WO2016007797A1 - Composition de vaccin stable multivalent et ses procédés de fabrication - Google Patents

Composition de vaccin stable multivalent et ses procédés de fabrication Download PDF

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WO2016007797A1
WO2016007797A1 PCT/US2015/039826 US2015039826W WO2016007797A1 WO 2016007797 A1 WO2016007797 A1 WO 2016007797A1 US 2015039826 W US2015039826 W US 2015039826W WO 2016007797 A1 WO2016007797 A1 WO 2016007797A1
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composition
antigen
vaccine
ricin
protein
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PCT/US2015/039826
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English (en)
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Robert N. Brey
Christopher Schaber
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Soligenix, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/07—Bacillus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine

Definitions

  • the invention relates to stable immunogenic compositions comprising more than one antigen and conferring increased immunity to an individual.
  • the invention also related to methods of making and using the stable immunogenic compositions described herein.
  • Vaccination is an important tool for handling health care programs both in developed and developing countries.
  • the number of recommended vaccines has increased significantly in recent years against individual infections.
  • vaccines present an antigen to the immune system without introducing viral particles, whole or otherwise.
  • a combination vaccine which can provide immunogenicity against large number of diseases is always advantageous over the monovalent vaccines. We cannot reduce the number of immunizations required in infants and children to protect them from various fatal diseases but the compliance can be increased by reducing the number of separate vaccinations.
  • a combination vaccine is advantageous over monovalent vaccine as it not only increases the compliance but it is also cost effective and convenient thereby reducing the chances of missing any vaccination.
  • due to complications associated with the preparation of such combination vaccines due to possible interaction between the antigens has always been a challenge before the scientific community. Complications include immune interference when vaccine antigens are administered concurrently at the same site in the body, and instability of the antigens together in combinations, impeding the simultaneous development of protective immunity.
  • Combination vaccines while greatly desired for ease of administration and increased compliance, pose difficulties in development due to factors including: physical and biochemical incompatibility between antigens and other components, immunological interference and stability.
  • adjuvants e.g. aluminum salt, oil-in- water emulsions
  • Adjuvants are often included in vaccines to enhance a recipient's immune response to a supplied antigen, while keeping the injected foreign material to a minimum.
  • a specific adjuvant may reduce the activity of one antigen and excessively increase the reactivity of another antigen.
  • Buffers used to minimize changes in acidity of a solution may also interact with other vaccine components.
  • Stabilizers are used to ensure vaccines maintain effectiveness during storage by counteracting the effects of temperature, pH and preservatives. Vaccine stability is essential, particularly where the cold chain is unreliable. However, stabilizers may also have an effect on other agents present in the vaccine. All these potential interactions between vaccine components can negatively effective vaccine potency reducing the immunogenicity of the vaccine.
  • the trend towards combination vaccines has the advantage of reducing discomfort to the recipient, facilitating scheduling, and ensuring completion of regiment. However, there is also the concomitant risk of reducing the vaccine's efficacy.
  • An immune response is generated to an antigen through the interaction of the antigen with the cells of the immune system.
  • the resultant immune response may be broadly distinguished into two extreme categories, being humoral or cell mediated immune responses (traditionally characterized by antibody and cellular effector mechanisms of protection, respectively). These categories of response have been termed Th2-type immune responses (humoral response) and Thl- type responses (cell-mediated response).
  • Thl -type immune responses may be characterized by the generation of antigen specific, haplotype-restricted cytotoxic T lymphocytes, and natural killer cell responses.
  • Thl -type responses are often characterized by the generation of antibodies of the IgG2a subtype, while in the human these correspond to IgGl type antibodies.
  • Th2-type immune responses are characterized by the generation of a broad range of immunoglobulin isotypes, including (in mice) IgGl, IgA, and IgM.
  • cytokines a number of identified protein messengers which serve to help the cells of the immune system and steer the eventual immune response to either a Thl or Th2 response.
  • Thl -type cytokines tend to favor the induction of cell mediated immune responses to the given antigen
  • Th2-type cytokines tend to result in the induction of humoral immune responses to the antigen.
  • Thl :Th2 balance of the immune response after a vaccination or infection includes direct measurement of the production of Thl or Th2 cytokines by T lymphocytes in vitro after re-stimulation with antigen, and/or the measurement of the IgGl :IgG2a ratio of antigen specific antibody responses.
  • a Thl -type adjuvant is one which stimulates isolated T-cell populations to produce high levels of Thl -type cytokines when re-stimulated with antigen in vitro, and induces antigen specific immunoglobulin responses associated with Thl -type isotype.
  • Suitable adjuvant systems which promote a predominantly Thl response include Monophosphoryl lipid A or a derivative thereof, particularly 3-de-O-acylated monophosphoryl lipid A, and a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL) together with an aluminum salt.
  • Adjuvants are molecules, compounds, or macromolecular complexes that boost the potency and longevity of specific immune response to antigens, but cause minimal toxicity or long-lasting immune effects on their own. Adjuvants can be used to enhance immunogenicity, modulate the type of immune response, reduce the amount of antigen or the number of immunizations required, and improve the efficacy of vaccines in newborns or elderly. To be maximally effective, adjuvants must be selected judiciously and formulated appropriately based on the desired immune response. However, the number of adjuvants with acceptable efficacy and safety profiles is limited, and these proprietary molecules/compounds are in the hands of a few companies, as is most of the formulation expertise.
  • Vaccines containing recombinant proteins require an adjuvant to elicit a durable immune response (Callahan, Shorter, et al., 1991, The importance of surface charge in the optimization of antigen-adjuvant interactions, Pharm Res, v8:851-8).
  • the default position in developing subunit protein immunogens for human vaccines is to utilize aluminum adjuvants as the starting point.
  • the use of aluminum adjuvants is thus fostered by the fact that the record of safety of newer formulations cannot match the long term acceptability of aluminum adjuvants in human vaccines.
  • Aluminum-salt adjuvants are currently the most widely used adjuvants for general use in humans. Aluminum adjuvants are considered relatively weak, effective in generation of neutralizing antibodies against certain bacterial antigens, but relatively ineffective at inducing cellular immune responses. There is some consensus that the more effective vaccines with aluminum are those in which antigen is bound to the aluminum surface, rather than free in solution (Lindblad, 2004, Aluminium adjuvants- -in retrospect and prospect, Vaccine, v22:3658-68).
  • Aluminum adjuvants have a point of zero charge (PZC) at a certain solution pH, but are charged at pHs above or below this value (White and Hem, 2000, Characterization of aluminium-containing adjuvants, Dev Biol (Basel), vl 03:217-28).
  • PZC point of zero charge
  • a solution pH is selected in which the protein and adjuvant have opposite charges.
  • a solution pH that provides optimal protein stability may not allow for appropriate binding of the vaccine to adjuvants.
  • a vaccine protein may have to be prepared at pH that is suboptimal for stability and lyophilized with appropriate stabilizing excipients to minimize degradation during long-term storage.
  • Adjuvant formulations consist of aqueous suspensions of vaccine particles, adsorption of antigens to salts of aluminum, oil-in-water emulsions of antigens, and nanovesicles such as liposomes or niosomes.
  • the surface charge on the adjuvant also can be modified by surface exchange reactions with buffer salts such as phosphate, succinate, and citrate (Hem and White, 1984, Characterization of aluminum hydroxide for use as an adjuvant in parenteral vaccines. J Parenter Sci Technol, 38(1): p. 2-10; Chang et al, 1997, Role of the electrostatic attractive force in the adsorption of proteins by aluminum hydroxide adjuvant. PDA J Pharm Sci Technol, 51(1): p. 25-9; and Rinella et al., 1996, Treatment of aluminium hydroxide adjuvant to optimize the adsorption of basic proteins. Vaccine, 14(4): p. 298-300.)
  • buffer salts such as phosphate, succinate, and citrate
  • adjuvant aids in delivery of the antigen to antigen- presenting cells (Lindblad 2004).
  • adjuvant serves as an immunostimulator and elicits Th2 cytokines (Grun and Maurer 1989, Different T helper cell subsets elicited in mice utilizing two different adjuvant vehicles: the role of endogenous interleukin 1 in proliferative responses, Cell Immunol, 121(1): 134-145)).
