Embodiments herein relate to compositions of and methods for live viruses. In certain embodiments, a live, attenuated virus composition includes, but is not limited to, one or more live, attenuated viruses and compositions to reduce inactivation and/or degradation of the live, attenuated virus. In other embodiments, the live, attenuated virus composition may be a vaccine composition. In yet other compositions, a live, attenuated virus composition may include at least one carbohydrate, at least one protein and at least one high molecular weight surfactants for reducing inactivation and/or degradation of the live, attenuated virus.

Description of the Invention

SUMMARY

Embodiments herein concern methods and compositions to reduce or prevent deterioration or inactivation of a live attenuated virus composition. Certain compositions disclosed can include combinations of components that reduce deterioration of a live attenuated virus. Other embodiments herein concern combinations of excipients that greatly enhance the stability of live attenuated viruses. Yet other compositions and methods herein are directed to reducing the need for lower temperatures (e.g. refrigerated or frozen storage) while increasing the shelf life of aqueous and/or reconstituted live attenuated virus.

In accordance with these embodiments, certain live attenuated viruses are directed to flaviviruses. Some embodiments, directed to compositions, can include, but are not limited to, one or more live, attenuated viruses, such as one or more live, attenuated flaviviruses in combination with one or more high molecular weight surfactants, proteins, and carbohydrates.

Compositions contemplated herein can increase the stabilization and/or reduce the inactivation and/or degradation of a live attenuated virus including, but not limited to, a live attenuated Flavivirus, Togavirus, Coronavirus, Rhabdovirus, Filovirus, Paramyxovirus, Orthomyxovirus, Bunyavirus, Arenavirus, Retrovirus, Hepadnavirus, Pestivirus, Picornavirus, Calicivirus, Reovirus, Parvovirus, Papovavirus, Adenovirus, Herpes virus, or Poxvirus.

Other embodiments concern live, attenuated virus compositions and methods directed to a vaccine compositions capable of reducing or preventing onset of a medical condition caused by one or more of the viruses contemplated herein. In accordance with these embodiments, medical conditions may include, but are not limited to, West Nile infection, dengue fever, Japanese encephalitis, Kyasanur forest disease, Murray valley encephalitis, Alkhurma hemorrhagic fever, St. Louis encephalitis, tick-borne encephalitis, yellow fever and hepatitis C virus infection.

In certain embodiments, compositions contemplated herein can be partially or wholly dehydrated or hydrated. In other embodiments, protein agents contemplated of use in compositions herein can include, but are not limited to, lactalbumin, human serum albumin, a recombinant human serum albumin (rHSA), bovine serum albumin (BSA), other serum albumins or albumin gene family members. Saccharides or polyol agents can include, but are not limited to, monosaccharides, disaccharides, sugar alcohols, trehalose, sucrose, maltose, isomaltose, cellibiose, gentiobiose, laminaribose, xylobiose, mannobiose, lactose, fructose, sorbitol, mannitol, lactitol, xylitol, erythritol, raffinose, amylase, cyclodextrins, chitosan, or cellulose. In certain embodiments, surfactant agents can include, but are not limited to, a nonionic surfactant such as alkyl poly(ethylene oxide), copolymers of poly(ethylene oxide) and poly(propylene oxide) (EO-PO block copolymers), poly(vinylpyrrolidone), alkyl polyglucosides (such as sucrose monostearate, lauryl diglucoside, or sorbitan monolaureate, octyl glucoside and decyl maltoside), fatty alcohols (cetyl alcohol or olelyl alcohol), or cocamides (cocamide MEA, cocamide DEA and cocamide TEA).

In other embodiments, the surfactants can include, but are not limited to, copolymer poloxamer 407; Pluronic F127.RTM., poloxamer 188; Pluronic F68.RTM., copolymer EO30PO70EO70; Pluronic P123.RTM., or other EO-PO block copolymers of greater than 3,000-4,000 MW.

In some embodiments, vaccine compositions can include, but are not limited to, one or more protein agent that is serum albumin; one or more saccharide agent that is trehalose; and one or more surfactant polymer agent that is the EO-PO block copolymer, copolymer poloxamer 407, Pluronic F127.RTM..

Some embodiments herein concern partially or wholly dehydrated live, attenuated viral compositions. In accordance with these embodiments, a composition may be 20% or more; 30% or more; 40% or more; 50% or more; 60% or more; 70% or more; 80% or more; or 90% or more dehydrated.

