Invented by Theodore Randolph, Amber Clausi, John F Carpenter, Daniel K. Schwartz, University of Colorado

The market for the method of preparing an immunologically-active adjuvant-bound dried vaccine composition is experiencing significant growth and is expected to continue expanding in the coming years. This innovative approach to vaccine development offers numerous advantages over traditional liquid vaccines, making it a promising solution for preventing various infectious diseases. The method involves combining an adjuvant, which enhances the immune response to the vaccine, with the antigenic components of the vaccine. These components can be proteins, peptides, or other molecules that stimulate the immune system to produce a protective response against specific pathogens. The adjuvant-bound dried vaccine composition is then formulated into a dry powder, which can be easily reconstituted with a suitable diluent before administration. One of the key advantages of this method is its improved stability and shelf life. Traditional liquid vaccines often require refrigeration to maintain their potency, which can be challenging in resource-limited settings or during transportation to remote areas. In contrast, the adjuvant-bound dried vaccine composition can be stored at room temperature for extended periods without compromising its efficacy. This makes it more accessible and easier to distribute, especially in regions with limited infrastructure. Furthermore, the dried vaccine composition offers enhanced convenience and ease of administration. Unlike liquid vaccines that require careful handling and cold chain storage, the dried formulation can be reconstituted with a diluent at the point of use. This eliminates the need for complex cold chain logistics and reduces the risk of vaccine wastage due to improper storage or handling. The convenience of the dried vaccine composition also facilitates mass vaccination campaigns, making it an ideal solution for controlling outbreaks or immunizing large populations. The market for this method of vaccine preparation is driven by several factors. Firstly, the increasing global demand for effective vaccines against infectious diseases is a primary driver. With the ongoing COVID-19 pandemic and the constant threat of emerging pathogens, there is a growing need for innovative vaccine technologies that can be rapidly developed and deployed. The adjuvant-bound dried vaccine composition offers a versatile platform that can be adapted to various pathogens, making it attractive for both established and emerging infectious diseases. Secondly, the market is fueled by the advantages offered by this method in terms of cost-effectiveness. The dried vaccine composition eliminates the need for expensive cold chain infrastructure, reducing the overall cost of vaccine distribution. Additionally, the stability of the dried formulation reduces the risk of vaccine wastage, further optimizing cost-efficiency. These factors make the method particularly appealing for low- and middle-income countries, where cost and logistics are significant barriers to vaccine accessibility. Lastly, the market is supported by ongoing research and development efforts in the field of vaccine adjuvants. Adjuvants play a crucial role in enhancing the immune response to vaccines, and continuous advancements in this area are driving the development of more potent and targeted adjuvant formulations. The combination of adjuvants with dried vaccine compositions opens up new possibilities for vaccine design and efficacy, attracting investments and collaborations from pharmaceutical companies, research institutions, and governments. In conclusion, the market for the method of preparing an immunologically-active adjuvant-bound dried vaccine composition is witnessing substantial growth due to its stability, convenience, and cost-effectiveness. This innovative approach to vaccine development has the potential to revolutionize immunization strategies, particularly in resource-limited settings and during outbreaks. With ongoing advancements in adjuvant research and increasing global demand for effective vaccines, the market is expected to expand further, contributing to improved public health outcomes worldwide.

The University of Colorado invention works as follows

The disclosure provides a method of preparing an immunologically-active adjuvant-bound freeze dried vaccine composition. In one embodiment, the stable vaccine composition comprises an aluminum-salt adhuvant, a Clostridium Botulinum Neurotoxin recombinant protein and a glass forming agent. These vaccine compositions can be used to treat humans and animals who are at risk of infection by Clostridium neurotoxin.

Background for Method of preparing an immunologically-active adjuvant-bound dried vaccine composition

Adjuvants are required for vaccines that contain recombinant protein to produce an immune response. (Callahan, et. al., 1991 The importance of surface charges in the optimization antigen-adjuvant interaction, Pharm. Res. 8(7):851-858). “Aluminum-salt adjuvants is currently the most widely used adjuvant in general use for humans.