  • Aluminum-salt adjuvants provide a well explored means to augment the immunogenicity of protein or peptide subunit vaccines.
  • exploratory formulations to enhance vaccines have been developed as more potent alternative to aluminum-salts adjuvants, but are not currently available in FDA- licensed human vaccines.
  • Formulations designed to enhance immune responses include a variety of compositions based on water-in-oil emulsions, oil-in-water emulsions, self-assembling macrostructures, cytokines, saponins, toll-like receptor agonists (TLR- 4, TLR-5, and TLR-9), immunostimulatory double stranded RNA species, unmethylated DNA oligonucleotides, and polymeric microparticles and nanostructures. Many of these compositions are directed towards improving the immunogenicity of injected vaccines, and some variations can be applied to altering routes of delivery for intranasal or oral vaccination.
  • bacterial DNA can be used because of direct immunostimulatory effects that activate lymphocytes. This is due to unmethylated CpG dinucleotides being present at the expected frequency in bacterial DNA but are under-represented and methylated in vertebrate DNA (Krieg et al., 1995). Activation may also be triggered by addition of synthetic oligodeoxynucleotides (ODN) that contain an unmethylated CpG dinucleotide in a particular sequence context.
  • ODN synthetic oligodeoxynucleotides
  • CpG DNA induces proliferation of almost all (>95%) B cells and increases immunoglobulin (Ig) secretion.
  • B cell activation by CpG DNA is T cell independent and antigen non-specific.
  • B cell activation by low concentrations of CpG DNA has strong synergy with signals delivered through the B cell antigen receptor for both B cell proliferation and Ig secretion (Krieg et al., 1995).
  • This strong synergy between the B cell signaling pathways triggered through the B cell antigen receptor and by CpG DNA promotes antigen specific immune responses.
  • CpG DNA In addition to its direct effects on B cells, CpG DNA also directly activates monocytes, macrophages, and dendritic cells to secrete a variety of cytokines, including high levels of IL-12 (Klinman et al, 1996; Halpern et al., 1996; Cowdery et al., 1996). These cytokines stimulate natural killer (NK) cells to secrete gamma-interferon (IFN. gamma.) and have increased lytic activity (Klinman et al, 1996, supra; Cowdery et al, 1996, supra; Yamamoto et al, 1992; Ballas et al., 1996). Overall, CpG DNA induces a Thl-like pattern of cytokine production dominated by IL-12 and IFN-gamma with little secretion of Th2 cytokines (Klinman et al., 1996).
  • flagellin the protein subunit comprising numerous bacterial flagella.
  • Flagellin is a TLR-5 ligand and triggers at least one of the biological functions of antigen presenting cells upon such binding.
  • Flagella are found on the surface of rod and spiral shaped bacteria, including members of the genera Escherichia, Salmonella, Proteus, Pseudomonas, Bacillus, Campylobacter, Vibrio, Treponema, Legionella, Clostridia, and Caulobacter.
  • the conserved regions of flagellins are important for TLR5 binding, while the polymorphic central region can be deleted without affecting binding to TLR5.
  • Flagellin sequences from numerous bacteria are available in the art, such as Genbank accession numbers D13689, YP.sub.-275549, YP.sub.-275550, AAU18718, AAU18717, ZP.sub.-00743095, EA052626, YP.sub.-315348, AAT28337, AAT28336, AAT28335, AAT28334, AAT28333, AAZ36356, AAZ33167, AAZ94424, AAZ91670, NP.sub.-414908, BAD18052, and BAD18051.
  • nontoxic (chemically synthesized or enzymatically modified) derivatives of gram negative lipopolysaccharides are potent adjuvants and act by stimulating lymphocytes through TLR-4 binding and activation.
  • monophosphoryl lipid A MPL is a derivative of the lipid A component of lipopolysaccharide and is a potent activator of pro-inflammatory cytokines.
  • MPL and its chemically synthesized analogues are not toxic but induce a compendium of host proinflammatory cytokines including IL-1, IL-6, and TNF-alpha.
  • adjuvants In addition, to enhance the immune response to subunits adsorbed to aluminum salts, it is likely that co-adjuvants will be required in order to generate effective antibody responses in humans after one or two doses.
  • adjuvant compounds that are compatible with aluminum salts have been evaluated as adjuvants in recent years. Primarily these adjuvants include Monophosphoryl Lipid A (MPL) and QS-21, and CpG sequences.
  • MPL and QS-21 have been also used with aluminum salts as well as in proprietary oil emulsion formulations being developed by Glaxo Smith Kline Biologies.
  • QS-21 has been evaluated in AIOH vaccines in humans and animal models with good evidence of tolerability and systemic safety.
  • QS-21 is thought to bind to aluminum salts through ionic and hydrophobic interactions, as well as some part of it remaining in solution (in aqueous vaccines) in a micellar form.
  • QS-21 is a saponin purified from tree bark with broad adjuvant effects to induce both antibody and cell mediated immunity. Though the mechanism is not understood, dose levels effective in conjunction with human vaccines have been evaluated.
  • QS-21 with aluminum has been evaluated in clinical studies and independent safety studies of QS-21 formulated with antigens have been studied.
  • QS-21 has been associated with stinging at the site of injection (that resolves), with very little evidence of systemic side effects (Waite, Jacobson et al., 2001, Three double-blind, randomized trials evaluating the safety and tolerance of different formulations of the saponin adjuvant QS-21, Vaccine, 19:3957-67).
  • Several studies in humans have shown that QS-21 enhances responses to antigens that are adsorbed to aluminum.
  • Lyophilization of proteins to stabilize structure and activity for storage and reconstitution has been commonly applied to recombinant protein therapeutic proteins. This has been usually accomplished by freeze drying in the presence of disaccharides such as trehalose and other excipients that promote a glass state during process and storage. Proteins can be stored for long term as long as the product is stored below its glass transition temperature (Tg), above which the material transitions into a rubbery state. Excipients are thought to stabilize protein in the amorphous state through interactions of the stabilizer with specific sites substituting for water during drying and by simultaneously suppressing translational and rotational motions of the protein molecule (a-relaxations) or portions of the molecule ( ⁇ -relaxations).
  • Tg glass transition temperature
  • Drying technology has been less frequently applied to long term storage of vaccines, especially in the case of vaccines adsorbed to aluminum phosphate or aluminum hydroxide adjuvants. Very little data is available on the storage of dried vaccines under elevated temperature conditions, as most of the attempts to generate dried vaccines have been to obtain inhalable powders or preparations able to survive moderate excursions in temperature. For example, because the yellow fever vaccine is used primarily in tropical climates, lyophilization in the presence of stabilizers (lactose, sorbitol) has been used to preserve viability of the live virus vaccine (Monath, 1996, Stability of yellow fever vaccine, Dev Biol Stand, 87:219-25).
  • stabilizers lactose, sorbitol
  • a lyophilized dried vaccine for the cattle disease rinderpest has also been developed and can be employed for up to a month after leaving the cold chain in African field conditions (House and Mariner, 1996, Stabilization of rinderpest vaccine by modification of the lyophilization process, Dev Biol Stand, 87:235-44).
  • formaldehyde As a stabilizer, small amounts of formaldehyde are occasionally added to vaccines, including the current AVA anthrax vaccine (Biothrax®), and may act by cross-linking proteins forming more immunogenic protein aggregates on the surface of aluminum crystals (Little, Ivins et al., 2007, Effect of aluminum hydroxide adjuvant and formaldehyde in the formulation of rPA anthrax vaccine, Vaccine, 25:2771-7).
  • Formaldehyde had been used historically as the stabilizer of choice in the older vaccines derived from culture supernatants, such as tetanus toxoid, botulinum toxoids, and others.
  • the current AVA vaccine is labeled for 3 year stability, where stability is a function of a number of biochemical evaluations and potency. Although a moderate amount of stability can be achieved with liquid suspension vaccines, it is not likely that all stability parameters can be met for longer storage periods that are required for vaccines to be stockpiled and distributed.
