonization of the mouse GI tract by the recombinant strain could be detected, the BB2646 titer in fecal samples declined within 7 days.

Example 16: Intranasal immunization with BB2646 spores

Ability of the BB2646 spore preparations from cells expressing the antigen cytoplasm. cally to immunize mice after intranasal inoculation was tested. In this case, very high levels of serum anti-TTFC antibodies were detected after three rounds of inoculation (one or two doses per round) at two-week intervals (Fig. 8). The titer after the third round of immunization was the same whether the mice received a total of six inoculations or three

(Fig 8) and was also as high as that generated by intramuscular inoculation with commercial DTaP vaccine.

These mice were completely protected from lethal toxin challenge (Fig. 8). Coadministration of cholera toxin (CT) as an adjuvant did not affect the observed immune response (Fig. 8), Mice inoculated with control spores (strain BB2643) that were isogenic to BB2646 and lacked the TTFC coding sequence gave no detectable antibody response and were fully sensitive to challenge by tetanus toxin (Fig, 8), The dose of spores between 3 x 10 and 3 x 10 per dose was observed to give protective immunity (Fig. 9).

Example 17: Mechanism of intranasal immunization by BB2646 spores

Without being limited by any particular theory or mechanism of action, a model the protective immunity afforded by spore preparations of BB2646 might be due to germination of the spores in the nasopharynx, followed by outgrowth of vegetative cells, expression of TTFC and presentation of the TTFC to cells of the nasopharyngeal immune system. However dissection of the nasopharynx of inoculated mice revealed the presence of spores but not of any detectable level of vegetative cells. Moreover, incubation of the spores at 80"C for 10 min or at 37⁰C for 5 weeks, conditions which do not affect spore viability, greatly reduced immunogenicity of the spore preparation (Fig. 10). In addition, introduction into strain BB2646 of a mutation that greatly reduced the ability of the spores to germinate had only a small effect on immunogenicity (Fig. 1 1). Finally, purification of the spores by density gradient centrifugal ion removed the ability to induce an immune response (data not shown). Taken together these results suggest strongly that the spore form of strain BB2646 was not responsible for generating the protective immunity that we had seen. Example 18: Immunization by vegetative cells of BB2646

During spore preparation, contaminating vegetative cells were removed by osmotic shock, treatment with Iysozyme and extensive washing to the extent that the level of contamination was no higher than 1% as measured by phase contrast microscopy.

Nonetheless, a low level of contamination could have been present. Since it is the vegetative form of BB2646 that expresses the TTFC antigen, whether such vegetative cells could account for the immunization obtained with spore preparations.

In fact, freshly grown vegetative cells harvested from growth medium and resuspended in PBS were observed to be very active inducers of protective immunity. Three doses of vegetative cclis of BB2646 with titers as low as 10⁷ cells per dose were observed to give a strong antibody response and protection against tetanus toxin (Fig. 12). Thus, vegetative cells contaminating the spore preparation could explain protective immunity observed herein. To explore the timing of development of immunogenic vegetative cells from a population of spores, spores of stain BB2646 were heated to 80⁰C for 10 min to kill any contaminating vegetative cells and the heated spores were then suspended in LB and incubated at 37⁰C. At timed intervals samples of the germinating spores were removed and tested for immunogenicity. The data show that unheated spore preparation gave a strong immune response at a dose of 10⁹; heating destroyed immunogenicity (Fig. 13), After 1 hr of incubation at 37°C, an amount of culture equivalent to 10⁹ original spores was highly immunogenic (Fig. 13); microscopic examination revealed that these spores had lost their refractility but had not yet grown out as vegetative cells. After 3 hr in LB, the spore population had been converted almost entirely to vegetative cells. A sample corresponding to 10 original spores generated very high levels of serum antibody and full protection against a tetanus toxin challenge (Fig. 13).

Example 19: Heat stability of TTFC expressed in B. subtilis vegetative cells

The rationale for using B. subtilis as a vaccine delivery system is that the spore form of the bacterium is highly resistant to a variety of environmental conditions, including high temperatures, to which conventional vaccines would be very sensitive. Since the active form of the vaccine strain engineered herein was observed to be the vegetative cell rather than the spore, ability of such vaccine strains to survive storage at elevated temperatures was determined. Resistance to high temperatures is particularly important for vaccine distribution and administration in areas of the world that lack consistent and widespread refrigeration.

