Indian Patents. 279320:"METHODS AND COMPOSITIONS FOR LIVE ATTENUATED VIRUSES"

Title of Invention	"METHODS AND COMPOSITIONS FOR LIVE ATTENUATED VIRUSES"
Abstract	One or more live, attenuated viruses and compositions to reduce inactivation and/or degradation of the live, attenuated vims, including a vaccine are disclosed. This composition may include at least one carbohydrate, at least one protein and at least one high molecular weight surfactant.

Full Text	Priority This application claims the benefit of priority of provisional U.S. patent application No. 60/910,579, filed on April 06, 2007, which is incorporated herein in its entirety. Field Embodiments herein relate to compositions and methods for stabilizing live, attenuated viruses. Other embodiments relate to compositions and methods for redi cing degradation of live, attenuated viruses. Still other embodiments relate to uses of these compositions in kits for portable applications and methods. Background Vaccines to protect against viral infections have been effectively used to rec uce the incidence of human disease. One of the most successful technologies for viral vacc ines is to immunize animals or humans with a weakened or attenuated strain of the virus (a " ive, attenuated virus"). Due to limited replication after immunization, the attenuated sti ain does not cause disease. However, the limited viral replication is sufficient to express the full repertoire of viral antigens and generates potent and long-lasting immune response;. to the virus. Thus, upon subsequent exposure to a pathogenic strain of the virus, the imn unized individual is protected from disease. These live, attenuated viral vaccines are amon \ the most successful vaccines used in public health. Ten of the sixteen viral vaccines approved for sale in the U.S. are live, attenuated viruses. Highly successful live viral vaccines include the yellow fever 17D virus, Sabin poliovirus types 1, 2 and 3, measles, mumps, rubella, varicella and vaccinia viruses. Use of the vaccinia virus vaccine to control smallpox outbreaks led to the first and only eradication of a human disease. The Sabin poliovirus vaccine has helped prevent crippling dis Recent technical advances, such as reassortment, reverse genetics and cold adaptation, have led to the licensure oflive, attenuated viruses for influenza and rolavirus. A number of live, viral vaccines developed with recombinant DNA technologies are ii human clinical testing, including vaccines for West Nile disease, dengue fever, malaria, tuberculosis and HIV. These recombinant viral vaccines rely on manipulation of well-characterized attenuated viral vaccines, such as adenovirus, vaccinia virus, yellow fever 17D or the dengue virus, DEN-2 PDK-53. The safe, attenuated viruses are genetically engineered to express protective antigens for other viral or bacterial pathogens. Several recombinant vira [ vaccines have been approved for animal use, including a canarypox/feline leukemia recombi nant virus, a canarypox/canine distemper recombinant virus, a canarypox/West Nile recombin; tnt virus and a yellow fever/West Nile recombinant virus. As a group, the live attenuated vi -us vaccines are amongst the most successful medical interventions in human history, second only to the advent of antibiotics and hold the promise to improve public health throaghout the world. In order for live, attenuated viral vaccines to be effective, they must be caps ble of replicating after immunization. Thus, any factors that inactivate the virus can cripp le the vaccine. For example, widespread distribution and use of the smallpox vaccine prior to World War II was limited because the virus was inactivated after only a few days a: ambient temperatures. In the 1920s, French scientists demonstration that freeze-dried vacci le provided long term stability and techniques for large-scale manufacture of freeze-d -ied vaccine were developed in the 1940s (see for example Collier 1955). In addition to freeze-drying, various additives have been identified that can help stabilize the viruses in live, attenuated viral vaccines (See for example Burke, Hsu et al 1999). These stabilizers typically include one or more of the following components: divalent cations, buffered salt solutions, chelators, urea, sugars (e.g. sucrose, lactose, trehalose), polyols (e.g., glycerol, mannitol, sorbitol, polyethylene glycol), amino acids, protein hydrolystates (e.g. casein hydrclysate, lactalbumin hydrolysate, peptone), proteins (e.g. gelatin, human serum albumin) or polymers (e.g. dextran). However, even with these stabilizing agents, many of the commonly used viccines still require refrigeration for stabilization. Other commonly used vaccines are sensitive to temperature extremes; either excessive heat or accidental freezing can inactivate thu vaccine. Maintaining this "cold chain" throughout distribution is particularly difficult in the developing world. Thus, there remains a need for improving the stability of both existing and newly developed live, attenuated viral vaccines. Fl aviviruses are amongst the most labile viruses. They are enveloped virusod vector and their vertebrate host species (often birds or mammals). Expanding urbanizatioi i, worldwide travel and environmental changes (such as deforestation or rain patterns i have lead to the emergence of several flavhiruses as threats to human public health. Sue h viruses include, but are not limited to, yellow fever virus, the dengue viruses, West Nile viius, Japanese encephalitis virus, and tick-borne encephalitis viruses. Through intensive mosquito control and vaccination efforts, yellow fever w is eliminated from much of North, Central and South America, the Caribbean and Euiope. However, in the last 20 years, the number of countries reporting cases has increases. Yellow fever virus is now endemic in major portions of Africa and South America and some Caribbean islands. The World Health Organization (WHO) estimates that 200,000 cases of yellow fever occur annually leading to 30,000 deaths. Since World War II, dengue flaviviruses have spread to tropical and subtropical regions throughout the world an d now threaten over 3.5 billion people, about half of the world's population. The WHO e jtimates that 50-100 million cases of dengue fever occur annually. 500,000 of these are the nore sever, life-threatening form of the disease, termed dengue hemorrhagic fever, that leads to more than 25,000 deaths per year. A particularly virulent form of West Nile virus \ /as introduced into the Western hemisphere, presumably by travel, in New York in 199 9. The mosquito-transmitted virus infected birds as the primary host, but also caused disea >e and mortality in humans and horses. West Nile virus spread throughout the United Sta :es and i nto Canada and Mexico. Since its introduction, West Nile virus has caused over 2i ),000 reported cases of West Nile disease leading to 950 deaths in the United States. Jap anese encephalitis virus causes 30,000 to 50,000 cases of neurological disease annually, primarily in eastern and southern Asia. 25-30% of the reported cases are fatal. The tick-borne encephalitis viruses are endemic to parts of Europe and Asia and continue to cause episodic outbreaks affecting thousands of individuals. Related viruses with more limited geographical spread include Kunjin virus (a close relative of West Nile) and Murray Valley enceihalitis virus in Australia and New Guinea, St. Louis encephalitis virus in North and South America, the Usutu, Koutango, and Yaonde viruses in Africa, and Cacipacore virus in South American. Live, attenuated viral vaccines have been developed that are safe and protect against flavivirus diseases, such as yellow fever and Japanese encephalitis. The livs, attenuated viral vaccine, 17D, has been widely used to prevent yellow fever. The c arrent flavivirus vaccines are lyophilized in the presence of stabilizers. Nonetheless, the vaccines require storage and shipment at 2 — 8° C, a requirement that is difficult to achieve it. the developing world and more remote regions of developed nations. Furthermore, upon reconstitution, the vaccines rapidly lose potency even when stored at 2 - 8° C. The measles vaccine is another example of a labile attenuated virus i hat is used worldwide to prevent disease. Measles virus is an enveloped, non-segmented negative strand RNA virus of the Paramyxovirus family. Measles is a highly contagious, seasonal disease that can affect virtually every child before puberty in the absence of vaccins.tion. In developing countries, mortality rates in measles-infected children can by as high as 2 to 15%. Indeed, despite efforts to institute worldwide immunization, measles still causes greater than 7,000 deaths in children per year. The measles vaccine is a live, attenuated virus that is manufactured in primary chicken fibroblast cells. The vaccine is stabilized with ge latin and sorbitol and is then lyophilized. The stabilized, lyophilized vaccine has a shelf life :>f 2 years or more if stored at 2 to 8° C. However, the lyophilized vaccine still requires a cold chain that is difficult to maintain in the developing world. Furthermore, upon reconstitutian, the vaccine loses 50% of its potency within 1 hour at room temperature (20 to 25° C). Thus, a need exists in the art for improved vaccine formulations. SUMMARY Embodiments herein concern methods and compositions to reduce o • 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 method;, 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 viruse s 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, attenuited 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 limi ed 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 val ey encephalitis, Alkhurma hemorrhagic fever, St. Louis encephalitis, tick-borne encep lalitis, 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 inclu ie, but are not limited to, monosaccharides, disaccharides, sugar alcohols, trehalose, sucrose, rialtose, isomaltose, cellibiose, gentiobiose, laminaribose, xylobiose, mannobiose, lactose, fiuctose, sorbitol, mannitol, lactitol, xylitol, erythritol, raffinose, amylse, cyclodextrins, chitcsan, 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(ethylenc oxide) and polypropylene oxide) (EO-PO block copolymers ), poly(vinyl pyrroloidone), alkyl polyglucosides (such as sucrose monostearate, lauryl diglucoside, or sorbitan mono aureate, 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 limite d to, the Pluronic F127, Pluronic F68, Pluronic P123, or other EO-PO block copolymers of greater than 3,000-4,000 MW. In some embodiments, vaccine compositions can include, but are nc t limited to, one or more protein agent that is serum albumin; one or more saccharide agent 1 hat is trehalose; and one or more surfactant polymer agent that is the EO-PO block copolymer Pluronic F127. 