The mechanisms of action of Aluminum-Salt Adjuvants is poorly understood but probably due to multiple mechanisms. (Lindblad 2004. Aluminium compounds as vaccine ingredients? Immunol. Cell. Biol. 82(5):497-505. Gupta & Siber, 1995. Adjuvants For Human Vaccines – Current Status, Problems, and Future Prospects. Vaccine 13(14).1263-1276. Gupta and Rost 2000. Aluminum Compounds As Vaccine Adjuvants. In O’Hagan D. editor Vaccine Adjuvants Preparation Methods and Vaccine Research Protocols. Totowa, N.J. Humana Press Inc. p. 65-89. Cox and Coulter 1997. Adjuvants. A classification and review of the modes of action. Vaccine 15.3.2448-2256

Commonly proposed mechanisms include the adjuvant acting as a depot on the injection site, where the antigen slowly releases after administration. (Cox & Coulter 1997). A second proposed mechanism is the adjuvant’s role in delivering antigens to antigen-presenting cell (Lindblad, 2004). Another proposed mechanism is the adjuvant acts as an immunostimulator, eliciting Th2 cytokines. (Grun and Maurer 1988, Different T Helper Cell Subsets Elicited in Mice Using Two Different Adjuvant Vehicles: The Role of Endogenous Interleukin-1 in Proliferative Responses. Cell Immunol, 121(1), 134-145. Another proposed mechanism is the adjuvant may destabilize protein antigens that are on the surface of adjuvants, making them more susceptible for proteolytic degradation. (Jones, et. al., 2005 Effects of Adsorption to Aluminum Salt Adjuvants on Structure and Stability of Model Protein Antigens. J Biol Chem, 280(14), 13406-13414. That et. al. (2004). ?Antigen stability controls antigen presentation? J. Biol. Chem. 279(48):50257-50266).

Vaccines that are based on recombinant antigens should be made with adjuvants to maximize their potency.” Adjuvants are used in the formulation of vaccines based on recombinant protein antigens (Singh & O’Hagan, 1999, Advances In Vaccine Adjuvants, Nat Biotechnol, 17(11), 1075-1081, and O’Hagan, et. al., 2001. Recent developments in adjuvants to prevent infectious diseases. Biomol Eng, 18(3), 69-85). Adjuvants approved by the FDA are only aluminum salt adjuvants. Aluminum hydroxide, and aluminum phosphate. According to some, in order to achieve adequate immunogenicity the antigens need be adsorbed onto the surface of adjuvant. (Gupta, et. al., Adjuvant Properties Of Aluminum And Calcium Compounds, 1995. Pharmaceutical Biotechnology. White and Hem 2000, Characterizations of Aluminium-containing Adjuvants in Dev Biol Basel 103: 217-88). This adsorption occurs primarily through electrostatic interactions. The pH of the formulation is chosen to ensure that the adjuvant and antigen are charged in opposite directions (Callahan and al. 1991). Surface exchange reactions can also be used to modify the surface charge of the adjuvant (Hem & White, 1984, Characterization Of Aluminum Hydroxide For Use As An Adjuvant In Parenteral Vaccines). J Parenter Sci Technol 38(1): 2-10. Chang et. al. 1997, Role electrostatic attraction force in the adsorption by aluminum hydroxide. PDA J Pharm Sci Technol 51(1): p.25-9 and Rinella, et. al. 1996, Treatments of aluminum hydroxide adjuvants to optimize the adsorption basic proteins. Vaccine, 14(4): p. 298-300.)

Although it is still not clear how the adjuvants work, the surface area, the surface charge and the morphology are likely to be important factors in dictating immune responses to the antigens that have been adsorbeed onto the adjuvants” (Hem & White, 1984). Maa et. al. (2003) suggest that the larger the adjuvant particle size, the greater the immunogenicity of the vaccine preparation. Stabilization of alum adjuvanted dry powder vaccine formulations: Mechanism and application. J Pharm Sci 92(2):319-332., Diminsky et al., 1999. Physical, chemical and immune stability of CHO derived hepatitis B antigen (HBsAg). Vaccine 18(1-2):3-17).

Lyophilization is often used to improve the long-term stability of protein preparations. When vaccines with aluminum-salts adjuvants undergo freezing and lyophilization in an effort to improve their stability, they often lose potency. According to previous studies, a freeze-dried product containing adjuvants cannot be manufactured due to the aggregation. (Diminsky et al., 1999; Maa et al., 2003).