  • Linear B and T cell epitopes contained therein can be also affected by oxidation (of methionine and cysteine residues) and deamidation (especially of asparagine residues). pH is one of the most critical formulation variables governing stability of therapeutic proteins.
  • Protein aggregation was minimized following lyophilization from a solution at pH greater than 6, although, deamidation occurred at an unacceptably high rate. Following lyophilization from a solution containing amounts of sucrose greater than 0.3 sucrose/protein mass ratio at pH 6.5, both destabilization pathways could be inhibited.
  • interleukin-2 (IL-2) had significantly greater structural perturbation during freeze-drying at pH 7, which resulted in higher levels of aggregation after storage and rehydration than samples lyophilized from solutions at pH 5 (Prestrelski, Pikal et al., 1995, Optimization of lyophilization conditions for recombinant human interleukin-2 by dried-state conformational analysis using Fourier-transform infrared spectroscopy, Pharm Res, 12: 1250-9).
  • sucrose to the pre-lyophilization solution formulation at pH 7 improved the stability of IL-2 during storage following lyophilization.
  • U.S. Pat. No. 6,890,512 (“Roser et al.”) teaches lyophilization methods that will prevent aggregation of aluminum particles and further discloses a method of preventing gross aggregation during dehydration and rehydration of particulates in suspension by adding to a particulate suspension of aluminum hydroxide in excess of 15% (w/v) of trehalose.
  • Trehalose, alpha.-D- glucopyranosyl-alpha-D-glucopyranoside is a naturally occurring disaccharide responsible for protection of plant cells from desiccation.
  • Trehalose has been shown to prevent denaturation of proteins during desiccation by forming sugar glasses that immobilize protein structure.
  • Multivalent vaccine compositions are known in the art and have been described in the literature.
  • WO 1993/024148 discloses an invention of multivalent vaccine containing antigens IPV- DPT-Hib-Hepatitis B wherein DPT is adsorbed to AIOH or aluminum phosphate and Hib is adsorbed to only aluminum phosphate, wherein the Hib antigen is used extemporaneously by mixing to the other antigens just prior to the administration.
  • WO 1997/00697 discloses a DPT-Hib and pertussis multivalent vaccine adsorbed to aluminum phosphate, in which one container has a freeze-dried vaccine and the other container comprises a second antigen.
  • WO 1998/000167 discloses a DTaP-IPV-Hib antigen vaccine and WO 1999/13906 describes a multiple component vaccine in which certain components may be reconstituted from a lyophilized state by the other components of the vaccine, or may exist in a single solution, and administers the vaccine in a specially designed container at the time when the vaccination is performed.
  • WO2000/07623 describes a multi-component vaccine composition having acellular pertussis vaccine components (PT and FHA), diphtheria toxoid (DT), tetanus toxoid (TT), a conjugate of a capsular polysaccharide of Haemophilus influenzae type b and tetanus toxoid or diphtheria toxoid (Hib), Hepatitis B Surface Ag (HBsAg) and inactivated poliovirus (IPV) which may be in a single solution, or certain components may be reconstituted from a lyophilized state by the other components of the vaccine.
  • PT and FHA pertussis vaccine components
  • DT diphtheria toxoid
  • TT tetanus toxoid
  • Hib Hepatitis B Surface Ag
  • IPV inactivated poliovirus
  • WO2002/000249 discloses a capsular polysaccharide of Haemophilus influenza type b not adsorbed onto an aluminum adjuvant salt, and two or more further bacterial polysaccharides which may include whole cell pertussis, tetanus toxoid, diphtheria toxoid, Hepatitis B surface antigen (HbsAg), and/or conjugate polysaccharides of N. meningitides type A, or B, or C as antigens in a single quadrivalent and/or trivalent vaccine.
  • HbsAg Hepatitis B surface antigen
  • WO2006/097851 discloses a multivalent vaccine which can be prepared extemporaneously at the time of use by mixing together two components the first component comprising D, T, wP and HBsAg antigens and a second component comprising a Hib conjugate and one or more meningococcal conjugates.
  • WO2007/054820 relates to a vaccine composition wherein the D, T, and aP antigens are specifically adsorbed on aluminum hydroxide and the Hib and the Hep B antigens are adsorbed onto aluminum phosphate which do not exist in a fully liquid stable composition.
  • WO2008/044611 discloses a method for the preparation of a mixed IPV-DPT vaccine comprising an inactivated poliovirus Sabin strains type I, II, and III grown in Vera cells, a protective antigen against Bordetella pertussis, a diphtheria toxoid and a tetanus toxoid, which involves the step of producing a poliovirus Sabin strain having a high titer.
  • Ricin is a plant toxin that is classified as a ribosome inactivating protein (RIP) and is a potent member of the AB family of toxins.
  • Ricin is a highly toxic, naturally occurring lectin (a carbohydrate-binding protein) produced in the seeds of the castor plant Ricinus communis.
  • Ricin is thought to be a bioterror threat because of its stability and high potency as well as the large worldwide reservoir created as a by-product of castor oil production.
  • Exposure to ricin results in local tissue necrosis, and general organ failure leading to death within several days of exposure. A dose of purified ricin powder the size of a few grains of table salt can kill an adult human.
  • the median lethal dose (LD50) of ricin if given by injection is around 22 micrograms per kilogram of body weight (1.78 mg for an average adult, around 1/228 of a standard aspirin tablet/0.4 g gross) in humans if exposure is from injection or inhalation.
  • Ricin is toxic by all routes of exposure, but is especially toxic by the aerosol route, resulting in necrosis of lung epithelia within hours of exposure, multifocal hemorrhagic edema and death within 24-36 hours, with an estimated aerosol LD50 of 5-8 micrograms per kilogram of body weight (0.4 mg - 0.64 mgs for an average adult).
  • the enzymatic A subunit is an RNA-N-glycosidase which cleaves a specific adenine residue with eukaryotic 28S ribosomal RNA, leading to protein synthesis arrest and cell death. Depurination of this residue results in an immediate cessation of ribosome progression, which subsequently inhibits protein synthesis.
  • RTA RNA-N-glycosidase
  • RTB B subunit
  • the ricin toxin B subunit binds with micromolar affinity to a(l-3)-linked galactose and N60 acetylgalactosamine residues that are expressed on the surface of all mammalian cell types. Binding of RTB to these receptors mediates internalization and retrograde transport of the ricin holotoxin to the endoplasmic reticulum (ER). In the ER, RTA dissociates from RTB and is retrotranslocated across the ER membrane into the cytosol where it gains access to rRNA targets. Ricin is therefore extremely potent, as it is able to internalize into almost all mammalian cell types.
  • ricin In addition to ribosome inactivating properties, ricin also elicits vascular leak syndrome (VLS), which primarily affects endothelial cells.
  • VLS vascular leak syndrome
  • Data based on murine monoclonal antibodies to ricin A chain have indicated that antibodies that neutralize ricin toxin in vitro by inhibiting cytotoxicity of ricin on cultured cells can confer protection to mice by passive transfer. Equally important, non-neutralizing monoclonal antibodies do not confer protection.
  • Immunodominant region II is located within folding domain I and contains a solvent-exposed a-helix (residues N97-F108) (O'Hara, Neal, et al, 2010, Folding domains within the ricin toxin A subunit as targets of protective antibodies, Vaccine), a target of the protective mAb R70 also known as Univax 70 (Lebeda and Olson, 1999, Prediction of a conserved, neutralizing epitope in ribosome-inactivating proteins, Int J Biol Macromol, v24: 19-26). Residues N97-F108 likely constitutes one of the most immunodominant regions on RTA.
  • Immunodominant region IV on RTA spans amino acids I170-T190. There are at least two linear B-cell epitopes within this region. Residues LI 61 to 1175, in particular, were identified as being a conserved target of serum Abs from Hodgkin's lymphoma patients who had been treated with deglycosylated RTA (RTA.dg) immunotoxin (Castelletti, Fracasso, et al., 2004, A dominant linear B-cell epitope of ricin A-chain is the target of a neutralizing antibody response in Hodgkin's lymphoma patients treated with an anti-CD25 immunotoxin, Clin. Exp. Immunol, vl36:365-72).