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 attenu* ted viruses with a composition capable of reducing inactivation of a live, attenuated viius including, but not limited to, one or more protein agents; one or more saccharides cr 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, Bunyavirui;, Arenavirus, Retrovirus, Hepadnavirus, Pestivirus, Picornavirus, Calicivirus, Reovirus, Parvovirus, Papovavirus, Adenovirus, Herpes virus, or a Poxvirus. Additionally, m ;thods and compositions disclosed herein can include freeze drying or other dehydrating methods for the combination. In accordance with these methods and compositions, the met! ods and compositions decrease inactivation of the freeze dried or partially or wholly dehydi ated live attenuated virus. In other methods, compositions for decreasing inactivation of a live attenuated virus may comprise an aqueous composition or may comprise a rehydrc ted 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 11 vaccine composition contemplated herein may include, but is not limited to, one or more Irs e, 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-4A30) 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° to about 25° C) or refrigeration temp matures (e.g. about 0° to about 10° C). In more particular embodiments, a combination composition is capable of maintaining about 100 percent of the live attenuated virus for greater ths n 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° to about 10° C). Compositions contemplated in these methods, can include, but are not limited to, one or more protein agent of serum albumin; on; or more saccharide agent of trehalose; and one or more EO-PO block copolymer agent of P uronic F127. In certain embodiments, the live, attenuated virus composition remains at at out 100% viral titer after 7 days at approximately 21° C and about 100% viral titer after 50 days at refrigeration temperatures around 4° C. Other embodiments herein may include liv<: attenuated virus composition remaining at about or viral titer aftei days approximately c and after refrigsration temperatures around c. other embodiments contemplated include live compositions to lox the concentration of affc several hours compared knov in art. for example figs. disclosed herein reduce degradation when is stored> 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 oi 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 thesi; embodiments, a kit composition may include one or more one protein agent 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, Corot.avirus, Rhabdovirus, Filovirus, Paramyxovirus, Orthomyxovirus, Bunyavirus, Arenavirus, Retrovirus, Hepadnavirus, Pestivirus, Picornavirus, Calicivirus, Reovirus, Parvovixis, Papovavirus, Adenovirus, Herpes virus, or Poxvirus. In certain embodiments, compositions herein can include trehalose as a saccharide agent. In accordance with these emboc intents, trehalose concentration may be equal to or greater than 5% (w/v). In certain emboc [intents, compositions herein can include polymer F127 as an EO-PO block copolymer ager t. In accordance with these embodiments, polymer F127 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 m ay have trace amounts or no calcium/magnesium (Ca+2/Mg+2). BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the instant specification and are included to further demonstrate certain aspects of particular embodiments herein. The embodiments n ay be better understood by reference to one or more of these drawings in combination wilh the detailed description presented herein. Fig.l represents an exemplary histogram of experiments using various compositions for testing the stability of an exemplary virus, DEN-2 PDK 53 flavivirus, in the compositions. Fig. 2 represents an exemplary graph of a kinetic analysis of an exenplary virus, DEN-2 PDK 53 flavivirus, for viral inactivation at 37° C in various exemplar/ compositions. Fig. 3 represents an exemplary histogram of an analysis of an exemi lary virus, DEN-2 PDK 53 virus, stored at 37°C for 21 hours. Values are expressed as a percentage of the viral titer remaining after incubation relative to the input titer. Formulation percentages refer to (w/v) of the respective excipient. Fig. 4 represents an exemplary histogram of an analysis of an exemt lary virus, DEN-2 PDK 53 virus, stored at 37° C for 23 hours in different compositions. Value* are expressed as a percentage of the viral titer remaining after incubation relative to the input titer. Fig. 5 represents an exemplary histogram of an analysis of an exemplary virus, DEN-2 PDK 53 virus, stored at 37° C for 23 hours in different compositions. Values are expressed as a percentage of the viral titer remaining after incubation relative to th Fig. 6 represents an exemplary histogram analysis of an exemplary virus, DEN-2 PDK 53 virus, after two freeze-thaw cycles when stored in different formulations. Values are expressed as a percentage of the viral titer remaining after freeze-thaw cycles relative to the input titer. Fig. 7 represents an exemplary graph of a kinetic analysis of an exemplary virus, DEN-2 PDK 53/WN recombina.nt flavivirus, in various exemplary compositions for viral inactivation at 25° C over several weeks of time. Fig. 8 represents an exemplary graph of a kinetic analysis of an exemplary virus, DEN-2 PDK 53/WN recombinant flavivirus, in various exemplary compositions for viral inactivation at 4° C over several weeks of time. Fig. 9 represents an exemplary histogram analysis of an exemplary v irus, DEN-2 PDK-53 virus, after lyophilization in various exemplary compositions. Vira1 inactivation was assessed as described above after two weeks at different temperatires. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Definitions As used herein, "a" or "an" may mean one or more than one of an item. As used herein, "about" may mean up to and including plus or mins five percent, for example, about 100 may mean 95 and up to 105. As used herein, "saccharide" agents can mean one or more monosac;harides, (e.g. glucose, galactose, ribose, mannose, rhamnose, talose, xylose, or allose arabin :>se.), one or more disaccharides (e.g. trehalose, sucrose, maltose, isomaltose, cellibiose, genti obiose, laminaribose, xylobiose, mannobiose, lactose, or fructose.), trisaccharides (e.g. acaibose, raffinose, melizitose, panose, or cellolriose) or sugar polymers (e.g. dextran, xanthi m, pullulan, cyclodextrins, amylose, amylopectin, starch, celloologosaccharides, cellu ose, maltooligosaccharides, glycogen, chitosan, or chitin). As used herein, "polyol" agents can mean any sugar alcohol (e.g. mannitol, sorbitol, arabitol, erythritol, maltitol, xylitol, glycitol, glycol, polyglycitol, polyethylene glycol, polypropylene glycol, or glycerol).As used herein, "high molecular weight surfactants" can mean a surface active, amphiphilic molecule greater than 1500 molecular weight. As used herein, "EO-PO block copolymer" can mean a copolymer c insisting of blocks of poly(ethylene oxide) and poly(propylene) oxide. In addition, as used 1 erein, "Pluronic" can mean EO-PO block copolymers in the EOx-POy-EOx. This configuration of EO-PO block copolymer is also referred to as "Poloxamer" or "Synperonic". As used herein, "attenuated virus" can mean a virus that demonstrati ;s reduced or no clinical signs of disease when administered to an animal. DETAILED DESCRIPTIONS In the following sections, various exemplary compositions and methods are described in order to detail various embodiments. It will be obvious to one skilled n 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 kncvTi methods or components have not been included in the description. Stability of flavivirus vaccines has been assessed for both the existir g 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 c itions was demonstrated to stabilize the lyophilized vaccine, so that the vaccine lost less than '. log of potency after incubation at 37° C for 14 days. Stabilizing lyophilized formulations lor the yellow fever vaccine have been described (see for example U. S. Pat. No 4,500,512). U.S. Patent 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 jn 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/1 CaC12 and 0.076 g/1 MgS04 were found to provide the best stability (see for example Adebay o, 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 exampl; 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 flavirus vaccine for protection against Japa lese 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 or primary hamster kidney cells and the cell supernatant is harvested and coarsely filtered. Or e 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° C for at least 1.5 years, but for only 4 months at room temperature and 10 days at 37° C. As with the yellow ft ver vaccine, the reconstituted vaccine is very labile and is stable for only 2 hours at rocm temperature (see for example Wanf, Yang et al 1990). In certain embodiments heroin, live, attenuated flavirus virus compositions for stabilizing or reducing the degradation o 'Japanese encephalitis are contemplated. No formulation for a live, attenuated flavivirus vaccine has been ide atified that provides long term stability of lyophilized formulations at temperatures greater tha:i 2 - 8° 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 basi; 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 a lergic reactions in immunized children and could be a cause of vaccine-related adverse e^ ents. U.S. patent 6,210,683 describes the substitution of recombinant human serum albumin f jr 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 pric r art. Certain compositions disclosed herein provide stability of aqueous viruses for up tc 2 hours; up to 3 hours; up to 4 hours and greater than 4 hours at or about 37° C. Certain compositions disclosed herein