Other embodiments concern methods for decreasing inactivation of a live attenuated viruses including, but not limited to, combining one or more live attenuated viruses with a composition capable of reducing inactivation of a live, attenuated virus including, but not limited to, one or more protein agents; one or more saccharides or polyols agents; and one or more high molecular weight surfactants, wherein the composition decreases inactivation of the live attenuated virus. In accordance with these embodiments, the live attenuated virus may include, but is not limited to, a Flavivirus, Togavirus, Coronavirus, Rhabdovirus, Filovirus, Paramyxovirus, Orthomyxovirus, Bunyavirus, Arenavirus, Retrovirus, Hepadnavirus, Pestivirus, Picornavirus, Calicivirus, Reovirus, Parvovirus, Papovavirus, Adenovirus, Herpes virus, or a Poxvirus. Additionally, methods and compositions disclosed herein can include freeze drying or other dehydrating methods for the combination. In accordance with these methods and compositions, the methods and compositions decrease inactivation of the freeze dried or partially or wholly dehydrated live attenuated virus. In other methods, compositions for decreasing inactivation of a live attenuated virus may comprise an aqueous composition or may comprise a rehydrated composition after dehydration. Compositions described herein are capable of increasing the shelf life of an aqueous or rehydrated live attenuated virus.

In certain particular embodiments, a live attenuated virus for use in a vaccine composition contemplated herein may include, but is not limited to, one or more live, attenuated flavivirus vaccines, including but not limited to, attenuated yellow fever viruses (such as 17D), attenuated Japanese encephalitis viruses, (such as SA 14-14-2), attenuated dengue viruses (such as DEN-2/PDK-53 or DEN-4.DELTA.30) or recombinant chimeric flaviviruses.

In certain embodiments, compositions contemplated herein are capable of decreasing inactivation and/or degradation of a hydrated live attenuated virus for greater than 24 hours at room temperatures (e.g. about 20.degree. to about 25.degree. C.) or refrigeration temperatures (e.g. about 0.degree. to about 10.degree. C.). In more particular embodiments, a combination composition is capable of maintaining about 100 percent of the live attenuated virus for greater than 24 hours. In addition, combination compositions contemplated herein are capable of reducing inactivation of a hydrated live attenuated virus during at least 2 freeze and thaw cycles. Other methods concern combination compositions capable of reducing inactivation of a hydrated live attenuated virus for about 24 hours to about 50 days at refrigeration temperatures (e.g. about 0.degree. to about 10.degree. C.). Compositions contemplated in these methods, can include, but are not limited to, one or more protein agent of serum albumin; one or more saccharide agent of trehalose; and one or more EO-PO block copolymer agent of copolymer poloxamer 407, Pluronic F127. In certain embodiments, the live, attenuated virus composition remains at about 100% viral titer after 7 days at approximately 21.degree. C. and about 100% viral titer after 50 days at refrigeration temperatures around 4.degree. C. Other embodiments herein may include live, attenuated virus composition remaining at about 90%, or about 80% viral titer after 7 days at approximately 21.degree. C. and about 90%, or about 80% viral titer after 50 days at refrigeration temperatures around 4.degree. C. Other embodiments contemplated include live, attenuated virus compositions remaining at about 3.times. to about 10.times. the concentration of viral titer after several hours (e.g. 20 hours) at approximately 37.degree. C. compared to other compositions known in the art. (see for example, FIGS. 4 and 5 (see Original Patent)). Compositions disclosed herein reduce degradation of the live, attenuated virus when the composition is stored at approximately 37.degree. C.

Other embodiments concern kits for decreasing the inactivation of a live, attenuated virus composition including, but not limited to, a container; and a composition including, but not limited to, one or more protein agents, one or more saccharide or polyol agents, and one or more EO-PO block copolymer agents, wherein the composition decreases inactivation and/or degradation of a live, attenuated virus. In accordance with these embodiments, a kit composition may include one or more protein agents of serum albumin; one or more saccharide agent of trehalose; and one or more EO-PO block copolymer agent. Additionally, a kit contemplated herein may further include one or more live, attenuated viruses including, but not limited to, a Flavivirus, Togavirus, Coronavirus, Rhabdovirus, Filovirus, Paramyxovirus, Orthomyxovirus, Bunyavirus, Arenavirus, Retrovirus, Hepadnavirus, Pestivirus, Picornavirus, Calicivirus, Reovirus, Parvovirus, Papovavirus, Adenovirus, Herpes virus, or Poxvirus. In certain embodiments, compositions herein can include trehalose as a saccharide agent. In accordance with these embodiments, trehalose concentration may be equal to or greater than 5% (w/v). In certain embodiments, compositions herein can include copolymer poloxamer 407, Pluronic F127.RTM.. as an EO-PO block copolymer agent. In accordance with these embodiments, copolymer poloxamer 407, Pluronic F127.RTM.. concentration may be about 0.1 to about 4 percent (w/v).