  • a murine IgGi mAb GDI 2 was effective in protecting mice against the effects of intraperitoneal and intragastric ricin challenges, thereby establishing that preexisting serum Abs directed against residues in immunodominant region IV are sufficient to confer both systemic and mucosal immunity to ricin, at least in rodents (Neal, O'Hara, et al., 2010, A monoclonal immunoglobulin G antibody directed against an immunodominant linear epitope on the ricin A chain confers systemic and mucosal immunity to ricin, Infect Immun, v78:552-61).
  • the GD12 epitope is situated within a-helix E, which runs through the core of RTA' s domain II and which terminates with two residues (Glul77 and Argl80) that are involved in RTA catalytic activity (O'Hara, Neal, McCarthy, Kasten- Jolly, Brey and Mantis, 2010, Folding domains within the ricin toxin A subunit as targets of protective antibodies, Vaccine; Katzin, Collins, et al, 1991, Structure of ricin A-chain at 2.5 A, Proteins., vl0:251-9; Li, Chiou, et al, 2009, A two-step binding model proposed for the electrostatic interactions of ricin a chain with ribosomes, Biochemistry, v48:3853-63).
  • Ingestion of whole castor beans results in severe abdominal pain, vomiting, diarrhea, and (depending on the number of beans and degree of mastication) death (Audi, Belson, et al, 2005, Ricin poisoning: a comprehensive review, JAMA, v294:2342-51; Bradberry, Dickers, et al, 2003, Ricin poisoning, Toxicological Reviews, v22:65- 70; Mantis, 2005, Vaccines against the category B toxins: Staphylococcal enterotoxin B, epsilon toxin and ricin, Advanced Drug Delivery Reviews, v57: 1424-39; Olsnes, 2004, The history of ricin, abrin and related toxins, Toxicon, v44:361-70).
  • Ricin administered orally can impair sugar absorption by rat small intestine, Chem Pharm Bull (Tokyo), v31 :3222-7; Ishiguro, Nakashima, et al., 1992, Interaction of toxic lectin ricin with epithelial cells of rat small intestine in vitro, Chem Pharm Bull (Tokyo), v40:441-5; Ishiguro, Tanabe, et al, 1992, Biochemical studies on oral toxicity of ricin. IV. A fate of orally administered ricin in rats, Journal of Pharmacobio-Dynamics, vl5: 147-56).
  • the inherent resistance of the gastrointestinal tract to ricin is likely due to a number of factors that impede toxin absorption, including intestinal proteases, digestive enzymes, mucus, and secretory IgA, whose galactose-rich oligosaccharide can competitively inhibit ricin attachment to the apical surfaces of intestinal epithelial cells (Mantis, Farrant, et al., 2004, Oligosaccharide side chains on human secretory IgA serve as receptors for ricin, Journal of Immunology, vl72:6838-45).
  • RTA subunits Efforts to create a vaccine against ricin have mostly focused on the RTA subunit.
  • Two mutant versions of RTA have been extensively studied for their ability to promote antibody mediated immunity to ricin.
  • One mutant, RVEc completely removes the third folding domain, which mediates binding to RTB but is only targeted by non-neutralizing antibodies.
  • Another mutant, used in RiVaxTM retains the entire structure of native RTA but contains two point mutations, Y80A and V76M, which completely remove both of RTA' s known toxicities, ribotoxicity and vascular leak induction respectively.
  • RiVaxTM is safe and immunogenic when administered by the intramuscular (i.m.) and intradermal (i.d.) routes.
  • i.m. intramuscular
  • i.d. intradermal
  • adsorption of RiVaxTM to aluminum salt adjuvants enhanced RiVaxTM-specific serum antibodies (Ab) titers.
  • the levels of anti-RiVaxTM Ab were neither robust nor long lasting.
  • levels of toxin-neutralizing Ab were also extremely low in RiVaxTM-immunized individuals.
  • Ricin neutralizing antibodies are likely the primary determinant of protective immunity to ricin. Collectively, these data defined the need to identify new adjuvants to boost the efficacy of RiVaxTM and other antigens derived from ribosome inactivating proteins.
  • Anthrax is an acute infectious disease that is easily transmitted to humans by environmentally durable spores produced by gram positive bacterium Bacillus anthracis. Because the spores are robust and contagious, anthrax is considered a Category A bioterror threat. Anthrax infection can occur in three forms: cutaneous (skin), inhalation, and gastrointestinal. Inhaled spores can cause a rapidly progressing form of anthrax since the spores are transported to lymph nodes near the lungs where they germinate, releasing vegetative bacteria into the bloodstream. After infection in the bloodstream, the bacteria synthesize a complex series of toxin components that make up anthrax toxin, resulting in overwhelming toxemia that causes shock and organ failure.
  • the bacterium secretes 3 proteins which can combine to form two binary toxins, which cause the major pathology during anthrax infection of humans and animals.
  • the cell binding component is Protective Antigen (PA), and once bound it is cleaved by a cell surface furin-like protease, leaving behind a 63 kDa fragment which then heptamerizes.
  • PA Protective Antigen
  • the surface of the heptamer can bind up to 3 copies of edema factor (EF) and/or lethal factor (LF), forming edema toxin or lethal toxin, respectively.
  • EF is an adenylate cyclase which increases intracellular cAMP concentrations.
  • LF is a zinc-dependent metalloprotease which cleaves members of the MAPKK pathway, leading to apoptosis.
  • Antibodies targeting PA can neutralize both toxins, and thus recombinant PA is the major antigen in the currently licensed anthrax vaccine adsorbed (AVA, Biothrax ® ).
  • AZA anthrax vaccine adsorbed
  • a six-dose vaccination series is required to sustain a high anti-PA IgG titers.
  • Treatment of anthrax involves long-term antibiotic therapy, since ungerminated spores can lie dormant in the lungs for up to 60 days. Only a few inhaled spores can cause inhalational anthrax poisoning.
  • PA is the dominant immunogen in AVA and target for protective immunity in pre-exposure and post exposure prophylaxis as a single component recombinant vaccine.
  • Aluminum-adjuvant PA vaccines have been shown to be immunogenic in relationship to AVA, but it is difficult to directly state with the current evidence that an aluminum-adsorbed PA vaccine will in fact be the successor to AVA (Brown, Cox, et al, Phase I study of safety and immunogenicity of an Escherichia coli-derived recombinant protective antigen (rPA) vaccine to prevent anthrax in adults, PLoS One, v5:el3849; Campbell, Clement, et al, 2007, Safety, reactogenicity and immunogenicity of a recombinant protective antigen anthrax vaccine given to healthy adults, Hum Vaccin, v3:205-l 1; Gorse, Keitel, et al, 2006, Immunogenicity and tolerance of ascending doses of a recombinant protective antigen (rPA102) anthrax vaccine: a randomized, double-blinded, controlled, multicenter trial, Vaccine, v24:5950-9).
  • This first generation product is now approved for a vaccination regimen that involves injection by the intramuscular route at 0, 4 weeks, 6 months, and booster doses at 12 and 18 months (Marano, Plikaytis, et al, 2008, Effects of a reduced dose schedule and intramuscular administration of anthrax vaccine adsorbed on immunogenicity and safety at 7 months: a randomized trial, JAMA, v300: 1532-43).
  • the changes were implemented following extensive study of AVA showing non inferiority of the 0, 4 i.m. regimen.
  • the vaccine is routinely used in the USA for military personnel and has been acquired by HHS for the stockpile for civilian use in the event of a terrorist event and could be given upon Emergency Use Authorization (EUA) for post exposure prophylaxis (PEP) in conjunction with antibiotic therapy, prior to the time that FDA licensure is achieved for that indication.
  • EUA Emergency Use Authorization
  • PEP post exposure prophylaxis
  • the ACIP has recommended the 0, 2, 4 week AVA subcutaneously for PEP along with a 60 day course of antibiotics.