provide stability of aqueous viruses for up to 1 day to about 1 wee c or more, at or about room temperature {e.g. 25c 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 sta bilize, reduce deterioration and/or prevent inactivation of dehydrated live, attenuated viral products in room temperature conditions (e.g. about 25 ° C). In other embodiments, compositions contemplated herein can stabilize, reduce deterioration and/or prevent inactivation of aqueous live, attenuated viral products at about 25 ° C or up to or about 37 ° C. Composition s and methods disclosed herein can facilitate the storage, distribution, delivery and admii istration of viral vaccines in developed and under developed regions. Other embodiments can include compositions for live attenuated vir is vaccines including, but not limited to, Picornaviruses (e.g., polio virus, foot and mc uth 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 en cephalitis 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 v rus, Newcastle disease virus, rinderpest vims), Orthomyxoviruses (e.g., human influen2 a viruses, avian influenza viruses, equine influenza viruses), Bunyaviruses (e.g., hantavirus, LaCrosse virus, Rift Valley fever virus), Arenaviruses (e.g., Lassa virus, Machupo virus), Re* >viruses (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 mamrr ary tumor virus), Hepadnaviruses (e.g., hepatitis B virus), Parvoviruses (e.g., human parvovins B, canine parvovirus, feline panleukopenia virus) Papovaviruses (e.g., human papillpn lavirases, SV40, bovine papillomaviruses), Adenoviruses (e.g., human adenovirus, canine adenovirus, bovine adenovirus, porcine adenovirus), Herpes viruses (e.g., herpes simplex virus* s, varicella-zoster virus, infectious bovine rhinotracheitis virus, human cytomegalovirus, human herpesvirus 6), and Poxviruses (e.g., vaccinia, fowlpoxviruses, raccoon poxvirus, slainkpox 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 :ulture passage, reassortment, incorporation of mutations in infectious clones, reverse genetics, other recombinant DNA or RNA manipulation. In addition, those skilled in the art will r jcognize that other embodiments relate to viruses that are engineered to express any other proteins or RNA including, but not limited to, recombinant flaviviruses, recombinant adenovirises, recombinant poxviruses, recombinant retroviruses, recombinant adeno-associated viruses and recombinant herpes viruses. Such vinises 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, compositio is herein can contain one or more enveloped, positive strand RNA virus of the Togavirus, Fli.vivirus, Coronavirus, or Retrovirus families. In certain embodiments, compositions can cor tain 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 formula tions that improve thermal viral stability and prevent freeze-thaw inactivation will improve products that are liquid, powdered, freeze-driecl 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 i >r oral administration. A variety of devices are known in the art for delivery of the vaccin 5 including, but not limited to, syringe and needle injection, bifurcated needle admin stration, 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 li ye 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 certai: 1 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 n tolecular 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 embodiment 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 (-CH2CH2O- designated EO) and polypropylene oxide (-CH2CHCH3O- designated PO). The PO block can be flanked by two EO blocks ii a EOx-POy-EOx 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 EOx-POy-EOx block structure. In aqueous solutions, the EO-PO block copolymers will self-assemble into micelles with a PO core and a cor ana 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, USA or inactivated vaccines (e.g. Todd, Lee el: al. 1998; Kabanov, Lemieux et al. 2002). A: 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, Smith son 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 moi e surfactants with molecular weight of 1500 or greater. In a certain embodiment, the surfactant is a non-ionic, hydrophilic, polyoxyethylene-polyoxypropylene block copolymer (cr 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. ,000 or greater. In further embodiments, compositions can include in part an EO-PO block copolymer Pluronic F127 or 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 cam 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 vertebra :e 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 predominant: y a-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 or e of two primary sites located in subdomains IIA and IIIA. Due to its lack of immunogenici .y, albumin is commonly used as a carrier protein in biological products. Since the prctein dose contained in a live, attenuated viral vaccine can be fractions of a microgram (deriv In certain embodiments, serum albumin may be from a human or otl ter mammalian source. For vaccines intended for human use, particular embodiments ran 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 sp ecific for each species may be used in animal vaccines (e.g. canine albumin for canine produ ;ts, bovine albumin for bovine products). In further embodiments, the protein is a recombinan t human albumin. Standard methods exist for expressing recombinant human albumin or pcrtions thereof in a variety of expression systems including bacteria, yeast, algae, plant, rm mmalian cell or transgenic animal systems. In addition, serum albumin or portions thereof an be produced in cell-free systems or chemically synthesized. Recombinant human albt min 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 mer ibers of the family (e.g. a-fetoprotein, vitamin D binding protein, or afamin) may substitute for albumin in compositions and methods contemplated herein. Those skilled in the ar t will also recognize that modifications can be made to albumin by any means known in the a: t, for example, by recombinant DNA technology, by post-translational modification, by ] iroteolytic cleavage and/or by chemical means. Those substitutions and alterations to albumir that provide essentially equivalent stabilizing function to serum albumin without substil utions and alterations are contemplated herein. In certain embodiments, compositions having a high molecular weig ht 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 c an include, but are not limited to, monosaccharides, (e.g. glucose, galactose, ribose, muinose, rhamno.se, talose, xylose or allose arabinose), disaccharides (e.g. trehalose, sucrose, maltose, isomaltose, cellibiose, gentiobiose, laminaribose, xylobiose, mannobiose, lactose, cr 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 chi :in). Polyols can include, but are not limited to, mannitol, sorbitol, arabitol, erythritol, rx altitol, xylitol, glycitol, glycol, polyglycitol, polyethylene glycol, polypropylene glycol, ar d 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 pharmaco ogically acceptable buffer. In certain embodiments, trehalose can be present at concentratic ns 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 nari ire to protect organisms from stress. Anhydrobiotic organisms that can tolerate low wate r conditions contain large amounts of trehalose. Trehalose has been shown to prevent both membrane fusion events and phase transitions that can cause membrane destabiliza tion during drying. Structural analysis suggests that trehalose fits well between the poh r head groups in lipid bylayers. Trehalose also prevents denaturation of labile proteins duing drying. It is thought that trehalose stabilizes proteins by hydrogen bonding with pc lar 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 educing power and is thus essentially non-rcactivc 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 ECK 'O 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 vi ais stability. In certain embodiments, compositions can be described that typicall y include a physiologically acceptable buffer. Those skilled in the art recognize that a variety i )f 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 pai n 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 equivalen t 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-bi iffered saline (PBS). Some live, attenuated "viral vaccine compositions herein concern cor ipositions that increase stability and/or reduce deterioration of live, attenuated virus in addition to having reduced immunogenicity or are non-immunogenic. In accordance with thes<: embodiments compositions can include one or more protein agents st ccharides polyols and high molecular weight surfactants wherein the composition decreases inactivation of live attenuated virus. therefore certain contemplated herein have reduced adverse reaction when administered to a subject. in some exemplary surfactant agent consists eo-po block copolymers are selected from group consisting lactalbumin serum albumin a-fetoprotein vitamin d binding afamin derived vertebrate species carbohydrate is saccharide anc polyol. trehalose sucrose chitosan sorbitol> mannitol. In certain more particular embodiments, in order to reduce immune reac:ion 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 embodirr ents, the carbohydrate agent is trehalose. In certain embodiments, at least one surfactant agent is the EO-PO block copolymer Pluonic F127. In some live, attenuated viral vaccine com] tositions at least one carbohydrate agent is trehalose. In certain live, attenuated viral vaccine compositions include, the EO-PO block copolymer Pluronic F127 where the conce ltration 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 ac ministered in which any toxic effects are outweighed by the therapeutic effects of the ac live 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 achiev<: a desired result. for example therapeutically active amount of compound may vary cording to factors such as the disease state age sex and weight individual ability formulations elicit response in individual. dosage regima be idjusted provide optimum therapeutic response.