In other embodiments, compositions contemplated herein may contain trace amounts or no divalent cations. For example, compositions contemplated herein may have trace amounts or no calcium/magnesium (Ca.sup.+2/Mg.sup.+2).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In the following sections, various exemplary compositions and methods are described in order to detail various embodiments. It will be obvious to one skilled in the art that practicing the various embodiments does not require the employment of all or even some of the specific details outlined herein, but rather that concentrations, times and other specific details may be modified through routine experimentation. In some cases, well known methods or components have not been included in the description.

Stability of flavivirus vaccines has been assessed for both the existing yellow fever and Japanese encephalitis live, attenuated viruses. When tested in 1987, only five of the twelve yellow fever vaccines manufactured at that time met minimal standards of stability. Subsequently, addition of a mixture of sugars, amino acids and divalent cations was demonstrated to stabilize the lyophilized vaccine, so that the vaccine lost less than 1 log of potency after incubation at 37.degree. C. for 14 days. Stabilizing lyophilized formulations for the yellow fever vaccine have been described (see for example U.S. Pat. No. 4,500,512). U.S. Pat. No. 4,500,512, describes a combination of lactose, sorbitol, the divalent cations, calcium and magnesium, and at least one amino acid. While this formulation may help to stabilize the lyophilized vaccine, it fails to provide stability to the vaccine in aqueous form. Another study examined the ability of several different formulations including the compositions described above and the effect of sucrose, trehalose and lactalbumin on the stability of the lyophilized yellow fever vaccine. Formulations consisting of 10% sucrose alone, 2% sorbitol with 4% inositol, or 10% sucrose with 5% lactalbumin, 0.1 g/l CaCl.sub.2 and 0.076 g/l MgSO.sub.4 were found to provide the best stability (see for example Adebayo, Sim-Brandenburg et al. 1998). However, in all cases after resuspension, yellow fever vaccine is still very unstable and must be discarded after only about one hour (see for example Monath 1996; Adebayo, Sim-Brandenburg et al. 1998). This leads to vaccine wastage and the potential to cause administration of ineffective vaccine under field conditions, if an unstable vaccine is used.

Another live, attenuated flavivirus vaccine for protection against Japanese encephalitis has been licensed and is in widespread use in China (see for example Halstead and Tsai 2004). The Japanese encephalitis vaccine strain, SA 14-14-2, is grown on primary hamster kidney cells and the cell supernatant is harvested and coarsely filtered. One previous composition included 1% gelatin and 5% sorbitol added as stabilizers. Using these stabilizers, the vaccine is lyophilized and then is stable at 2 to 8.degree. C. for at least 1.5 years, but for only 4 months at room temperature and 10 days at 37.degree. C. As with the yellow fever vaccine, the reconstituted vaccine is very labile and is stable for only 2 hours at room temperature (see for example Wanf, Yang et al 1990). In certain embodiments herein, live, attenuated flavivirus compositions for stabilizing or reducing the degradation of Japanese encephalitis are contemplated.

No formulation for a live, attenuated flavivirus vaccine has been identified that provides long term stability of lyophilized formulations at temperatures greater than 2-8.degree. C. In addition, no formulation has been described that prevents loss of titer, stabilizes or reduces degradation of aqueous vaccines for greater than a few hours.

Formulations for other live, attenuated viruses have also been described (see for example Burke, Hsu et al. 1999). One common stabilizer, referred to as SPGA is a mixture of 2 to 10% sucrose, phosphate, potassium glutamate and 0.5 to 2% serum albumin (see for example Bovarnick, Miller et al. 1950). Various modifications of this basic formulation have been identified with different cations, with substitutions of starch hydrolysate or dextran for sucrose, and with substitutions of casein hydrolysate or poly-vinyl pyrrolidone for serum albumin. Other formulations use hydrolyzed gelatin instead of serum albumin as a protein source (Burke, Hsu et al 1999). However, gelatin can cause allergic reactions in immunized children and could be a cause of vaccine-related adverse events. U.S. Pat. No. 6,210,683 describes the substitution of recombinant human serum albumin for albumin purified from human serum in vaccine formulations.