To evaluate antigenic stability to heating, B. subtilis vegetative cells were incubated at 60⁰C for 1 hi^* in either the wet state (in PBS) or after drying in a Speed-Vac or freeze- drying in a lyophilizer. In the latter cases, cells were resuspended in sterile H₂O after heating.

Mice that were immunized with vegetative cells that had been heated to 6O⁰C in the wet state showed no increase in serum anti-TetC titers and were indistinguishable from mice inoculated with control cells that do not express TTFC (Fig. 14 panel A). When the cells were heated in the dry state, however, generated very strong immune reactions similar to those obtained with fresh, unhealed vegetative cells, demonstrating that the TTFC in dried vegetative cells was still highly immunogenic after heat treatment. The mice immunized with cells that were heated in the dry state were completely protected against lethal tetanus toxin challenge (Fig. 14 panel B).

Similar studies were carried out with strain BB2645 that displays the TTFC on the surface of spores by fusion to the spore coat protein CotC. The immunogcnicity of dried spores of this strain was entirely resistant to incubation at 60⁰C for 60 min (Fig. 15).

To assess long-term heat stability at a temperature that is near the limit of ambient conditions anywhere in the inhabited world, the survival of immunogcnicity in dried preparations of vegetative cells and spores kept at 45°C for 30 days was tested. For this example, strains that carried three copies of the Ϋspac-ietC (BB3059) or cotC-lelC (BB3184) construct were used to increase overall antigen delivery. In both cases, dried cells or spores were completely resistant to high temperature, generating very high serum antibody responses at doses of 10⁷- 10⁸ per round of inoculation (Figs. 16 and 17).

Example 20: Recombinant B. subtilis vegetative cells induced a balanced ThI and Th2 immune response

Ratios of lgG2a and IgGl subclasses in host serum indicate the bias towards a ThI or Th2 type immune response. Mice inoculated intranasally with recombinant B. sublilis vegetative cells showed increased levels of both IgGl and IgG2a, giving ratios near unity, whereas the mice receiving the conventional DTaP vaccine given i.m. had increased levels of IgGl but not of IgG2a, indicative of a Th-2 type immune response (Fig, ! 8). These results indicate that recombinant B. subtilis vegetative cells induced a balanced immune response.

Example 21 : Recombinant B. subtilis spores induced increased JgA levels Mice were immunized intranasally with spores of strain BB2666, which expressed bovine VP6 under the control of spac promoter, or the control strain BB2643. See Examples 1 and Figs. 1-4, and 21. Fecal samples were collected two-weeks after the third round of inoculation and assayed for IgA-type antibodies by ELISA. Mice inoculated with rotavirus vaccine spores showed increased IgA level compared to mice inoculated with control spores (Figs. 19 and 20).

Claims

What is claimed is:

1. A method of immunizing a subject to an infectious agent, the method comprising: sporalating a vegetative host bacterial cell comprising an isolated nucleotide sequence encoding an antigen of the infectious agent, wherein the nucleotide sequence is operably linked to a promoter for cytoplasmic vegetative expression of the antigen, wherein at least one of the vegetative cells and spores are associated with the antigen; and, contacting the subject with a composition comprising at least one of the vegetative ceils and the spores, wherein the antigen immunizes the subject to the infectious agent.

2. A method of immunizing a subject to an infectious agent, the method comprising: sporulating a vegetative host bacterial cell comprising an isolated nucleotide sequence encoding an antigen of the infectious agent, wherein the nucleotide sequence is operably linked to a promoter for expression of the antigen as a fusion to a spore coat protein, wherein spores are associated with the antigen; and, contacting the subject witli a composition comprising the spores, wherein the antigen immunizes the subject Io the infectious agent.

3. The method according to either of claims 3 and 2, wherein the infectious agent is viral or bacterial.

4. The method according to either of claims 1 and 2, wherein the infectious agent is at least one bacterium selected from the group of consisting of Bacillus anthracis, Clostridium tetcmi, Corynebacterium diphtheriae, Bordetella pertussis, Mycobacterium tuberculosis, Salmonella typhimurium, Staphylococcus aureus, Streptococcus pneumoniae, Treponema pallidum, Neisseria gonorrhoeae, and the like.

5. The method according to either of claims 1 and 2, wherein the infectious agent is at least one virus selected from the group consisting of human immunodeficiency virus (HIV), influenza, polio, herpes, smallpox, measles, mumps, rubella, rotavirus, chicken pox, rabies, West Nile virus, eastern equine encephalitis, neurovirus, and the like.

6, The method according to either of claims 1 and 2, wherein the antigen is a rotavirus antigen, for example of bovine or murine origin, or a Clostridium telani antigen.