> In some embodiments, composition (e.g. pharmaceutical chemical, protein, peptide of an embodiment) may be administered in a convenient manne:- 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 en tbodiment, 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 aqueaus buffer solutions. The active agent may also be administered parenterally or intrap ;ritoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, ani mixtures thereof and in oils. Under ordinary conditions of storage and use, these prepar itions may contain a preservative to prevent the growth of microorganisms. Pharmaceutical compositions suitable for injectable use may be ac ministered 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 md can be fluid to the extent that easy syringability exists. It may further be preserved iigainst the contaminating action of microorganisms such as bacteria and fungi. The pharm aceutically acceptable carrier can be a solvent or dispersion medium containing, for example, water, cthanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene 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 ngredients 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 fo rmulations 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 mici ©particles, 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 c an include about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to ] .0 or even about 1 to 10 gram per dose. Single dose or multiple doses can also be administ ;red 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 inhala nts 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 miy 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 Vo 2%. Oral formulations include such normally employed excipients as, fcr example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. In certain embodiments, oral phaimaceutical compositions can include an inert diluent or assimilable edible carrier, or may be i snclosed in hard or soft shell gelatin capsule, or may be compressed into tablets, or may be ir corporated 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, bu xal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compo jitions 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 1 >e 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 tie 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 on s 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, :he 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. EXAMPLES The following examples are included to demonstrate certain embodiments presented herein. It should be appreciated by those of skill in the art that the techniques disclosed in the Examples which follow represent techniques discovered to function well in the practices disclosed herein, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are Example 1 Base stability of DEN-2 PDK 53 flavivirus in liquid phase In one illustrative method, the thermal stability for flaviviruses in lie uid phase was investigated. In accordance with this method, the base stability of the DEN-2 IDK 53 parental vaccine vector, stored in phosphate buffered saline (PBS), at different temperatures was determined (Table 1). In one example, lxlO4 pfu of DEN-2 PDK 53 virus in a total volume of 0.5ml PBS was incubated, in 2ml screw capped vials at either 4° C, room temperature (~ 21° C) or 37° C. After 24 hours of incubation viral titer and activity was determined by a Neutral Red agarose overlay plaque titration assay in Vero cells. A s illustrated in Table 1, incubation of DEN-2 PDK 53 in PBS at 4° C results in an awrage fourfold decrease in viral titer and complete loss in viral activity when incubated at 37° C for the same period. These results demonstrate the relatively poor stability of the DEN-2 PDK 53 flavivirus in the absence of stabilizing excipients. Table 1 Stability of Den-2 PDK53 vims stored for 24 hours at different temperatures. (Table Removed) Exampile 2 Stabiliz ng Effects of Compositions In certain exemplary compositions, pharmaceutically acceptable exc ipients contemplated herein that aid in thermal stability of live viral vaccines are known in the art. In one exemplary method, PBS was used as a base composition to assess the stabilizing effects of different excipients. In these examples, a stock solution of each excipient was mide in PBS and the pH adjusted to approximately 7.1 with NaOH, except for chitosan whore the pH of the stock solution was adjusted to approximately 6.8. Excipients were diluted in PBS to the final concentrations indicated (w/v) (Table 2). In accordance with this method, lxl d4 pfu of DEN-2 PDK 53 virus, in serum-free medium, was added to 0.5ml of each composiiion and stored a: 37° C for 24 hours. Following incubation, viral activity and titer was deteimined by plaque titration in Vero cells, as described above. As illustrated in Table 2, the stabilizing effects of compositions including a single excipient, at various concentrations comparable to previous experimental examples, was minimal. However, some excipients for example, trehalose and recombinant human serum albumin (rHSA), were more effective thai others at stabilizing DEN-2 PDK 53 virus at 37° C. Results of the study represented in Table 2 also revealec. that increased stabilizing effects of several excipients, including rHSA and trehalose, can be obtained within certain ranges of concentrations of these excipien :s. In this particular example, trehalose was more effective at concentrations above 15% (w/v) and F127 at concentrations between 0.5 and 3%. Table 2 Effects of different excipients; on DEN-2 PDK53 stability when stored at 37° C for 24 hours (Table Removed) Example 3 Stabilizing effects of compositions including specific combinations of excipients In the following illustrative procedure, compositions including mult, pie excipients in differing combinations and concentrations were tested for stabilizing a pluronic co-polymer non-ionic surfactant and a protein were optimal at improving DEN-2 PDK 53 stability at 37° C. Formulations including trehalose, F127 and rHSA had tie greatest stabilizing effects. Unexpectedly, the combined stabilizing effect of these three excipients was much greater than the sum of that observed with each individual component suggesting synergism between the components. Improved thermal stability of the DEN-2 PDK 53 flavi vims was obtained through the synergistic activities of the combination of trehalose, F127 and rHSA could not have been anticipated based on prior art examples. Figs '. and 2 also illustrate that the stabilizing effect of the trehalose/F127/rHSA mixture was fuither enhanced by the addition of 0.05% chitosan. Fig. 2 shows that the rate of viral inacivation when stored over a 48 hour period at 37° C is significantly reduced by composition! containing trehalose, F127 and rHSA. Examples in the art suggest that the stability of flaviviruses can be enhanced by formulations containing Ca 2+ and Mg 2+ divalent cations. However, as represented in Figs. 1 and 2, the addition of Ca 2+ (0.0009M) and Mg '+ (0.0005M) to a formulation confers no additional stabilizing benefits. The results fiom Fig. 2 suggest that addition of divalent cations may have a negative impact to long term li :mid phase viral stability in the context of particular embodiments. In one exemplary method, a composition including trehalose, F127 rnd rHSA was assessed for its stabilizing properties with multiple flaviviruses. The stability o f chimeric DEN-2 flaviviruses expressing the membrane and envelope proteins from either W;st Nile (DEN-2/WN), Dengue 1 (DEN-2/D1), Dengue 3 (DEN-2/D3, or Dengue 4 (DEN-2 /D4) viruses was determined as described for Example 1. Illustrative results in Table 3 rsveal greatly improved liquid phase stability of all the chimeric flaviviruses when stored n a composition including trehalose, F127 and rHSA. The different chimeras express d fferent envelope and membrane proteins from five serologically distinct flaviviruses. In ac dition, West Nile virus and the dengue viruses are significantly divergent. This result suggests that compositions herein may be useful for liquid phase stabilization of diverse member j of the family of Flaviviradae as well as other virus families. The ability to stabilize flaviviruses at room temperature (~21° C) and at 4° C was examined by representative procedures as outlined for Example 1. The exemplary results, illustrated in Table 4, reveal that a composition including trehalose, F127 and rHSA effectively preserves viral activity for 7 days at 21 ° C and for 48 days at 4° C. Table 3. Stability of different chimeric flaviviruses stored at 37° C for 21 hours in 5BS or a composition (Fl) including 15% trehalose, 2% F127 and 1% rHSA. (Table Removed) Table 4. Stability of flaviviruses stored at different temperatures for 7 or 48 days in PBS or a compos ition (F1) including 15% trehalose, 2% F127 and 1 % rHSA. (Table Removed) Examplle 4 Use of alternate components Another exemplary method was used to compare the stabilizing effe cts of bovine serum albumin (BSA) and, gelatin, to that of rHSA and of different pluroni F127. Thus, while proteins other than rHSA may be used in combination with treru lose and Fl 27 to aid in stabilization of flaviviral vaccines, the use of a serum albumin or closely related proteins is more suitable in accordance with this exemplary method. In addition, Fig. 3 illustrates that, as isolated excipients, the polymer Pluronic PI23 is comparable to Pluronic Fl27 in its ability to stabilitze the DEN-2 PDK-53 virus. However, in this exemph ry method. PI23 does not appear to be an effective subsititute for F127 in compositions also containing trehalose and serum albumin. As exemplified in Fig. 4, compositions containing trehalose, rHSA and other commonly used pharmaceutical surfactants such as Poly sorbate 20 (Tween 20), instead of a pluronic co-polymer, are not effective in stabilizing DEN-2 PDK 53 relative to formulations containing a pluronic co-polymer. These exemplary methc ds suggest better stabilizing efficiencies of formulations containing distinct high molecular we ight pluronic: co-polymer surfactants. Exemplary data is further illustrated in Fig 4. Fig. 4. represents stability of the DEN-2 PDK 53 virus in compositions containing different surfactants. DEN-2 PD C 53 was stored at 37° C for 23 hours in each formulation. Surfactants evaluated in this example include n-octyl-p-D-glucopyranoside (P -OG), Polysorbate 20 (P 20), Polysorbate i 10 (P 80 ) and F127 (F). Other formulation components include trehalose (T) and