Embodiments herein disclose compositions that enhance the stability of and/or reduce deterioration of live, attenuated virus vaccines compared to those in the prior art. Certain compositions disclosed herein provide stability of aqueous viruses for up to 2 hours; up to 3 hours; up to 4 hours and greater than 4 hours at or about 37.degree. C. Certain compositions disclosed herein provide stability of aqueous viruses for up to 1 day to about 1 week or more, at or about room temperature (e.g. 25.degree. C.). Embodiments contemplated herein provide increased protection of a live, attenuated virus from for example, freezing and/or thawing, and/or elevated temperatures. In certain embodiments, compositions herein can stabilize, reduce deterioration and/or prevent inactivation of dehydrated live, attenuated viral products in room temperature conditions (e.g. about 25.degree. C.). In other embodiments, compositions contemplated herein can stabilize, reduce deterioration and/or prevent inactivation of aqueous live, attenuated viral products at about 25.degree. C. or up to or about 37.degree. C. Compositions and methods disclosed herein can facilitate the storage, distribution, delivery and administration of viral vaccines in developed and under developed regions.

Other embodiments can include compositions for live attenuated virus vaccines including, but not limited to, Picornaviruses (e.g., polio virus, foot and mouth disease virus), Caliciviruses (e.g., SARS virus, and feline infectious peritonitis virus), Togaviruses (e.g., sindbis virus, the equine encephalitis viruses, chikungunya virus, rubella virus, Ross River virus, bovine diarrhea virus, hog cholera virus), Flaviviruses (e.g., dengue virus, West Nile virus, yellow fever virus, Japanese encephalitis virus, St. Louis encephalitis virus, tick-borne encephalitis virus), Coronaviruses (e.g., human coronaviruses (common cold), swine gastroenteritis virus), Rhabdoviruses (e.g., rabies virus, vesicular stomatitis viruses), Filoviruses (e.g., Marburg virus, Ebola virus), Paramyxoviruses (e.g., measles virus, canine distemper virus, mumps virus, parainfluenza viruses, respiratory syncytial virus, Newcastle disease virus, rinderpest virus), Orthomyxoviruses (e.g., human influenza viruses, avian influenza viruses, equine influenza viruses), Bunyaviruses (e.g., hantavirus, LaCrosse virus, Rift Valley fever virus), Arenaviruses (e.g., Lassa virus, Machupo virus), Reoviruses (e.g., human reoviruses, human rotavirus.), Birnaviruses (e.g., infectious bursal virus, fish pancreatic necrosis virus), Retroviruses (e.g., HIV 1, HIV 2, HTLV-1, HTLV-2, bovine leukemia virus, feline immunodeficiency virus, feline sarcoma virus, mouse mammary tumor virus), Hepadnaviruses (e.g., hepatitis B virus), Parvoviruses (e.g., human parvovirus B, canine parvovirus, feline panleukopenia virus) Papovaviruses (e.g., human papillomaviruses, SV40, bovine papillomaviruses), Adenoviruses (e.g., human adenovirus, canine adenovirus, bovine adenovirus, porcine adenovirus), Herpes viruses (e.g., herpes simplex viruses, varicella-zoster virus, infectious bovine rhinotracheitis virus, human cytomegalovirus, human herpesvirus 6), and Poxviruses (e.g., vaccinia, fowlpoxviruses, raccoon poxvirus, skunkpox virus, monkeypoxvirus, cowpox virus, musculum contagiosum virus).

Those skilled in the art will recognize that compositions or formulas herein relate to viruses that are attenuated by any means, including but not limited to, cell culture passage, reassortment, incorporation of mutations in infectious clones, reverse genetics, other recombinant DNA or RNA manipulation. In addition, those skilled in the art will recognize that other embodiments relate to viruses that are engineered to express any other proteins or RNA including, but not limited to, recombinant flaviviruses, recombinant adenoviruses, recombinant poxviruses, recombinant retroviruses, recombinant adeno-associated viruses and recombinant herpes viruses. Such viruses may be used as vaccines for infectious diseases, vaccines to treat oncological conditions, or viruses to introduce express proteins or RNA (e.g., gene therapy, antisense therapy, ribozyme therapy or small inhibitory RNA therapy) to treat disorders.