A number theories have been put forward to explain the possible mechanisms that are responsible for the potency loss following lyophilization in vaccines with aluminum-salt as adjuvants. The aggregation after freezing and defrosting of gels containing aluminum hydroxycarbonate or magnesium hydroxide has been attributed, for example, to the formation of ice crystals which force particles together and result in irreversible aggregates. (Zapata, et. al., 1984. Mechanism of freeze-thaw stability of aluminum hydroxycarbonate gels and magnesium hydroxide). J Pharm Sci 73(1):3-8). Maa et. al., 2003 have echoed this explanation. They propose that faster cooling results in a higher rate of ice nucleation, and smaller ice crystalline formation, which wouldn’t force alum particle into an aggregate. Nygaard et al. The immunological response to polystyrene in mice was dominated by the particle diameter and surface area, not the mass or volume (Nygaard, et al. 2004). Since then, many of these mechanisms have been proven to be incorrect.

Roser et al., U.S. Pat. No. No. However, Roser et al. “Freezing rate” is not discussed by Roser et al.

The ability of particles to cause allergic sensitization can be predicted by the number of particles and their surface area rather than by mass. Toxicol Sci 82(2):515-524). Moorefield et al. Moorefield et. al.,2005) showed that the amount of antigen internalization in adjuvant particles was inversely proportional to the size of the aggregated adjuvants. Role of aluminum-containing adhuvants in the internalization of antigen by dendritic cell in vitro Vaccine 23(13):1588-1595). The particle size may be an important parameter in determining immunogenicity. However, there is no comprehensive study that examines the PSD as a function formulation and cooling rates.

The botulinum-neurotoxin is a highly poisonous substance, and has therefore been identified as an important biological weapon (Gill 1982: Bacterial Toxins: A Table of Lethal Amounts, Microbiol Review 46(1):86-94, Caya, 2001: Clostridium Botulinum and The Ophthalmologist: An overview of botulism and biological warfare implications of botulinum-toxin, Surv Ophthalmol, 46(1):25-34) Toxin in bloodstream can be lethal at 10-9 mg/kg of body weight. The neurotoxin is produced by Clostridium Botulinum and comes in seven different but structurally identical variants. These are identified as A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, S, T, U, V, W, Z, and Y (Oguma et. al. 1995, Structure & function of Clostridium Botulinum Toxins, Microbiol Immunol There is very little cross-reactivity of antibodies between the seven BoNT Serotypes. They are composed of a 100kDa heavy chains containing the binding and internalization regions, and a lighter 50kDa chain that contains the catalytic region, which is connected by a Disulfide Bond. Botulinum toxin blocks nerve transmission from the muscles resulting in flaccid parlysis. The toxin can lead to respiratory failure and death when it reaches the airway muscles or respiratory muscles. (Amon, 1986, J. Infect. Dis. 154:201-206).

The four different types of botulism are based on how the toxin is introduced into the bloodstream. Ingestion of botulin toxin-containing food that has been improperly stored and heated can cause food-borne botulism. (MacDonald et al., 1986, Am. J. Epidemiol. 124:79). C. botulinum penetrates traumatized tissues and produces toxin which is then absorbed by the bloodstream. (Swartz 1990, ‘Anaerobic Spore Forming Bacilli: the Clostridia? pp. 633-646, in B. D. Davis et al., (eds. ), Microbiology, 4th edition, J. B. Lippincott Co.). C. botulinum colonization in the infant’s intestine leads to the production of toxin, which is then absorbed into the bloodstream. The bacterium is most likely to enter the body when spores ingested germinate. (Amon 1986). When the toxin inhaled, it results in Inhalation Botulism. The cause of inhalation botulism was reported to be accidental laboratory exposure (E. Holzer). Klin. Klin. 473-476).

The toxin produced by different strains of Clostridium Botulinum is antigenically unique and designated A-G. Types B, E, and F are also implicated but in smaller numbers. (Sugiyama 1980, Clostridium botulinum neurotoxin, Microbiol. Rev. 44:419-448). Smith et. al. describe the sequence variation between C. botulinum serotypes. (Smith et. al., The impact of sequence variation on antibody binding and neutralization. Infection Immun. 2005, 73 (9): 5450-5457).