rHSA (A). Values are expressed as a percentage of the viral titer remaining after incubation relative to the input titer. Examplle 5 Comparison of the stabilizing effects of different compositions The stabilizing properties of one exemplary composition were compared to that of compositions known in the art. A stabilizing composition for live flaviviral vaccines, disclosed in the art (U.S. Pat. No. 4,500,512), includes 4% lactose, 2 % sorbitol, 0. g/L CaCl2, 0.076 MgS04 and amino acids on the order of 0.0005M to 0.05M in PBS. Another composition reported by Adebayo et al (1998) consists of 10% sucrose, 5% lactalbumin, 0. lg/L CaCb, and 0.076 g/L MgSO Adebayo, et al. lxlO4 pfu of DEN-2 PDK 53 vaccine virus were incubated at 37° C in 0.5ml of each composition for 23 hours, after which viral activity and titer was assayed as described in Example 1. As exemplified in Fig. 5, some embodiments, for example formulation Fl, represents a significant improvement over those previously described compositions. In the example shown, virtually no viral activity was recovered after storage in the formu ations known in the art (formulations F3 and F4), whereas upwards of 50% of the initial viral titer was recovered after storage in a composition disclosed herein. These results reveal that previous formulations are ineffective at promoting live viral vaccine stability durin % liquid phase storage. Examplle 6 Preservation of viral activity after multiple freeze-thaws In one exemplary method, the ability of select compositions to preserve viral activity after freeze-thaw cycles was demonstrated. lxlO4 pfu of DEN-2 PDK 53 vuccine virus was suspended in 0.5 ml of each composition in screw cap vials. For the first freeze-thaw cycle vials were frozen at -80° C for 24 hours and thawed rapidly at 37° C. This was immediately followed by a second freeze-thaw cycle where the vials were frozen al -80° C for 1 hour and thawed rapidly at 37° C. Viral titer and activity was then assessed by a plaque titration assay as described in Example 1. As illustrated in Fig. 6, particular compof itions that include trehalose, F127 and rHSA effectively preserved full viral activity through two freeze-thaw cycles. Additionally, compositions including these three excipients were more effective than those containing just a single excipient. The results of this particular illustrative experiment suggest the compositions and methods disclosed herein are an effective cryoprotectant for flaviviral vaccines and may facilitate viral preservation during fr;eze-drying, spray-drying, or other dehydration techniques. Example 7 Stabilization of other live, attenuated viruses. Examples illustrated previously reveal effective liquid phase stabiliz ition of several live, attenuated flaviviruses in compositions including trehalose, F127 and iHSA. It is anticipated that embodiments disclosed herein may also be effective at stabilizing o ther live, attenuated viruses. For example, a formulation including trehalose, F127 and rHSA may be used to stabilize live attenuated measles virus, an attenuated sindbis virus, an attenuated influenza virus, a recombinant, attenuated adenovirus or a recombinant, attenuated vaccinia virus. In one exemplary method, these non-flaviviral viruses can be suspended and maintained in liquid phase, in a composition including trehalose, F127, and rHSA directly after hai-vesting from cell culture. In another illustrative method, non-flaviviral vin ses can be suspended in a composition prior to, or subsequent to, freeze or spray-drying. Statistically improved viral stability may demonstrate that the formulation of this embodiment is applicable to other attenuated viral vaccines outside of the Flavivirus family. Those skilled in the art recognize that application may then be extended to other live, attenuated vir ises. Example 8 Safety and in vivo immunogenicity. Molecular interactions between excipients and molecular or cellular components may serve, not only to enhance stability of viral vaccines, but also to ciuse increased cell or tissue damage in vivo. Formulations may decrease the immunogenicity of these viral vaccines in live animals. In this example, it is demonstrated that exemp ary compositions are safe after subcutaneous injection and are essentially immunologically inert. Four different exemplary compositions were selected for testing in mice as follow:. Formulation 1: 15% Trehalose, 2% F-127, 1 % rHSA Formulation 2: 15% Trehalose, 2% F-127, 1% rHSA, ImM CaCl2/ 0.5mM MgS04 Formulation 3: 15% Trehalose, 2% F-127, 1% rHSA, 0.5% chitosan Formulation 4: 22.5% Trehalose, 3% F-127, 1.5% rHSA Formulation 5: PBS In certain methods described herein, groups of 8 or 9 NIH Swiss mice were immunized by subcutaneous injection with 1x10s pfu of a formulated DEN-2 PDK- 53/WN recombinant flavivirus vaccine at day 0 (d0), were boosted with the same formulate d vaccine at d29 and were then challenged with 103 pfu on a pathogenic West Nile strain (N"V 99) on d45. Control mice (four groups of 8) received formulations 1—4 alone with no vir is. No adverse events after administration in any of the immunized mice were observed. Thus, in this example, no apparent adverse events are caused by the exemplary formulations with or without vaccine virus. Sera were collected prior to immunization at dO, prior to boost at d28, prior to challenge at d44 and post-challenge at d75. West Nile neutralizing antibod y titers in the sera were determined by plaque reduction neutralization test (PRNT). The results of the study are represented in Table 5. Table 5: Neutralizing antibody and protection induced by formulated DEN2/\^N vaccines 1 GMT = geometric mean titer; titers of 10. A majority of the animals receiving the DEN-2/WN vaccine sero-co nverted after the first dose regardless of whether no formulation (Formulation 5) or one of the exemplary formulations (Formulations 1 - 4) was used. In addition, all of the vacc nated animals sero-converted after the booster administration. Geometric mean PRNT titers (GMT) demonstrate few differences between the vaccine groups. Titers were low * iter the primary immunization, increased 3-10 fold after the boost and then showed a drariatic anamnestic response upon challenge. 100% of all the vaccinated animals survived challenge, again independent of vaccine formulation. Only 22% of the control animals survived; those that did survive showed evidence of potent neutralizing antibody responses after challenge. One advantage is that this example demonstrates that the exemplary formulations d 3 not reduce the ability of an exemplary recombinant DEN-2/WN vaccine to prevent Wert Nile disease in a mice. Example 9 In another example, liquid compositions were used containing trehalose, rHSA and F127 to stabilize a West Nile chemeric flavivirus stored for various periods at e ither 25°C or 4°C. IxlO4 pfu of chimeric DEN-2/WN vaccine virus were incubated at each ten perature and viral activity was assessed at one or two week intervals as described in Exampl; 1. As illustrated in Figs.7 and 8, formulations containing trehalose, rHSA and F127 signiiicantly improved the thermal stability of the DEN-2/WN vaccine virus during storage at 2*°C and 4°C, respectively. At 25°C loss of viral activity was less than one log over 7 days. \t 4°C viral inactivation was negligible for periods up to 12 weeks when stored in exempl try formulations including trehalose, F127 and rHSA. Example 10 In another exemplary method, stabilizing effects of compositions were demonstrated including trehalose, rHSA and a pluronic co-polymer with dehydrated DEN-2 PDK 53 vaccines, lxl 04 pfu of DEN-2 PDK 53 vaccine virus formulated in accordance with procedures disclosed herein. Formulated vaccines were placed in serum vials and s lbjected to conventional lyophilzation procedures. Dried vaccines were stoppered under vacuum, stored at either 37°C or 4°C for 14 days followed by reconstitution of the vaccine to its original liquid volume by addition of sterile water. Viral activity of the reconstituted vaccina was assessed as outlined earlier. At 37°C, in the presence of compositions containing trehalose, rHSA and a pluronic co-polymer formulated in phosphate buffered saline, an average viral titer loss of 1 log was observed (Fig. 9). No loss in viral activity was observed for formulated dehydrated DEN-2 PDK 53 viral vaccines stored at 4°C for 14 days. These results demonstrate effective preservation of a dehydrated viral vaccine utilizing compositions disclosed herein. Fig. 9. represents stability of lyophilized DEN-2 PDK 53 at differen; temperatures. Log titer loss of formulated lyophilized DEN-2 PDK 53 vaccine virus following incubation at 37°C or 4°C for 2 weeks as indicated. Formulations Fl (15% trehalose, 2% F127, 1% rHSA) and F2 (15% trehalose, 2% F127, 0.01% rHSA) were formulated in phosphate buffered saline. Formulation F3 (15% trehalose, 2% F127. 0.01% rHSA) was formulated in 10 mM Tris base. All of the COMPOSITIONS and METHODS disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods have been described in terms of preferred embodiments, it is apparent to those of skill in the art that variations maybe applied to the COMPOSITIONS and METHODS and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope herein. More specifically, cen ain agents that are both chemically and physiologically related may be substituted for the agei ts described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept as defined by the appended claims. All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety. We claim: 1. A live attenuated virus composition comprising: one or more live, attenuated viruses; one or more high molecular weight surfactant agents wherein the high molecular weight surfactant agents have a molecular weight of 1500 or greater; one or more proteins agents, the one or more protein agents are selected from the group consisting of lactalbumin, serum albumin, α-fetoprotein, vitamin D binding protein, afamin derived from a vertebrate species and a combination thereof, and one or more carbohydrate agents, the one or more carbohydrate agent are selected from the group consisting of trehalose, sucrose, chitosan, sorbitol, mannitol and a combination thereof, wherein the composition is capable of reducing the inactivation of the live attenuated virus. 