7. The method according to cither of claims 1 and 2, wherein the rotavirus antigen is a viral virion protein.

8. The method according to either of claims 1 and 2, wherein the viral virion protein is selected from at least one of the group consisting of VP2, VP4, VP6, VP7, NSP4, and a portion or a derivative thereof.

9. The method according to either of claims 1 and 2, wherein the subject is a vertebrate animal .

10. The method according to claim 9, wherein the vertebrate animal is selected from at least one of the group consisting of an agricultural animal, a high value zoo animal, a research animal, a human, and a wild animal in a dense human environment.

1 1. The method according to either of claims 1 and 2, wherein contacting the subject further comprises administering the composition by a route selected from at least one of the group consisting of intravenous, intramuscular, intraperitoneal, intradermal, mucosal, and subcutaneous.

12. The method according to either of claims 1 and 2, wherein contacting the subject is intranasal administration.

13. The method according to claim 12, wherein the intranasal administration further comprises inhalation or nose drops.

34. The method according to either of claims 1 and 2, wherein an immunizing host bacterial cell is a Bacillus cell.

15. The method according to claim 14, wherein the Bacillus is Bacillus subiilis.

16. The method according to either of claims 1 and 2, wherein the composition further comprises an adjuvant.

17. The method according to claim 16, wherein the adjuvant is selected from at least one of the group consisting of cholera toxin, a non-toxic variant of Escherichia coli labile toxin, and a portion or a derivative thereof.

18. The method according to either of claims 1 and 2, further comprising observing resistance of the composition to at least one condition selected from the group of heat, drying, freezing, deleterious chemicals and radiation.

19. The method according to cither of claims 1 and 2, further comprising prior to contacting, lyophilizing the composition.

20. The method according to either of claims 18 or 19, wherein resistance to heat comprises observing resistance at 6O⁰C for one hour and 45⁰C for at least 30 days.

21. The method according to claim 20, wherein observing resistance comprises observing an heat treated composition maintaining full protective immunity.

22. The method according to either of claims 1 and 2, further comprising: measuring an antibody titer in serum of an infected subject, wherein an increase in antibody for the antigen in comparison to a control serum is an indication of efficacy of the immunogenicity of the composition.

23. The method according to either of claims 1 and 2, further comprising: measuring an amount of viral shedding in the subject having been afflicted by the infectious agent, wherein a decrease in fecal virus as compared to that in a control also afflicted by the infectious agent and not contacted with the composition, is a measure of efficacy of the immunogenicity of the composition.

24. A thermally-stable vaccine composition for immunizing a subject with an antigen from an infectious agent, the composition comprising at least one of vegetative cells and spores from a Bacillus cell, wherein the cells comprise an isolated nucleotide sequence encoding the antigen, the nucleotide sequence being genetically engineered and having been integrated into the host bacterial chromosome or carried on a plasmid and provided with appropriate transcriptional and translational regulatory sequences, whereby the cells express the antigen cytoplasmically during vegetative growth, or the cells express the antigen during sporulation as a genetic fusion to a spore coat piOtein whereby upon sporulation by the cells the antigen is associated with the vegetative cells, the spores, or both, and wherein the composition is effective to immunize the subject.

25. The composition according to claim 24, wherein the antigen is a viral protein or a portion or a derivative thereof.

26. The composition according to claim 25, wherein the viral protein is a viral virion protein.

27. The composition according to claim 26, wherein the viral virion protein is selected from at least one of the group consisting of VP2, VP4, VP6, VP7, NSP4, and a portion or a derivative thereof.

28. The composition according to claim 24, wherein the Bacillus is Bacillus subtilis.

29. The composition according to claim 24, further comprising an adjuvant.

30. The composition according to claim 29, wherein the adjuvant is selected from at least one of the group consisting of cholera toxin, a non-toxic variant of Escherichia coli labile toxin, and a portion or a derivative thereof.

31. The composition according to claim 24, further comprising the isolated nucleotide sequence encoding the antigen which is from a strain that is bovine or murine.

32. The composition according to any of claims 24-31, wherein components are treated to remove substantially all water.

33. The composition according to claim 32, wherein components are treated by at least one of centrifugation under vacuum, lyopMKzation, spray drying and the like.

34. A vaccination kit comprising a unit dose of the composition according to any of claims 24-33, a container, and instructions for use.

35. The vaccination kit according to claim 34, wherein the instructions include storage at a room temperature from about 4°C to about 45"C and the like.