  • peak antibody titers are reached by week six through eight (after two injections), similar to peak titers reached using the former 0, 2, 4 week subcutaneous vaccination regimen (Marano, Plikaytis, Martin, Rose, Semenova, Martin, Freeman, Li, Mulligan, Parker, Babcock, Keitel, El Sahly, Tru, Jacobson, Keyserling, Soroka, Fox, Stamper, McNeil, Perkins, Messonnier and Quinn, 2008, Effects of a reduced dose schedule and intramuscular administration of anthrax vaccine adsorbed on immunogenicity and safety at 7 months: a randomized trial, JAMA, v300: 1532-43).
  • a notable improvement in the AVA vaccine has been reported by adding immunostimulatory CpG sequences to AVA (Rynkiewicz, Rathkopf, et al., 2011, Marked enhancement of the immune response to BioThrax® (Anthrax Vaccine Adsorbed) by the TLR9 agonist CPG 7909 in healthy volunteers, Vaccine, v29:6313-20; Klinman, Xie, et al, 2006, CpG oligonucleotides improve the protective immune response induced by the licensed anthrax vaccine, Ann N Y Acad Sci, vl082: 137-50; Klinman, Xie, et al, 2004, CpG oligonucleotides improve the protective immune response induced by the anthrax vaccination of Rhesus macaques, Vaccine, v22:2881-6).
  • Vaccination with modified product resulted in a demonstrably accelerated immune response in relationship to AVA achieving titers of TNA equivalent to peak AVA titers in approximately 21 days (after two doses of AVA7909 vaccine) (Rynkiewicz, Rathkopf, Sim, Waytes, Hopkins, Giri, DeMuria, Ransom, Quinn, Nabors and Nielsen, 2011, Marked enhancement of the immune response to BioThrax ® (Anthrax Vaccine Adsorbed) by the TLR9 agonist CPG 7909 in healthy volunteers, Vaccine, v29:6313-20).
  • U.S. Pat. No. 7,037,503 discloses dominant negative inhibitor (DNI) mutations of recombinant PA in which mutations of PA result in inhibition of its pore-forming ability, in which the mutations result in a PA molecule that is therapeutic for the treatment of toxemia caused by the secretion of anthrax toxins by bacteria circulating in the bloodstream of animals and humans afflicted with anthrax disease.
  • DNI dominant negative inhibitor
  • DNI dominant negative inhibitor
  • DNI was originally proposed for use as an intravenous agent for post-aerosol exposure to anthrax spores to block holotoxin, since one subunit of DNI could block PA-dependent pore formation after heptamerization. It has been shown that animals vaccinated with the DNI antigen induced higher levels of antibodies to toxin and maintained high levels of protective antibody titers for up to one year without booster injections of antigen. The hyperimmunogencity of DNI has been attributed to the defects in pore formation which may enable more efficient antigen presentation in the context of class II MHC.
  • the anthrax DNI variant of recombinant PA was originally isolated by Collier et al. at Harvard in a screen for mutants of PA that were defective the steps of binding, transporting or internalization from the endosome (Sellman, Mourez, et al., 2001, Dominant-negative mutants of a toxin subunit: an approach to therapy of anthrax, Science, v292:695-7; Mourez, Kane, et al., 2001, Designing a polyvalent inhibitor of anthrax toxin, Nat Biotechnol, vl9:958-61).
  • the DNI protein is activated normally, undergoes heptamerization, and binds LF and EF.
  • the assembled complexes are endocytosed and trafficked to an acidic compartment similar to wild type PA. Upon exposure to acidic conditions, however, the complexes do not undergo membrane insertion or pore formation, and are not able to translocate the LF and EF into the cytosol. Thus, toxicity is completely blocked.
  • the inactive, dead-end anthrax toxin complexes are believed to be trafficked to lysosomes and metabolized.
  • DNI works in a dominant fashion meaning that DNI can be present in significantly lower concentrations than wt PA and still be effective in blocking toxin action.
  • DNI protein was developed on the basis that it could be used as a post exposure prophylaxis, and confirmatory studies were reported rats and then in rabbits with spore challenge.
  • the present invention provides for a stable immunogenic composition capable of eliciting a robust and durable immune response yielding a measurable increase in neutralizing antibodies at least 200 days post-administration, comprising at least one antigen consisting of a ribosome inactivating protein and at least one antigen comprising a toxin derived from bacterial spores.
  • the immunogenic composition comprises a first antigen comprising a ribosome inactivating protein and a second antigen comprising a toxin derived from bacterial spores.
  • the composition yields a measurable increase in neutralizing antibodies to the first antigen comprising the ribosome inactivating protein.
  • the composition is capable of yielding a measurable increase in neutralizing antibodies to the second antigen comprising a toxin derived from bacterial spores. In yet another embodiment, the composition is capable of yielding a measurable increase in neutralizing antibodies to both the first and the second antigen.
  • the composition of the present invention elicits a measurable increase in neutralizing antibodies to the first antigen at least 200 days post-administration.
  • the composition of the present invention elicits a measurable increase in neutralizing antibodies to the second antigen at least 200 days post-administration.
  • the composition of the present invention elicits a measurable increase in neutralizing antibodies to the first and the second antigen at least 200 days post-administration.
  • One additional aspect of the present invention provides for a method of eliciting a stable immune response yielding a measurable increase in neutralizing antibodies at least 200 days post- administration, comprising providing an immunogenic composition comprising at least one antigen comprising a ribosome inactivating protein and at least one antigen comprising a toxin derived from bacterial spores and administering the immunogenic composition to an individual.
  • the present invention provides for a process for formulating a vaccine composition
  • a process for formulating a vaccine composition comprising: providing a ricin A chain derived from ricin toxin; providing an effective amount of dominant negative inhibitor (DNI) protein derived from Bacillus anthracis; formulating (a) and (b) as a dried product for reconstitution using an excipient selected from the group consisting of mannitol and sucrose and a buffer; and providing at least one adjuvant to (a) and (b).
  • DNI dominant negative inhibitor
  • Figure 1 consists of CD spectroscopy data of stability samples at 24 months.
  • Figure 2 consists of a representative shift in peak tryptophan fluorescence as a function of time and temperature conditions.
  • Figure 3 shows intrinsic fluorescence peak position at the center of mass as a function of temperature for the DNI samples incubated at 4 °C (A), 40 °C (B) and 70 °C (C) for 16 week. Bars are average Tm values for fluorescence peak position of duplicate runs and error bars are standard deviation of mean.
  • Figure 4 depicts reciprocal anti-DNI antibody (A) and neutralizing (B) titers of liquid and lyophilized vaccines without any high temperature storage and with 1, 4, 8 and 16 weeks of storage at 40°C.
  • Figure 5 shows 7 week old Balb/c mice immunized by s.c. injection on days 0 and day 21 with 5 ⁇ g of DNI protein in PBS (DNI), SE (DNI + SE) or GLA/SE (DNI + GLA-SE).
  • DNI DNI
  • SE DNI + SE
  • GLA/SE DNI + GLA-SE
  • Serum TNA A
  • B total ELISA reactive IgGl, IgG, and IgG2a antibodies
  • Figure 6 shows the flow chart for formulating ricin A chain vaccine (RiVaxTM) in conjunction with aluminum hydroxide adjuvant and subsequent lyophilization.
  • the present invention is directed towards a combination vaccine that comprises vaccine components that are suitable for the prevention, amelioration and treatment of exposure to toxins consisting of ribosome inactivating proteins and concurrently exposure to toxins elaborated by spore forming bacteria.
  • the vaccine of the present invention elicits protective immunity to each of the antigenic components without causing immune interference after administration to an individual.
  • the combination vaccine is administered prior to exposure to the toxin or infectious agent.
  • the vaccine can be administered after exposure to the toxin or infectious agent.
  • the vaccine can be administered after exposure to the infectious agent or toxin in combination with therapies intended to cure or ameliorate the effects of the toxins that are responsible for the morbidity of disease exposure.
  • the combination vaccine is formulated with adjuvants that preferentially induce neutralizing antibodies against the corresponding antigenic vaccine ingredients.
  • the combination vaccine is formulated with antigen derived from ricin toxin, consisting of the purified ricin A chain combined with antigen consisting of the protective antigen (PA) derived from Bacillus anthracis.