The current protein therapies are ineffective against these toxins due to limited availability, high costs of production, and possible side effects. (Eubanks and al., 2007, High-throughput identification and analysis of antagonists of botulinum Neurotoxin A, Proceedings of the National Academy of Sciences. Nat. Acad. Sci., 104(8) 2602-2607) In an effort to induce immunity against the botulin toxins, subjects have been immunized with toxin-preparations. A C. botulinum vaccine comprising chemically inactivated (i.e., formaldehyde-treated) type A, B, C, D and E toxin is commercially available for human usage. This vaccine preparation is not without its drawbacks. The efficacy is variable. (For example, only 78% recipients produced protective levels of antitype B antibodies after administration of the initial series.) Second, the immunization process is painful. Deep subcutaneous inoculation must be used for administration. Adverse reactions are common. Third, the preparation of inactivated vaccines is hazardous as active toxin has to be handled by lab workers.

Recombinant proteins derived from the Clostridium Botulinum Neurotoxin can be used as immunogens in the preparation of vaccine compositions according to the disclosure.” U.S. Pat. describes several of these recombinant proteins, recombinant techniques, and immunological tests. No. No. 5,9196665, which is incorporated by reference herein. As part of the development for a heptavalent neurotoxin vaccine, recombinant antigens of the seven serotypes were created (Smith, 1998, Development for recombinant neurotoxins, Toxicon, 36(11), 1539-48, and Smith, et. al., 2004, “Roads from vaccines into therapies, Mov. Disord. 19 Suppl. 8: S48-52). These protein antigens (identified as rBoNTA(Hc)-rBoNTG(Hc)) consist of 50 kDa portions of the C-terminal domain of the heavy chains and have no neurotoxin activity (DePaz et al., 2005, Formulation of Botulinum Neurotoxin Heavy Chain Fragments for Vaccine Development: Mechanisms of Adsorption to an Aluminum-Containing Adjuvant, Vaccine 23: 4029-4035).

The U.S. Government has classified the paralysis-inducing Neurotoxins produced by Clostridium Botulinum as Category A Bioagents. It is important to continue improving vaccine stability and safety without compromising vaccine immunogenicity. There is a need to develop safe, effective and stable vaccine compositions to be administered to people at risk of exposure C. botulinum toxins.

One way to accomplish these goals is to develop methods of production of stable, immunologically-active freeze dried vaccine preparations which may incorporate recombinant antigens.

The disclosure describes a method for producing immunologically-active, stable freeze-dried vaccine preparations. The disclosure further provides a method of production of immunologically-active, stable, freeze dried vaccine preparations in which the vaccine antigens are recombinant antigens. The disclosure also includes a stable immunologically-active vaccine composition containing a recombinant Clostridium Botulinum Neurotoxin Protein.

In one embodiment, the disclosure provides a method of preparing an immunologically-active adjuvant-bound dried vaccine composition, the method comprising: combining at least one aluminum-salt adjuvant, at least one buffer system, at least one glass-forming agent, and at least one antigen to create a liquid vaccine formulation; freezing the liquid vaccine formulation to create a frozen vaccine formulation; and lyophilizing the frozen vaccine formulation to create a dried vaccine composition, where the composition is capable of eliciting an immune response in a subject. The subject’s immune response may be a humoral or cell-mediated immune response specific to the antigen. In one aspect, one or more adjuvants consisting of aluminum salts are selected from the group consisting aluminum phosphate, aluminum hydroxide and aluminum sulfate. Aluminum hydroxide is used as an aluminum-salt adjuvant in another aspect. In another aspect, one or more buffer system is selected from a group consisting acetates, succinates, citrates, prolamines, histidines, borates, carbonates, and phosphates buffer systems. In one aspect, one or more buffer system is selected from phosphate and succinate buffer systems. In another aspect, one or more glass-forming agent is selected from trehalose (sucrose), ficoll (dextran), maltotriose (lactose), mannitol (hydroxyethyl a starch), glycine(cyclodextrin), cyclodextrin potassium salts, cyclodextrin cyclodextrin and dextran. In another aspect, the glass forming agent is trehalose. In one aspect of the liquid vaccine formulation, the glass-forming agents trehalose are present at a weight to volumes concentration ranging from approximately 5% to around 20%. In a second aspect, the glass-forming agents trehalose are present in the liquid formulation in a concentration ranging from about 7% up to 15%. In another aspect, the freezing process can be one of four types: tray freezing, shelf-freezing, spray-freezing, and shell-freezing. Spray-freezing is one way to freeze.