2. The virus composition of claim 1, wherein the live, attenuated viruses are selected from the group consisting of Flavivirus, Togavirus, Coronavirus, Rhabdovirus, Filovirus, Paramyxovirus, Orthomyxovirus, Bunyavirus, Arenavirus, Retrovirus, Hepadnavirus, Herpesvirus, Poxvirus families and combinations thereof. 3. The virus composition of claim 1, wherein the live, attenuated viruses are Flaviviruses. 4. The virus composition of claim 1, wherein the composition is in aqueous form. 5. The virus composition of claim 1, wherein the composition is partially or wholly dehydrated. 6. The virus composition of claim 1, wherein the one or more high molecular weight surfactant agents further comprises one or more co-polymers wherein the molecular weight of the high molecular weight surfactant agent is 3000 or greater. 7. The virus composition of claim 1, wherein the surfactant agent(s) consists of one or more EO-PO block copolymers. 8. The virus composition of claim 1, wherein the protein agent(s) is serum albumin derived from a vertebrate species. 9. The virus composition of claim 1, wherein one of the one or more carbohydrate agent is trehalose. 10. The virus composition of claim 1, wherein at least one high molecular weight surfactant agent is an EO-PO block copolymer, at least one protein agent is serum albumin, and at least one carbohydrate agent is trehalose. 11. The virus composition of claim 10, wherein the EO-PO block copolymer concentration is less than from 4% (w/v), wherein the one or more protein concentration is from 0.001 to 3% (w/v) and the one or more carbohydrate concentration is from 5 to 50% (w/v). 12. A method for decreasing inactivation of a live, attenuated virus composition comprising, combining one or more live attenuated viruses with a composition comprising one or more high molecular weight surfactant agents wherein the high molecular weight surfactant agents have a molecular weight of 1500 or greater, one or more protein agents, the one or more protein agents are selected from the group consisting of lactalbumin, serum albumin, α-fetoprotein, vitamin D binding protein, afamin derived from a vertebrate species and a combination thereof, and one or more carbohydrates, the one or more carbohydrate agent are selected from the group consisting of trehalose, sucrose, chitosan, sorbitol, mannitol and a combination thereof, wherein the composition is capable of reducing inactivation of the live, attenuated virus. 13. The method of claim 12, wherein the live, attenuated viruses are selected from the group consisting of Flavivirus, Togavirus, Coronavirus, Rhabdovirus, Filovirus, Paramyxovirus, Orthomyxovirus, Bunyavirus, Arenavirus, Retrovirus, Hepadnavirus, Herpesvirus, Poxvirus families and combinations thereof. 14. The method of claim 12, further comprising partially or wholly dehydrating the combination. 15. The method of claim 14, further comprising partially or wholly re-hydrating the composition prior to administration. 16. The method of claim 12, wherein the composition increases the shelf life of a virus composition when the composition is maintained at room temperature or lower compared to a control composition. 17. The method of claim 12, wherein the composition decreases inactivation of an aqueous live, attenuated virus for 24 hours or greater. 18. The method of claim 12, wherein the composition decreases inactivation of an aqueous live, attenuated virus during one or more freeze and thaw cycles compared to a control composition. 19. The method of claim 12, wherein at least one high molecular weight surfactants is an EO-PO block copolymer polyxamer 407, at least one of the protein agents is serum albumin, and at least one of the carbohydrate agents is trehalose. 20. The method of claim 12, wherein the virus composition reduces the onset of or prevents a health condition selected from the group consisting of West Nile infection, Dengue fever, Japanese encephalitis, Kyasanur Forest disease, Murray Valley encephalitis, St. Louis encephalitis, Tick-borne encephalitis, Yellow fever and Hepatitis C Virus Infection. 21. A kit for decreasing the inactivation of a live, attenuated virus composition comprising: at least one container; and a composition comprising one or more protein agents, the one or more protein agents are selected from the group consisting of lactalbumin, serum albumin, α-fetoprotein, vitamin D binding protein, afamin derived from a vertebrate species and a combination thereof, one or more carbohydrate agents, the one or more carbohydrate agents are selected from the group consisting of trehalose, sucrose, chitosan, sorbitol, mannitol and a combination thereof, and one or more high molecular weight surfactants wherein the high molecular weight surfactant agents have a molecular weight of 1500 or greater, wherein the composition decreases inactivation of a live, attenuated virus. 22. The kit of claim 21, wherein at least one surfactant agent is an EO-PO block copolymer poloxamer 407, at least one protein agent is serum albumin, and at least one carbohydrate agent is trehalose. 23. The kit of claim 22, wherein the trehalose concentration is from 5 to 50% (w/v). 24. The kit of claim 22, wherein the EO-PO block copolymer poloxamer 407 concentration is from 0.1 to 4% (w/v). 25. The kit of claim 22, wherein the serum albumin concentration is from 0.001 to 3% (w/v). 26. The kit of claim 21, wherein the composition further comprises one or more live, attenuated viruses. 27. The kit of claim 26, wherein the live, attenuated viruses are selected from the group consisting of Flavivirus, Togavirus, Coronavirus, Rhabdovirus, Filovirus, Paramyxovirus, Orthomyxovirus, Bunyavirus, Arenavirus, Retrovirus, Hepadnavirus, Herpesvirus, Poxvirus families and combinations thereof.

Full Text

Priority
This application claims the benefit of priority of provisional U.S. patent application No. 60/910,579, filed on April 06, 2007, which is incorporated herein in its entirety.
Field
Embodiments herein relate to compositions and methods for stabilizing live, attenuated viruses. Other embodiments relate to compositions and methods for redi cing degradation of live, attenuated viruses. Still other embodiments relate to uses of these compositions in kits for portable applications and methods.
Background
Vaccines to protect against viral infections have been effectively used to rec uce the incidence of human disease. One of the most successful technologies for viral vacc ines is to immunize animals or humans with a weakened or attenuated strain of the virus (a " ive, attenuated virus"). Due to limited replication after immunization, the attenuated sti ain does not cause disease. However, the limited viral replication is sufficient to express the full repertoire of viral antigens and generates potent and long-lasting immune response;. to the virus. Thus, upon subsequent exposure to a pathogenic strain of the virus, the imn unized individual is protected from disease. These live, attenuated viral vaccines are amon \ the most successful vaccines used in public health.
Ten of the sixteen viral vaccines approved for sale in the U.S. are live, attenuated viruses. Highly successful live viral vaccines include the yellow fever 17D virus, Sabin poliovirus types 1, 2 and 3, measles, mumps, rubella, varicella and vaccinia viruses. Use of the vaccinia virus vaccine to control smallpox outbreaks led to the first and only eradication of a human disease. The Sabin poliovirus vaccine has helped prevent crippling dis Recent technical advances, such as reassortment, reverse genetics and cold adaptation, have led to the licensure oflive, attenuated viruses for influenza and rolavirus. A
number of live, viral vaccines developed with recombinant DNA technologies are ii human clinical testing, including vaccines for West Nile disease, dengue fever, malaria, tuberculosis and HIV. These recombinant viral vaccines rely on manipulation of well-characterized attenuated viral vaccines, such as adenovirus, vaccinia virus, yellow fever 17D or the dengue virus, DEN-2 PDK-53. The safe, attenuated viruses are genetically engineered to express protective antigens for other viral or bacterial pathogens. Several recombinant vira [ vaccines have been approved for animal use, including a canarypox/feline leukemia recombi nant virus, a canarypox/canine distemper recombinant virus, a canarypox/West Nile recombin; tnt virus and a yellow fever/West Nile recombinant virus. As a group, the live attenuated vi -us vaccines are amongst the most successful medical interventions in human history, second only to the advent of antibiotics and hold the promise to improve public health throaghout the world.
In order for live, attenuated viral vaccines to be effective, they must be caps ble of replicating after immunization. Thus, any factors that inactivate the virus can cripp le the vaccine. For example, widespread distribution and use of the smallpox vaccine prior to World War II was limited because the virus was inactivated after only a few days a: ambient temperatures. In the 1920s, French scientists demonstration that freeze-dried vacci le provided long term stability and techniques for large-scale manufacture of freeze-d -ied vaccine were developed in the 1940s (see for example Collier 1955). In addition to freeze-drying, various additives have been identified that can help stabilize the viruses in live, attenuated viral vaccines (See for example Burke, Hsu et al 1999). These stabilizers typically include one or more of the following components: divalent cations, buffered salt solutions, chelators, urea, sugars (e.g. sucrose, lactose, trehalose), polyols (e.g., glycerol, mannitol, sorbitol, polyethylene glycol), amino acids, protein hydrolystates (e.g. casein hydrclysate, lactalbumin hydrolysate, peptone), proteins (e.g. gelatin, human serum albumin) or polymers (e.g. dextran).
However, even with these stabilizing agents, many of the commonly used viccines still require refrigeration for stabilization. Other commonly used vaccines are sensitive to temperature extremes; either excessive heat or accidental freezing can inactivate thu vaccine. Maintaining this "cold chain" throughout distribution is particularly difficult in the developing world. Thus, there remains a need for improving the stability of both existing and newly developed live, attenuated viral vaccines.