In some embodiments, compositions herein can contain one or more viruses with membrane envelopes (e.g., enveloped viruses) of the Togavirus, Flavivirus, Coronavirus, Rhabdovirus, Filovirus, Paramyxovirus, Orthomyxovirus, Bunyavirus, Arenavirus, Retrovirus, Hepadnavirus, Herpesvirus or Poxvirus families. In certain embodiments compositions contain one or more enveloped RNA viruses of the Togavirus, Flavivirus, Coronavirus, Rhabdovirus, Filovirus, Paramyxovirus, Orthomyxovirus, Bunyavirus, Arenavirus, or Retrovirus families. In other embodiments, compositions herein can contain one or more enveloped, positive strand RNA virus of the Togavirus, Flavivirus, Coronavirus, or Retrovirus families. In certain embodiments, compositions can contain one or more live, attenuated Flaviviruses (e.g., dengue virus, West Nile virus, yellow fever virus, or Japanese encephalitis virus).

Some embodiments herein relate to compositions for live, attenuated viruses in aqueous or lyophilized form. Those skilled in the art will recognize that formulations that improve thermal viral stability and prevent freeze-thaw inactivation will improve products that are liquid, powdered, freeze-dried or lyophilized and prepared by methods known in the art. After reconstitution, such stabilized vaccines can be administered by a variety routes, including, but not limited to intradermal administration, subcutaneous administration, intramuscular administration, intranasal administration, pulmonary administration or oral administration. A variety of devices are known in the art for delivery of the vaccine including, but not limited to, syringe and needle injection, bifurcated needle administration, administration by intradermal patches or pumps, needle-free jet delivery, intradermal particle delivery, or aerosol powder delivery.

Embodiments can include compositions consisting of one or more live attenuated viruses (as described above) and a mixture of one or more high molecular weight surfactants and one or more proteins in a physiological acceptable buffer. In certain embodiments, compositions include, but are not limited to one or more live attenuated viruses, one or more high molecular weight surfactants, one or more proteins, and one or more carbohydrates, in a physiological acceptable buffer.

In other embodiments, compositions can contain one or more high molecular weight surfactants that increase the thermal stability of live, attenuated viruses. Surfactants have been incorporated into vaccine formulations to prevent material loss to surfaces such as glass vials (see for example Burke, Hsu et al. 1999). However, certain embodiments herein include high molecular weight surfactants with some unusual biochemical properties of utility for compositions and methods disclosed herein. The EO-PO block copolymers can include blocks of polyethylene oxide (--CH.sub.2CH.sub.2O-- designated EO) and polypropylene oxide (--CH.sub.2CHCH.sub.3O-- designated PO). The PO block can be flanked by two EO blocks in a EO.sub.x-PO.sub.y-EO.sub.x arrangement. Since the PO component is hydrophilic and the EO component is hydrophobic, overall hydrophilicity, molecular weight and the surfactant properties can be adjusted by varying x and y in the EO.sub.x-PO.sub.y-EO.sub.x block structure. In aqueous solutions, the EO-PO block copolymers will self-assemble into micelles with a PO core and a corona of hydrophilic EO groups. EO-PO block copolymer formulations have been investigated as potential drug delivery agents for a variety of hydrophobic drugs and for protein, DNA or inactivated vaccines (e.g. Todd, Lee et al. 1998; Kabanov, Lemieux et al. 2002). At high concentrations (for example: >than 10%) certain of the higher molecular weight EO-PO block copolymers will undergo reverse gelation, forming a gel as the temperature increases. Gel formation at body temperatures permits use of the EO-PO block copolymer gels to act as a depot in drug and vaccine delivery applications (see for example Coeshott, Smithson et al. 2004). In addition, due to their surfactant properties, these polymers have been used in adjuvant formulations, and as an emulsifier in topically applied creams and gels. The EO-PO block copolymers have also been shown to accelerate wound and burn healing and to seal cell membranes after radiation or electroporation-mediated damage.