  • PA protective antigen
  • the vaccine of the invention provides protective immune responses to toxins with no interference or immune competition among the antigens that are present in the vaccine.
  • a single shot will confer immunogenicity simultaneously in a single administration against anthrax diseases and exposure to ricin toxin.
  • the vaccine is easier to administer in fewer injections to achieve protective and long lasting immunity to ribosomal inactivating toxins and the toxins causing pathology against anthrax disease upon exposure to spores of Bacillus anthracis. Since a single shot would afford immunity against anthrax and ribosome inactivating proteins, the cost of vaccination would be reduced.
  • the vaccine of the present invention would be advantageous by reducing the number of visits required for full vaccination and the numbers of vaccine administrations necessary to achieve full protection.
  • the present invention provides a vaccine that is more acceptable for rapid onset of protective immunity.
  • Ricin A chain is structurally unstable, with improvements being necessary to achieve the objective of long lasting and rapid onset immunity using 2 vaccine doses or fewer.
  • the ricin A chain is extremely labile in aqueous buffers without stabilizers, leading to unfolding and aggregation of the protein in solution. Protein unfolding also occurs on the surface of aluminum adjuvant particles in the liquid suspension vaccines. The summation of the studies with liquid aluminum-adsorbed vaccine in mice, rabbits, humans and macaques indicate that improvements will be necessary to achieve the objective of long lasting and rapid onset immunity using two vaccine doses or fewer.
  • RiVaxTM a ricin A chain mutant vaccine candidate.
  • the purified RiVaxTM is stored in 50% glycerol for further formulation development and processing in the generation of RiVaxTM, AIOH adsorbed or RiVax -TR, (aluminum-adsorbed, lyophilized for reconstitution).
  • RiVax is stored in glycerol at -20°C, and is subject to dialysis or ultrafiltration to remove glycerol before adsorption to AIOH and further processing and vial filling.
  • glycerol The rationale for the use of glycerol concerns identifying conditions for the retention of RiVaxTM structure during storage prior to aluminum adsorption under conditions in which the protein does not significantly aggregate or unfold. These conditions were identified in extensive screens of conditions to examine tertiary conformation, secondary protein structure aggregation and the influence of generally regarded as safe (GRAS) excipients on possible unfolding events . Through screens, it was found that the most efficient stabilizer was glycerol for the retention of native structure. It may be possible to further scale up runs to eliminate the glycerol holding/storage step in favor of direct absorption to AIOH, which may stabilize protein structure and immunogenicity.
  • GRAS generally regarded as safe
  • a recombinant E. coli process was developed for the manufacture of RiVaxTM according to principles of rational design. This process is suitable for implementation within a GMP manufacturing environment and has been implemented in 7, 100 liter (L) runs, 4 of which were conducted as process development runs to scale up from 10 L, and 2 were conducted in a cGMP environment as engineering or demonstration runs, and one run was conducted under cGMP. All runs were conducted at the Cambrex/Lonza facility in Baltimore, MD. Material generated from one of the process development runs has been established as a reference protein, and material generated from one of the engineering runs has been used extensively in adjuvant formulation characterization
  • the pET28a-Y80A/V76M construct was transformed into BLR(DE3) and HMS174(DE3), which are recA- and do not shed phage particles, and evaluated for product expression.
  • Eight colonies from each transformation were streaked onto LB Kan agar and four well-isolated colonies from each transformation were grown out in 0.5X vegan TB with 50 mg/L kanamycin for IPTG-inducible RiVaxTM expression analysis by SDS-PAGE. Post-induction samples were taken 3.0 to 3.5 hours after the addition of IPTG and samples were analyzed by SDS-PAGE for product expression. A protein of approximately 30 kDa was clearly induced in each of these cultures.
  • the insert sequence from both the BLR 3 and HMS174 A clones was 100% homologous with the predicted sequence.
  • Fermentation development and optimization was conducted with the goals of increasing the cell mass and the fermentation titer of RiVaxTM, as well as developing scalable methods for RiVaxTM recovery in a soluble form using cGMP compliant medium and process. Fermentations were performed in B. Braun fermenters at the 10 L scale in batch and fed-batch mode with varying inducer (IPTG) concentration. The main variables that were examined included 1) sources of peptone from non-animal origin 2) concentration of IPTG inducer and 3) carbon source. Batch fermentations were performed for one clone from each of the E. coli host strains: Clone BLR 3 and Clone HMS A.
  • Starter cultures of ECPM medium (25 mL) were inoculated with 0.25 mL of either BLR 3 or HMS A frozen stock culture and grown 10 hours at 37°C, 200 RPM in a gyratory shaker. These cultures were used to inoculate 200 mL of Batch Fermentor Medium in 1000 mL baffled shake flasks. The 200 mL cultures were allowed to grow at 37°C, 200 RPM for nine hours then used to inoculate 10 L of Batch Fermentation Medium in 14 L BIOFLO 3000 vessels (New Brunswick Scientific). Casamino Acids and other non-animal origin nitrogen sources were screened for production of cell mass with the result that yeast extract was capable of supporting high levels of cell mass.
  • the yields in the soluble fraction as measured on a cation exchange column ranged between 1.2 g/L and 1.8 g/L in the soluble fraction.
  • the purification process comprises capture chromatography using cation exchange resin, endotoxin removal using a charged filter followed by hydrophobic interaction chromatography (HIC) and finally ultrafiltration/diafiltration (UF/DF) to affect buffer exchange in the final formulation for storage of bulk protein.
  • the processing steps include Poros ® HS50 Chromatography, Endotoxin Removal by Mustang ® E Filtration, Butyl-S Sepharose ® 6 FF Chromatography and Final Ultrafiltration/Diafiltration to obtain the final drug substance in 50% w/w glycerol.
  • the tangential flow filtrate (TFF) permeate from cell lysis was chromatographed with Poros ® HS50.
  • Poros ® HS50 chromatography was performed using a K'40 chromatography skid 4.
  • the elution product was collected from 0.1 OD A280 on the ascending slope to 0.06 OD A280 on the descending slope of the peak.
  • the product from the Poros ® HS column was pumped through a Pall Mustang ® E filter to remove endotoxin.
  • the Mustang ® filtered pool was stored for 2 hours at 2 - 8 °C prior to the Butyl S chromatography.
  • the filtered Poros ® HS50 pool was conditioned by the addition of 3.6 L of 25 mM sodium phosphate, 0.6 M NaCl, 3.0 M Ammonium Sulfate, pH 6.5 and 2.5 L of 25 mM sodium phosphate, 4.0 M NaCl, 0.5 M Ammonium Sulfate, pH 6.5.
  • the conditioned Butyl load had a conductivity of 151 mS/cm.
  • the Butyl S purification was performed using a K'40 chromatography skid. For lot 190-0306-005, the pre-elution eluate from 0.1 OD A280 to 0.725 OD A280 was collected as 1 L fractions in 2 L PETG bottles.
  • the combined product was diluted 1 : 1 with formulation buffer (17 mM Histidine, 238 mM NaCl, 15 % Glycerol, pH 6.0). The concentration/diafiltration was performed immediately following the dilution.
  • the cGMP batch was released for clinical evaluation and final formulation with Alhydrogel ® adjuvant.
  • a robust and scalable process with an overall yield of approximately 3-5 g of purified protein from 100 L fermentations, with reproducible batch yield. The fermentation yield has been calculated to be in the order of 700-1000 mgs of soluble protein per L of lysate.
  • Measurable fluorescence spectra can be detected in vaccine prepared with concentrations of RiVaxTM in excess of approximately 50 ⁇ g/mL with 0.85 mgs of AIOH.
  • Kinetics of the movement of the peak emission while the protein is adsorbed to Alhydrogel ® can be determined using a front face triangular geometry cuvette system. This system is a method to monitor changes of protein configuration under a variety of conditions. For example, the following data have been generated for a reference batch of vaccine stored at 2-4°C and 40°C (Fig. 2).