Fl aviviruses are amongst the most labile viruses. They are enveloped virusod vector and their vertebrate host species (often birds or mammals). Expanding urbanizatioi i, worldwide travel and environmental changes (such as deforestation or rain patterns i have lead to the emergence of several flavhiruses as threats to human public health. Sue h viruses include, but are not limited to, yellow fever virus, the dengue viruses, West Nile viius, Japanese encephalitis virus, and tick-borne encephalitis viruses.
Through intensive mosquito control and vaccination efforts, yellow fever w is eliminated from much of North, Central and South America, the Caribbean and Euiope. However, in the last 20 years, the number of countries reporting cases has increases. Yellow fever virus is now endemic in major portions of Africa and South America and some Caribbean islands. The World Health Organization (WHO) estimates that 200,000 cases of yellow fever occur annually leading to 30,000 deaths. Since World War II, dengue flaviviruses have spread to tropical and subtropical regions throughout the world an d now threaten over 3.5 billion people, about half of the world's population. The WHO e jtimates that 50-100 million cases of dengue fever occur annually. 500,000 of these are the nore sever, life-threatening form of the disease, termed dengue hemorrhagic fever, that leads to more than 25,000 deaths per year. A particularly virulent form of West Nile virus \ /as introduced into the Western hemisphere, presumably by travel, in New York in 199 9. The mosquito-transmitted virus infected birds as the primary host, but also caused disea >e and mortality in humans and horses. West Nile virus spread throughout the United Sta :es and i nto Canada and Mexico. Since its introduction, West Nile virus has caused over 2i ),000 reported cases of West Nile disease leading to 950 deaths in the United States. Jap anese encephalitis virus causes 30,000 to 50,000 cases of neurological disease annually, primarily in eastern and southern Asia. 25-30% of the reported cases are fatal. The tick-borne encephalitis viruses are endemic to parts of Europe and Asia and continue to cause episodic outbreaks affecting thousands of individuals. Related viruses with more limited geographical spread include Kunjin virus (a close relative of West Nile) and Murray Valley enceihalitis
virus in Australia and New Guinea, St. Louis encephalitis virus in North and South America, the Usutu, Koutango, and Yaonde viruses in Africa, and Cacipacore virus in South American.
Live, attenuated viral vaccines have been developed that are safe and protect against flavivirus diseases, such as yellow fever and Japanese encephalitis. The livs, attenuated viral vaccine, 17D, has been widely used to prevent yellow fever. The c arrent flavivirus vaccines are lyophilized in the presence of stabilizers. Nonetheless, the vaccines require storage and shipment at 2 — 8° C, a requirement that is difficult to achieve it. the developing world and more remote regions of developed nations. Furthermore, upon reconstitution, the vaccines rapidly lose potency even when stored at 2 - 8° C.
The measles vaccine is another example of a labile attenuated virus i hat is used worldwide to prevent disease. Measles virus is an enveloped, non-segmented negative strand RNA virus of the Paramyxovirus family. Measles is a highly contagious, seasonal disease that can affect virtually every child before puberty in the absence of vaccins.tion. In developing countries, mortality rates in measles-infected children can by as high as 2 to 15%. Indeed, despite efforts to institute worldwide immunization, measles still causes greater than 7,000 deaths in children per year. The measles vaccine is a live, attenuated virus that is manufactured in primary chicken fibroblast cells. The vaccine is stabilized with ge latin and sorbitol and is then lyophilized. The stabilized, lyophilized vaccine has a shelf life :>f 2 years or more if stored at 2 to 8° C. However, the lyophilized vaccine still requires a cold chain that is difficult to maintain in the developing world. Furthermore, upon reconstitutian, the vaccine loses 50% of its potency within 1 hour at room temperature (20 to 25° C).
Thus, a need exists in the art for improved vaccine formulations. SUMMARY
Embodiments herein concern methods and compositions to reduce o • 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 method;, 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 viruse s 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, attenuited 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 limi ed 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 val ey encephalitis, Alkhurma hemorrhagic fever, St. Louis encephalitis, tick-borne encep lalitis, 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 inclu ie, but are not limited to, monosaccharides, disaccharides, sugar alcohols, trehalose, sucrose, rialtose, isomaltose, cellibiose, gentiobiose, laminaribose, xylobiose, mannobiose, lactose, fiuctose, sorbitol, mannitol, lactitol, xylitol, erythritol, raffinose, amylse, cyclodextrins, chitcsan, 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(ethylenc oxide) and polypropylene oxide) (EO-PO block copolymers ), poly(vinyl pyrroloidone), alkyl polyglucosides (such as sucrose monostearate, lauryl diglucoside, or sorbitan mono aureate, 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 limite d to, the Pluronic F127, Pluronic F68, Pluronic P123, or other EO-PO block copolymers of greater than 3,000-4,000 MW.
In some embodiments, vaccine compositions can include, but are nc t limited to, one or more protein agent that is serum albumin; one or more saccharide agent 1 hat is trehalose; and one or more surfactant polymer agent that is the EO-PO block copolymer Pluronic F127.
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 attenu* ted viruses with a composition capable of reducing inactivation of a live, attenuated viius including, but not limited to, one or more protein agents; one or more saccharides cr 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, Bunyavirui;, Arenavirus, Retrovirus, Hepadnavirus, Pestivirus, Picornavirus, Calicivirus, Reovirus, Parvovirus, Papovavirus, Adenovirus, Herpes virus, or a Poxvirus. Additionally, m ;thods and compositions disclosed herein can include freeze drying or other dehydrating methods for the combination. In accordance with these methods and compositions, the met! ods and compositions decrease inactivation of the freeze dried or partially or wholly dehydi ated live attenuated virus. In other methods, compositions for decreasing inactivation of a live attenuated virus may comprise an aqueous composition or may comprise a rehydrc ted 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 11 vaccine composition contemplated herein may include, but is not limited to, one or more Irs e, 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-4A30) 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° to about 25° C) or refrigeration temp matures (e.g. about 0° to about 10° C). In more particular embodiments, a combination composition is capable of maintaining about 100 percent of the live attenuated virus for greater ths n 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° to about 10° C). Compositions contemplated in these methods, can include, but are not limited to, one or more protein agent of serum albumin; on; or more saccharide agent of trehalose; and one or more EO-PO block copolymer agent of P uronic F127. In certain embodiments, the live, attenuated virus composition remains at at out 100% viral titer after 7 days at approximately 21° C and about 100% viral titer after 50 days at refrigeration temperatures around 4° C. Other embodiments herein may include liv<: attenuated virus composition remaining at about or viral titer aftei days approximately c and after refrigsration temperatures around c. other embodiments contemplated include live compositions to lox the concentration of affc several hours compared knov in art. for example figs. disclosed herein reduce degradation when is stored> 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 oi 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 thesi; embodiments, a kit composition may include one or more one protein agent 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, Corot.avirus, Rhabdovirus, Filovirus, Paramyxovirus, Orthomyxovirus, Bunyavirus, Arenavirus, Retrovirus, Hepadnavirus, Pestivirus, Picornavirus, Calicivirus, Reovirus, Parvovixis, Papovavirus, Adenovirus, Herpes virus, or Poxvirus. In certain embodiments, compositions herein can include trehalose as a saccharide agent. In accordance with these emboc intents, trehalose concentration may be equal to or greater than 5% (w/v). In certain emboc [intents, compositions herein can include polymer F127 as an EO-PO block copolymer ager t. In accordance with these embodiments, polymer F127 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 m ay have trace amounts or no calcium/magnesium (Ca+2/Mg+2).
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the instant specification and are included to further demonstrate certain aspects of particular embodiments herein. The embodiments n ay be better understood by reference to one or more of these drawings in combination wilh the detailed description presented herein.
Fig.l represents an exemplary histogram of experiments using various compositions for testing the stability of an exemplary virus, DEN-2 PDK 53 flavivirus, in the
compositions.
Fig. 2 represents an exemplary graph of a kinetic analysis of an exenplary virus, DEN-2 PDK 53 flavivirus, for viral inactivation at 37° C in various exemplar/
compositions.
Fig. 3 represents an exemplary histogram of an analysis of an exemi lary virus, DEN-2 PDK 53 virus, stored at 37°C for 21 hours. Values are expressed as a percentage of the viral titer remaining after incubation relative to the input titer. Formulation percentages refer to (w/v) of the respective excipient.
Fig. 4 represents an exemplary histogram of an analysis of an exemt lary virus, DEN-2 PDK 53 virus, stored at 37° C for 23 hours in different compositions. Value* are
expressed as a percentage of the viral titer remaining after incubation relative to the input
titer.
Fig. 5 represents an exemplary histogram of an analysis of an exemplary virus, DEN-2 PDK 53 virus, stored at 37° C for 23 hours in different compositions. Values are expressed as a percentage of the viral titer remaining after incubation relative to th Fig. 6 represents an exemplary histogram analysis of an exemplary virus, DEN-2 PDK 53 virus, after two freeze-thaw cycles when stored in different formulations. Values are expressed as a percentage of the viral titer remaining after freeze-thaw cycles relative to the input titer.