In other embodiments, vaccine compositions can include one or more surfactants with molecular weight of 1500 or greater. In a certain embodiment, the surfactant is a non- ionic, hydrophilic, polyoxyethylene-polyoxypropylene block copolymer (or EO-PO block copolymer). While EO-PO block copolymers have been used as adjuvants and delivery vehicles for inactivated vaccines, protein vaccines or DNA vaccines, their use to prevent inactivation of a live virus is not anticipated in the art. In a particular embodiment, a formulation can contain one or more EO-PO polymers with a molecular weight of 3,000 or greater. In further embodiments, compositions can include in part an EO-PO block copolymer poloxamer 407, Pluronic F127.RTM. or copolymer EO30PO70EO70, Pluronic P123. Those skilled in the art will recognize that modifications of the surfactants can be chemically made. It is contemplated herein any essentially equivalent surfactant polymers are considered.

Embodiments herein can include compositions of one or more live, attenuated viruses, one or more surfactants and one or more proteins. In certain embodiments, a protein can be an albumin. Serum albumin is one of the most common proteins in vertebrate blood and has multiple functions. The protein is 585 amino acids with a molecular weight of 66500. Human serum albumin is not glycosylated and has a single free thiol group implicated in some of its myriad binding activities. Serum albumin is predominantly .alpha.-helix with three structural domains, each subdivided into two subdomains. Albumin is known to specifically bind a variety of molecules, including drugs such as aspirin, ibuprofen, halothane, propofol and warfarin as well as fatty acids, amino acids, steroids, glutathione, metals, bilirubin, lysolecithin, hematin, and prostaglandins. The different structural domains are implicated in drug binding; most small molecule drugs and hormones bind to one of two primary sites located in subdomains IIA and IIIA. Due to its lack of immunogenicity, albumin is commonly used as a carrier protein in biological products. Since the protein dose contained in a live, attenuated viral vaccine can be fractions of a microgram (derived from 10.sup.3 to 10.sup.5 viral particles), an inert carrier protein is used to prevent loss due to absorption and non-specific binding to glass, plastic or other surfaces. However, as demonstrated herein, an unexpected improvement in stability was observed with the combination of an albumin and EO-PO block copolymers suggesting interactions between the two components and/or between the components and the viral particles. In addition, enhanced stabilization of viruses in the presence of albumin is not likely due to function as a general carrier protein: other proteins such as gelatin and lactoferrin fail to improve virus stability.

In certain embodiments, serum albumin may be from a human or other mammalian source. For vaccines intended for human use, particular embodiments can include human albumin or other human products as needed in order to reduce or eliminate adverse immune responses. Those skilled in the art will recognize that albumins specific for each species may be used in animal vaccines (e.g. canine albumin for canine products, bovine albumin for bovine products). In further embodiments, the protein is a recombinant human albumin. Standard methods exist for expressing recombinant human albumin or portions thereof in a variety of expression systems including bacteria, yeast, algae, plant, mammalian cell or transgenic animal systems. In addition, serum albumin or portions thereof can be produced in cell-free systems or chemically synthesized. Recombinant human albumin produced in these or in any similar system is incorporated herein. Those skilled in the art will recognize that other proteins can substitute for albumin. For example, albumin is a member of a multi-gene family. Due to their structural and sequence similarities, other members of the family (e.g. .alpha.-fetoprotein, vitamin D binding protein, or afamin) may substitute for albumin in compositions and methods contemplated herein. Those skilled in the art will also recognize that modifications can be made to albumin by any means known in the art, for example, by recombinant DNA technology, by post-translational modification, by proteolytic cleavage and/or by chemical means. Those substitutions and alterations to albumin that provide essentially equivalent stabilizing function to serum albumin without substitutions and alterations are contemplated herein.

In certain embodiments, compositions having a high molecular weight surfactant, a protein and a carbohydrate in a pharmaceutically acceptable buffer are described. In some embodiments, the carbohydrate is a sugar or a polyol. Sugars can include, but are not limited to, monosaccharides, (e.g. glucose, galactose, ribose, mannose, rhamnose, talose, xylose or allose arabinose), disaccharides (e.g. trehalose, sucrose, maltose, isomaltose, cellibiose, gentiobiose, laminaribose, xylobiose, mannobiose, lactose, or fructose.), trisaccharides (e.g. acarbose, raffinose, melizitose, panose, or cellotriose) or sugar polymers (e.g. dextran, xanthan, pullulan, cyclodextrins, amylose, amylopectin, starch, celloologosaccharides, cellulose, maltooligosaccharides, glycogen, chitosan, or chitin). Polyols can include, but are not limited to, mannitol, sorbitol, arabitol, erythritol, maltitol, xylitol, glycitol, glycol, polyglycitol, polyethylene glycol, polypropylene glycol, and glycerol.