  • Example IV Manufacture of the Dominant Negative Inhibitor (DNI) for pre- and postexposure vaccination
  • the DNI protein was developed as a post-exposure therapeutic. A purification process was developed and cGMP lots were manufactured at the 300-600 Liter fermentation scale. These runs resulted in hundreds of grams of protein that were formulated with excipients to stabilize the protein prior to vaccine formulation and combination.
  • the protective antigen (PA) gene of Bacillus anthracis was cloned into E. coli BL21 (DE3) using a pET22-b(+) vector (Novagen, Inc), and mutated by site directed mutagenesis.
  • the resulting double mutant gene (K397D, D425K) encodes the DNI protein and resides on a 2.3 kb Nde I - Xho I fragment.
  • the 5' end of this fragment contains the 23 amino acid pelB leader sequence, including its cleavage signal, which directs the secretion of the DNI protein to the periplasm.
  • a ten amino acid N-terminal extension lies between the pelB signal cleavage site and a GAA codon (glu) that marks position #1 of the mature 83 kD PA as excreted by B. anthracis.
  • This ten amino extension is a cloning artifact derived from pET22 vector sequence and does not affect the function of the active DNI protein since it is located on the 20 kb fragment that is cleaved at the cell surface.
  • Two in-frame STOP codons, TAA TGA terminate transcription immediately upstream of the 6X His-Tag encoded by the pET22-b(+) vector.
  • the original expression plasmid carried the bla gene encoding resistance to ampicillin.
  • this gene construct was recloned into pET24-based vectors encoding the NPT II gene for resistance to kanamycin for periplasmic and cytoplasmic expression of DNI.
  • the DNI gene insert was recovered from the original DNI gene construct from Harvard University Medical School by digestion with Nde I and Xho I. Transformants were characterized by simple restriction analysis and used to purify sufficient plasmid DNA to isolate the Nde I-Xho I fragment containing the DNI gene. In addition to the DNI coding region, this fragment contained the pelB leader sequence and the 10 amino acid N-terminal extension.
  • the Nde I-Xho I fragment was ligated into a pET 24 vector cut with Nde I and Xho I, and the ligation products used to transform E. coli BLR (DE3) to kanamycin resistance. Transformants were characterized by simple restriction analysis. The complete amino acid sequence of the DNI protein is shown in Table 2.
  • Competent E. coli BLR (DE3) cells were obtained from Novagen, Inc. Ligation mixtures were used to transform competent E. coli BLR (DE3) cells to construct the recombinant DNI production strain following the procedures described by the manufacturer (Novagen, Inc.). Clones were selected for expression of the correct size protein band on SDS-PAGE gels with Coomassie staining. The fermentation and purification parameters were developed following standard processes. Both batch and fed-batch parameters were analyzed as were combinations of purification resins and combinations in order to maximize yield while maintaining a high level of purity and a low level of endotoxin, host cell protein (HCP) and DNA contamination.
  • the DNI Drug Substance is an 83 kd protein consisting of 745 amino acids.
  • the full length DNI protein is not capable of having a quaternary structure. However, once DNI is cleaved by furin, the resulting 63 kd protein forms either monomeric heptamers or hetero-heptamers with wt PA.
  • the DNI protein is expressed in the periplasm of E coli after induction with IPTG. The product is susceptible to degradation by a neutral protease and the inclusion of EDTA in the buffers during processing seems to limit proteolysis.
  • the cells are cultured in a medium that contains glycerol as a carbon source and Casamino Acids as the principal complex nitrogen source.
  • the fermentation is performed in a fed-batch mode with a simple linear feeding protocol.
  • the fermentor is inoculated with a seed culture with a temperature shift from 38°C to 30°C for induction.
  • the culture is induced with IPTG one hour after the temperature shift.
  • After the culture reaches an OD600 of 10 the fermentation is fed glycerol at a designated feed rate.
  • the temperature is shifted 30 rpm, and the culture is harvested by continuous centrifugation.
  • OD600 with IPTG for 4 hours.
  • the fermentor is chilled to 15°C, airflow is discontinued and agitation is set to 50 rpm at 15°C, airflow is discontinued and agitation is set to 50 rpm, and the culture is harvested by continuous centrifugation.
  • the downstream purification process for DNI involves the use of three column chromatography steps, a Mustang ® filtration step and two UF/DF steps. Capture of DNI is achieved by Q Sepharose ® FF chromatography. The fractions collected from the Q eluate are pooled based on in-process analysis by SDS-PAGE and passed through a Mustang ® filter. The Mustang ® filtered Q eluate pool is then diafiltered into phosphate buffer prior to loading onto a ceramic hydroxyapatite (CHT) column. Fractions collected from the CHT eluate are pooled based on in-process analysis by SDS-PAGE.
  • CHT ceramic hydroxyapatite
  • the CHT eluate pool is adjusted to the appropriate ammonium sulfate concentration by dilution, filtered, and loaded onto the Phenyl Sepharose ® HP column for final polishing and fractions are collected and pooled based on purity by SDS-PAGE. Final diafiltration into formulation buffer is performed on the Phenyl eluate pool.
  • the formulated bulk is aseptically filtered using a Millipak ® 0.2um filter and stored in 1000 ml Nalgene ® Teflon, PFA Narrow Mouth Bottles at -70°C.
  • Example V Formulation of DNI as a Dried Product for Reconstitution
  • the DNI Drug Product has been formulated using only mannitol and sucrose as excipients and disodium hydrogen phosphate as a buffer.
  • Table 3 lists the quantitative composition of the DNI Drug Product.
  • the bulk drug substance is thawed under 2-8°C conditions prior to formulation.
  • a 20 mM disodium hydrogen phosphate solution is formulated with disodium hydrogen phosphate and water. The solution is adjusted to a pH of 8.4 - 8.5.
  • the calculated amounts of mannitol and sucrose are added to the bulk drug substance and mixed until in solution.
  • the solution is adjusted to a pH of 8.1 - 8.5.
  • Table 4 lists additional elements for inclusion into various emulsion formulation embodiments.
  • SE animal derived sources
  • SE non-animal derived sources
  • Vitamin E Vitamin E
  • the anthrax DNI is an analog of rPA containing two mutations that prevent pore formation and translocation of the holotoxin in the cytosol (Sellman, Mourez and Collier, 2001, Dominant-negative mutants of a toxin subunit: an approach to therapy of anthrax, Science, v292:695-7).
  • DNI binds to the same cell-surface receptors with the same affinity as PA and can form self-assembled heptamers on the surface of cells.
  • the absence of the translocation step prevents DNI-dependent transport of Edema Factor and/or Lethal Factor into cell cytoplasm.
  • complexes of DNI with LF or EF are nontoxic.
  • DNI was originally proposed for use as an intravenous agent for post-aerosol exposure to anthrax spores to block holotoxin, since one subunit of DNI could block PA-dependent pore formation after heptamerization. It has been shown that animals vaccinated with the DNI antigen induced higher levels of antibodies to toxin and maintained high levels of protective antibody titers for up to one year without booster injections of antigen (Aulinger, Roehrl, et al, 2005, Combining anthrax vaccine and therapy: a dominant-negative inhibitor of anthrax toxin is also a potent and safe immunogen for vaccines, Infection and Immunity, v73:3408-14).
  • the hyperimmunogencity of DNI has been attributed to the defects in pore formation which may enable more efficient antigen presentation in the context of class II MHC.
  • the anthrax DNI is an analog of rPA containing two mutations that prevent pore formation and translocation of the holotoxin in the cytosol.
  • the DNI protein has been produced at scale as a native protein in E. coli fermentation and a complete battery of release and process control tests has been implemented during its manufacture.
  • GLA Glycopyranoside Lipid A
  • MPLa monophosphoryl Lipid A
  • the DNI vaccine candidate was formulated in a 9.5 w/v% trehalose 10 mM ammonium acetate buffer pH 7.
  • Aluminum hydroxide adjuvant (Alhydrogel ® ) and aluminum hydroxide with GLA were co-formulated as adjuvants. Lyophilized vaccines were stored at 40°C for 0, 1, 4, 8, and 16 weeks for immunogenicity studies and also at 70°C for structural studies. Vaccines were lyophilized to increase their stability at high temperature storage. Liquid vaccines of the same formulations were stored at 40°C for 0 or 8 weeks.