In one aspect, the antigen is selected from or derived from the group consisting of Rotavirus, Foot and mouth disease virus, influenza A virus, influenza B virus, influenza C virus, H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, human parainfluenza type 2, herpes simplex virus, Epstein Barr virus, varicella virus, porcine herpesvirus 1, cytomegalovirus, Lyssavirus, Poliovirus, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis E, distemper virus, venezuelan equine encephalomyelitis, feline leukemia virus, Reovirus, Respiratory syncytial virus, Lassa fever virus, polyoma tumor virus, canine parvovirus, Papilloma virus, tick borne encephalitis virus, Rinderpest virus, human rhinovirus species, Enterovirus species, Mengo virus, Paramyxovirus, avian infectious bronchitis virus, Human T-cell leukemia-lymphoma virus 1, Human immunodeficiency virus-1, Human immunodeficiency virus-2, lymphocytic choriomeningitis virus, Parovirus B19, Adenovirus, rubella virus, yellow fever virus, dengue virus, Bovine respiratory syncitial virus, Corona virus, Bordetella pertussis, Bordetella bronchiseptica, Bordetella parapertussis, Brucella abortus, Brucella melitensis, Brucella suis, Brucella ovis, Brucella species, Escherichia coli, Salmonella species, salmonella typhi, Streptococci, Vibrio cholerae, Vibrio parahaemolyticus, Shigella, Pseudomonas, tuberculosis, avium, Bacille Calmette Guerin, Micobacterium leprae, Pneumococci, Staphylococci, Enterobacter species, Rochalimaea henselae, Pasteurella haemolytica, Pasteurella multocida, Chlamydia trachomatis, Chlamydia psittaci, Lymphogranuloma venereum, Treponema pallidum, Haemophilus species, Mycoplasma bovigenitalium, Mycoplasma pulmonis, Mycoplasma species, Borrelia burgdorferi, Legionella pneumophila, Colstridium botulinum, Corynebacterium diphtheriae, Yersinia entercolitica, Rickettsia rickettsii, Rickettsia typhi, Rickettsia prowazekii, Ehrlichia chaffeensis, Anaplasma phagocytophilum, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Schistosomes, Trypanosomes, Leishmania species, Filarial nematodes, Trichomoniasis, Sarcosporidiosis, Taenia saginata, Taenia solium, Leishmania, Toxoplasma gondii, Trichinella spiralis, Coccidiosis, Eimeria tenella, Cryptococcus neoformans, Candida albicans, Apergillus fumigatus, Coccidioidomycosis, Neisseria gonorrhoeae, Malaria circumsporozoite protein, Malaria merozoite protein, Trypanosome surface antigen protein, Pertussis, Alphaviruses, Adenovirus, Diphtheria toxoid, Tetanus toxoid, meningococcal outer membrane protein, Streptococcal M protein, Influenza hemagglutinin, cancer antigen, tumor antigens, toxins, exotoxins, Neurotoxins, cytokines, cytokine receptors, monokines, monokine receptors, plant pollens, animal dander, and dust mites.

The antigen in a specific aspect is derived by one or more of the Clostridium Botulinum Neurotoxins A, B C, D E F G. In another specific aspect the antigen consists of recombinant neurotoxin (SEQID NO: 1) The antigen in another specific aspect is recombinant Botulinum Neurotoxin C. (SEQID NO: 2) The antigen in a second specific aspect is recombinant Botulinum Neurotoxin (SEQID NO: 3). In another specific aspect, the recombinant neurotoxin A (SEQID NO: 3) is the antigen. Another specific aspect is the antigen, which is recombinant Botulinum Neurotoxin B (SEQID NO: 4).

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