In a particular embodiment, formulations can contain a combination of one or more EO-PO block copolymers, one or more proteins, and trehalose in a pharmacologically acceptable buffer. In certain embodiments, trehalose can be present at concentrations ranging from 5 to 50% (w/v). Trehalose has been used to enhance the stability of protein formulations. It is widely known in the art as a cryopreservative and is used in nature to protect organisms from stress. Anhydrobiotic organisms that can tolerate low water conditions contain large amounts of trehalose. Trehalose has been shown to prevent both membrane fusion events and phase transitions that can cause membrane destabilization during drying. Structural analysis suggests that trehalose fits well between the polar head groups in lipid bylayers. Trehalose also prevents denaturation of labile proteins during drying. It is thought that trehalose stabilizes proteins by hydrogen bonding with polar protein residues. Trehalose is a disaccharide consisting of two glucose molecules in a 1:1 linkage. Due to the 1:1 linkage, trehalose has little or no reducing power and is thus essentially non-reactive with amino acids and proteins. This lack of reducing activity may improve the stabilizing affect of trehalose on proteins. In certain embodiments, trehalose provides stability to live, attenuated viruses. This activity of trehalose may be due to its ability to stabilize both the membranes and coat proteins of the viruses.

In further embodiments, compositions can include one or more EO-PO block copolymers, one or more proteins and one or more carbohydrates, where one of the carbohydrates is chitosan, in a physiological acceptable buffer to provide improved stability to live, attenuated viruses. In certain embodiments, compositions can include chitosan at concentrations ranging from 0.001 to 2% (e.g at a pH of about 6.8). Chitosan is a cationic polysaccharide derived by deacetylation of chitin, the structural polymer of crustacean exoskeletons. It is a polymer of N-acetyl-glucosamine and glucosamine; the content of the two carbohydrates depends on the extent of deacetylation. Chitosan's positive charge allows it to bind to negatively charged surfaces and molecules. Thus, it binds musosal surfaces and is thought to promote mucosal absorption. Chitosan also can bind and form nanoparticles with DNA, RNA and other oligonucleotides and has been used in non-viral gene delivery. Certain embodiments herein demonstrate that chitosan increases live, attenuated virus stability.

In certain embodiments, compositions can be described that typically include a physiologically acceptable buffer. Those skilled in the art recognize that a variety of physiologically acceptable buffers exist, including, but not limited to buffers containing phosphate, TRIS, MOPS, HEPES, bicarbonate, other buffers known in the art ad combinations of buffers. In addition, those skilled in the art recognize that adjusting salt concentrations to near physiological levels (e.g., saline or 0.15 M total salt) may be optimal for parenteral administration of compositions to prevent cellular damage and/or pain at the site of injection. Those skilled in the art also will recognize that as carbohydrate concentrations increase, salt concentrations can be decreased to maintain equivalent osmolarity to the formulation. In certain embodiments, a buffering media with pH greater than 6.8 is contemplated; some live, attenuated viruses (e.g. flaviviruses) are unstable at low pH. In another embodiment, physiologically acceptable buffer can be phosphate-buffered saline (PBS).

Some live, attenuated viral vaccine compositions herein concern compositions that increase stability and/or reduce deterioration of live, attenuated virus in addition to having reduced immunogenicity or are non-immunogenic. In accordance with these embodiments, compositions can include one or more protein agents; one or more saccharides or polyols agents; and one or more high molecular weight surfactants, wherein the composition decreases inactivation of the live attenuated virus. Therefore, certain compositions contemplated herein have reduced adverse reaction when administered to a subject. In some exemplary compositions, the surfactant agent(s) consists of one or more EO-PO block copolymers; the protein agent(s) are selected from the group consisting of lactalbumin, serum albumin, .alpha.-fetoprotein, vitamin D binding protein, afamin derived from a vertebrate species; and the carbohydrate agent(s) is one or more of a saccharide and/or a polyol. In certain embodiments, compositions can include one or more of the carbohydrate agent(s) selected from the group consisting of trehalose, sucrose, chitosan, sorbitol, and mannitol. In certain more particular embodiments, in order to reduce immune reaction to a vaccine, the serum albumin can be derived from a vertebrate species or in other embodiments, from the same source as the subject (e.g. human). In other embodiments, the carbohydrate agent is trehalose. In certain embodiments, at least one surfactant agent is the EO-PO block copolymer copolymer poloxamer 407, Pluonic F127.RTM.. In some live, attenuated viral vaccine compositions at least one carbohydrate agent is trehalose. In certain live, attenuated viral vaccine compositions include, the EO-PO block copolymer poloxamer 407, Pluronic F127 .RTM. where the concentration is from 0.1 to 4% (w/v); and/or serum albumin concentration from 0.001 to 3% (w/v) and/or the trehalose concentration can be from 5 to 50% (w/v).