  • Example VIII Additional Characterization Studies with Lyophilized DNI Vaccine: Particle Size, Glass Transition Temperature, and Adsorption/Desorption Studies
  • Lyophilized vaccines were characterized for glass transition temperature, protein adsorption, and particle size.
  • the onset glass transition temperature was found to be 115.5°C ⁇ 1.6 for lyophilized Alhydrogel ® vaccine formulations and 117.3°C ⁇ 3.8 for lyophilized Alhydrogel ® /GLA formulations. Both formulations have high glass transition temperatures similar to pure trehalose, showing minimal water in the lyophilized cakes.
  • Example IX Antibody Responses and Temperature Stability of Lyophilized Adsorbed DNI Vaccine
  • Newly made liquid (T 0) and lyophilized vaccines produced equivalent immune responses demonstrating that lyophilization does not decrease the immunogenicity of the vaccine. Lyophilized vaccines remained immunogenic even after being stored at 40°C for 16 weeks When synthetic MPL is added to the lyophilized formulations during processing, the resulting lyophilized DNI vaccine generated robust immune responses and elicited neutralizing antibodies that are enhanced in relationship to the vaccine that does not contain the synthetic TLR-4 agonist GLA (Tables 3 and 4). The TNA titers after 2 vaccinations with GLA TLR-4 lyophilized as well as liquid vaccine not exposed to temperature stress were 5 to 10-fold higher than in sera from mice vaccinated with the corresponding Alhydrogel ® vaccines.
  • Example X DNI/GLA-SE Induces High Titer Toxin Neutralizing Antibodies
  • mice were vaccinated with 5 micrograms of DNI admixed with SE-GLA. SE, or with buffer alone (FIG. 5).
  • Anti-rPA serum titers were determined in serum collected one week after the second vaccination and antibody titers measured by ELISA and neutralizing antibodies were determined by TNA.
  • TNA TNA
  • SE and GLA-SE there was a skewing of the antibody responses towards IgG2a, indicative of admixed Thl/Th2 response and the generation of antibodies that are associated with Fey component of anthrax toxin neutralization.
  • Vaccination with the DNI protein alone resulted in negligible IgG2a.
  • the SE-GLA vaccine induced the highest titers of toxin neutralizing antibodies (1 : 12,800) in contrast to mice that were immunized with protein alone that did not develop measurable neutralizing antibodies.
  • mice Groups of ten female Balb/c mice (Jackson Laboratories, Bar Harbor, ME) with 10 ⁇ g of either: RiVaxTM, DNI, or a combination of the two, in the presence of the adjuvant Alum (0.85 mg/mL) in PBS.
  • a control group received Alum only (Table 7).
  • Lyophilized dominant negative inhibitor (DNI) was manufactured by Baxter Pharmaceutical Solutions LLC (Bloomington, IN). It was stored for 8 years at 4 °C (date of manufacture: 12/15/2003, batch number: 803918A). On the day of use, the lyophilized powder was reconstituted in sterile water.
  • RiVaxTM (lot 190-100L-GLP- FF-090105) and aluminum hydroxide (Alhydrogel®) were provided by Soligenix, Inc. (Princeton, NJ). Alum was provided in histidine buffer (lOmM histidine, 144mM NaCl, pH 6.0), while RiVaxTM was in 50% glycerol, 50%> histidine buffer. All mouse experiments were approved by the Wadsworth Center's Institutional Animal Care and Use Committee (IACUC).
  • IACUC Institutional Animal Care and Use Committee
  • a, second challenge, b, mouse 2B-5 died unexpectedly before blood was collected on day 20.
  • Each antigen was mixed with aluminum hydroxide and allowed to adsorb while rotating at 4°C for 3 hours prior to immunization. Antigens in dual immunizations were adsorbed to Alum separately and mixed before injection. Mice were given a prime immunization, 400uL i.p., and a boost 2 weeks later. Their immune response was characterized 1 week after the boost, and again 6 months later, by collecting blood from the tail vein.
  • ELISAs for determining endpoint titers were performed. Plastic plates were coated with either ricin or PA, the blocked with 2% goat serum. Immune serum was then serially diluted twofold across the plate in duplicate. Secondary antibody detected bound murine -IgG, was visualized by the HRP/TMB calorimetric reaction, and absorbance was detected at 450nm on a VersaMax ® spectrophotometer (Molecular Devices, Sunnyvale, CA). Endpoint titer was defined as the highest dilution at which the absorbance was still higher than 3 times the background absorbance.
  • mice given RiVaxTM alone were very high, with a geometric mean inverse titer of 204,800.
  • mice given DNI alone also had very high anti-PA titers, with a geometric mean of 187,802. Neither of these groups had any detectable titers against the opposite antigen (Table 8).
  • the titer when no titers are detectable, the titer is assigned as 1 for geometric mean calculation
  • mice that received the dual immunization with 10 ⁇ of each antigen had high titers against both antigens, although the absolute levels were around half of the mice given each antigen by itself.
  • Anti-ricin titers in this group had a geometric mean of 126,069, while anti-PA titers had a geometric mean of 95,543. Therefore, at the endpoint titer level, there is clearly some immune interference from each antigen that impairs the response to the opposite antigen.
  • Immune serum was mixed with toxin at a 1 : 100 serum dilution, and diluted 2 fold into toxin containing media.
  • Vera cell assays the toxin/serum mixture was allowed to incubate with cells at 37°C for 2 hours, at which point the media was changed. Cell Titer Glo ® was then used to determine cell viability 48 hours later.
  • the media was not changed after toxin/serum mixture addition, and cell viability was determined with Cell Titer Glo ® 24 hours later. Table 9
  • colloidal suspensions of aluminum adjuvant particles are unstable and freezing-induced concentration of adjuvant suspensions cause aggregation during freeze-thawing, meaning that conventional lyophilization techniques cannot be successfully applied to vaccines that employ aluminum adjvuants.
  • aggregation of colloidal aluminum hydroxide suspensions can be inhibited by reducing the extent of their freeze-concentration by using formulations that contain high concentrations of glass-forming excipients, and by limiting the time over which the freeze-concentration occurs by using formulations that contain high concentrations of glass-forming excipients, as well as limiting the time over which the freeze-concentrated suspensions can aggregate by using rapid cooling procedures to maximize the kinetics of glass formation. This process has been developed from examination of a number of parameters and as outlined in FIG 6. Material Dialysis and Concentration

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Abstract

L'invention concerne une composition immunogène stable capable d'induire une réponse immunitaire robuste et durable, donnant une augmentation mesurable des anticorps neutralisants au moins 200 jours après administration, comprenant au moins un antigène constitué d'une protéine d'inactivation de ribosome et au moins un antigène comprenant une toxine dérivée de spores bactériennes. L'invention concerne également un procédé fabrication et d'utilisation d'une composition immunogène stable capable d'induire une réponse immunitaire stable produisant une augmentation mesurable des anticorps neutralisants au moins 200 jours après administration, comprenant la fourniture d'une composition immunogène comprenant au moins un antigène comprenant une protéine d'inactivation de ribosome et au moins un antigène comprenant une toxine dérivée de spores bactériennes et l'administration de la composition immunogène à un individu.
PCT/US2015/039826 2014-07-09 2015-07-09 Composition de vaccin stable multivalent et ses procédés de fabrication WO2016007797A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130280293A1 (en) * 2011-01-05 2013-10-24 Bharat Biotech International Limited Combination heptavalent vaccine
US20130309273A1 (en) * 2012-05-17 2013-11-21 Kimberly Hassett Thermostable Vaccine Compositions and Methods of Preparing Same

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130280293A1 (en) * 2011-01-05 2013-10-24 Bharat Biotech International Limited Combination heptavalent vaccine
US20130309273A1 (en) * 2012-05-17 2013-11-21 Kimberly Hassett Thermostable Vaccine Compositions and Methods of Preparing Same

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SCHABER, C ET AL., ANNUAL REPORT ON FORM 10-K., 26 March 2014 (2014-03-26), pages 9 *

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