Pharmaceutical Compositions

Embodiments herein provide for administration of compositions to subjects in a biologically compatible form suitable for pharmaceutical administration in vivo. By "biologically compatible form suitable for administration in vivo" is meant a form of the active agent (e.g. live, attenuated virus composition of the embodiments) to be administered in which any toxic effects are outweighed by the therapeutic effects of the active agent. Administration of a therapeutically active amount of the therapeutic compositions is defined as an amount effective, at dosages and for periods of time necessary to achieve a desired result. For example, a therapeutically active amount of a compound may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability formulations to elicit a desired response in the individual. Dosage regimen may be adjusted to provide the optimum therapeutic response.

In some embodiments, composition (e.g. pharmaceutical chemical, protein, peptide of an embodiment) may be administered in a convenient manner such as subcutaneous, intravenous, by oral administration, inhalation, transdermal application, intravaginal application, topical application, intranasal or rectal administration. In a more particular embodiment, the compound may be orally or subcutaneously administered. In another embodiment, the compound may be administered intravenously. In one embodiment, the compound may be administered intranasally, such as inhalation.

A compound may be administered to a subject in an appropriate carrier or diluent, co-administered with the composition. The term "pharmaceutically acceptable carrier" as used herein is intended to include diluents such as saline and aqueous buffer solutions. The active agent may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use may be administered by means known in the art. For example, sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion may be used. In all cases, the composition can be sterile and can be fluid to the extent that easy syringability exists. It may further be preserved against the contaminating action of microorganisms such as bacteria and fungi. The pharmaceutically acceptable carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

Sterile injectable solutions can be prepared by incorporating active compound in an amount with an appropriate solvent or with one or a combination of ingredients enumerated above, as required, followed by sterilization.

Upon formulation, solutions can be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above. It is contemplated that slow release capsules, timed-release microparticles, and the like can also be employed for administering compositions herein. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.

The active therapeutic agents may be formulated within a mixture can include about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 1 to 10 gram per dose. Single dose or multiple doses can also be administered on an appropriate schedule for a predetermined situation. In some embodiments, doses can be administered before, during and/or after exposure to a virus contemplated herein.

In another embodiment, nasal solutions or sprays, aerosols or inhalants may be used to deliver the compound of interest. Additional formulations that are suitable for other modes of administration include suppositories and pessaries. A rectal pessary or suppository may also be used. In general, for suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% 2%.

Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. In certain embodiments, oral pharmaceutical compositions can include an inert diluent or assimilable edible carrier, or may be enclosed in hard or soft shell gelatin capsule, or may be compressed into tablets, or may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 75% of the weight of the unit, or preferably between 25-60%. The amount of active compounds in such therapeutically useful compositions is such that a suitable dosage can be obtained.

Kits

Further embodiments concerns kits for use with methods and compositions described herein. Compositions and live virus formulations may be provided in the kit. The kits can also include a suitable container, live, attenuated virus compositions detailed herein and optionally one or more additional agents such as other anti-viral agents, anti-fungal or anti-bacterial agents.

The kits may further include a suitably aliquoted composition of use in a subject in need thereof. In addition, compositions herein may be partially or wholly dehydrated or aqueous. Kits contemplated herein may be stored at room temperatures or at refrigerated temperatures as disclosed herein depending on the particular formulation.

The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a composition may be placed, and preferably, suitably aliquoted. Where an additional component is provided, the kit will also generally contain one or more additional containers into which this agent or component may be placed. Kits herein will also typically include a means for containing the agent, composition and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
 

Claim 1 of 30 Claims

1. A live attenuated virus composition comprising: one or more live, attenuated viruses; one or more poly(ethylene oxide) and poly(propylene oxide) (EO-PO) block copolymers, the one or more EO-PO block copolymers include poloxamer 407, one or more proteins agents, the one or more protein agents include one or more albumins selected from the group consisting of lactalbumins and serum albumins, and one or more carbohydrate agents, the one or more carbohydrate agents include trehalose, wherein the composition is capable of reducing the inactivation of the live attenuated virus.

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