Fig. 7 represents an exemplary graph of a kinetic analysis of an exemplary virus, DEN-2 PDK 53/WN recombina.nt flavivirus, in various exemplary compositions for viral inactivation at 25° C over several weeks of time.
Fig. 8 represents an exemplary graph of a kinetic analysis of an exemplary virus, DEN-2 PDK 53/WN recombinant flavivirus, in various exemplary compositions for viral inactivation at 4° C over several weeks of time.
Fig. 9 represents an exemplary histogram analysis of an exemplary v irus, DEN-2 PDK-53 virus, after lyophilization in various exemplary compositions. Vira1 inactivation was assessed as described above after two weeks at different temperatires.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
Definitions
As used herein, "a" or "an" may mean one or more than one of an item.
As used herein, "about" may mean up to and including plus or mins five percent, for example, about 100 may mean 95 and up to 105.
As used herein, "saccharide" agents can mean one or more monosac;harides, (e.g. glucose, galactose, ribose, mannose, rhamnose, talose, xylose, or allose arabin :>se.), one or more disaccharides (e.g. trehalose, sucrose, maltose, isomaltose, cellibiose, genti obiose, laminaribose, xylobiose, mannobiose, lactose, or fructose.), trisaccharides (e.g. acaibose,
raffinose, melizitose, panose, or cellolriose) or sugar polymers (e.g. dextran, xanthi m, pullulan, cyclodextrins, amylose, amylopectin, starch, celloologosaccharides, cellu ose, maltooligosaccharides, glycogen, chitosan, or chitin).
As used herein, "polyol" agents can mean any sugar alcohol (e.g. mannitol, sorbitol, arabitol, erythritol, maltitol, xylitol, glycitol, glycol, polyglycitol, polyethylene glycol, polypropylene glycol, or glycerol).As used herein, "high molecular weight surfactants" can mean a surface active, amphiphilic molecule greater than 1500 molecular
weight.
As used herein, "EO-PO block copolymer" can mean a copolymer c insisting of blocks of poly(ethylene oxide) and poly(propylene) oxide. In addition, as used 1 erein, "Pluronic" can mean EO-PO block copolymers in the EOx-POy-EOx. This configuration of EO-PO block copolymer is also referred to as "Poloxamer" or "Synperonic".
As used herein, "attenuated virus" can mean a virus that demonstrati ;s reduced or no clinical signs of disease when administered to an animal.
DETAILED DESCRIPTIONS
In the following sections, various exemplary compositions and methods are described in order to detail various embodiments. It will be obvious to one skilled n 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 kncvTi methods or components have not been included in the description.
Stability of flavivirus vaccines has been assessed for both the existir g 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 c itions was demonstrated to stabilize the lyophilized vaccine, so that the vaccine lost less than '. log of potency after incubation at 37° C for 14 days. Stabilizing lyophilized formulations lor the yellow fever vaccine have been described (see for example U. S. Pat. No 4,500,512). U.S. Patent 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 jn 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/1 CaC12 and 0.076 g/1 MgS04 were found to provide the best stability (see for example Adebay o, 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 exampl; 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 flavirus vaccine for protection against Japa lese 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 or primary hamster kidney cells and the cell supernatant is harvested and coarsely filtered. Or e 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° C for at least 1.5 years, but for only 4 months at room temperature and 10 days at 37° C. As with the yellow ft ver vaccine, the reconstituted vaccine is very labile and is stable for only 2 hours at rocm temperature (see for example Wanf, Yang et al 1990). In certain embodiments heroin, live, attenuated flavirus virus compositions for stabilizing or reducing the degradation o 'Japanese encephalitis are contemplated.
No formulation for a live, attenuated flavivirus vaccine has been ide atified that provides long term stability of lyophilized formulations at temperatures greater tha:i 2 - 8° 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 basi; 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 a lergic reactions in immunized children and could be a cause of vaccine-related adverse e^ ents. U.S. patent 6,210,683 describes the substitution of recombinant human serum albumin f jr 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 pric r art. Certain compositions disclosed herein provide stability of aqueous viruses for up tc 2 hours; up to 3 hours; up to 4 hours and greater than 4 hours at or about 37° C. Certain compositions disclosed herein provide stability of aqueous viruses for up to 1 day to about 1 wee c or more, at or about room temperature {e.g. 25c 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 sta bilize, reduce deterioration and/or prevent inactivation of dehydrated live, attenuated viral products in room temperature conditions (e.g. about 25 ° C). In other embodiments, compositions contemplated herein can stabilize, reduce deterioration and/or prevent inactivation of aqueous live, attenuated viral products at about 25 ° C or up to or about 37 ° C. Composition s and methods disclosed herein can facilitate the storage, distribution, delivery and admii istration of viral vaccines in developed and under developed regions.
Other embodiments can include compositions for live attenuated vir is vaccines including, but not limited to, Picornaviruses (e.g., polio virus, foot and mc uth 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 en cephalitis 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 v rus, Newcastle disease virus, rinderpest vims), Orthomyxoviruses (e.g., human influen2 a viruses, avian influenza viruses, equine influenza viruses), Bunyaviruses (e.g., hantavirus, LaCrosse virus, Rift Valley fever virus), Arenaviruses (e.g., Lassa virus, Machupo virus), Re* >viruses
(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 mamrr ary tumor virus), Hepadnaviruses (e.g., hepatitis B virus), Parvoviruses (e.g., human parvovins B, canine parvovirus, feline panleukopenia virus) Papovaviruses (e.g., human papillpn lavirases, SV40, bovine papillomaviruses), Adenoviruses (e.g., human adenovirus, canine adenovirus, bovine adenovirus, porcine adenovirus), Herpes viruses (e.g., herpes simplex virus* s, varicella-zoster virus, infectious bovine rhinotracheitis virus, human cytomegalovirus, human herpesvirus 6), and Poxviruses (e.g., vaccinia, fowlpoxviruses, raccoon poxvirus, slainkpox 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 :ulture passage, reassortment, incorporation of mutations in infectious clones, reverse genetics, other recombinant DNA or RNA manipulation. In addition, those skilled in the art will r jcognize that other embodiments relate to viruses that are engineered to express any other proteins or RNA including, but not limited to, recombinant flaviviruses, recombinant adenovirises, recombinant poxviruses, recombinant retroviruses, recombinant adeno-associated viruses and recombinant herpes viruses. Such vinises 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, compositio is herein can contain one or more enveloped, positive strand RNA virus of the Togavirus, Fli.vivirus, Coronavirus, or Retrovirus families. In certain embodiments, compositions can cor tain 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 formula tions that improve thermal viral stability and prevent freeze-thaw inactivation will improve products that are liquid, powdered, freeze-driecl 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 i >r oral administration. A variety of devices are known in the art for delivery of the vaccin 5 including, but not limited to, syringe and needle injection, bifurcated needle admin stration, 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 li ye 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 certai: 1 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 n tolecular 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 embodiment 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 (-CH2CH2O- designated EO) and polypropylene oxide (-CH2CHCH3O- designated PO). The PO block can be flanked by two EO blocks ii a EOx-POy-EOx 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 EOx-POy-EOx block structure. In aqueous solutions, the EO-PO block copolymers will self-assemble into micelles with a PO core and a cor ana 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, USA or inactivated vaccines (e.g. Todd, Lee el: al. 1998; Kabanov, Lemieux et al. 2002). A: 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, Smith son 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 moi e surfactants with molecular weight of 1500 or greater. In a certain embodiment, the surfactant is a non-ionic, hydrophilic, polyoxyethylene-polyoxypropylene block copolymer (cr 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. ,000 or greater. In further embodiments, compositions can include in part an EO-PO block copolymer Pluronic F127 or 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 cam 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 vertebra :e 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 predominant: y a-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 or e of two primary sites located in subdomains IIA and IIIA. Due to its lack of immunogenici .y, albumin is commonly used as a carrier protein in biological products. Since the prctein dose
contained in a live, attenuated viral vaccine can be fractions of a microgram (deriv In certain embodiments, serum albumin may be from a human or otl ter mammalian source. For vaccines intended for human use, particular embodiments ran 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 sp ecific for each species may be used in animal vaccines (e.g. canine albumin for canine produ ;ts, bovine albumin for bovine products). In further embodiments, the protein is a recombinan t human albumin. Standard methods exist for expressing recombinant human albumin or pcrtions thereof in a variety of expression systems including bacteria, yeast, algae, plant, rm mmalian cell or transgenic animal systems. In addition, serum albumin or portions thereof an be produced in cell-free systems or chemically synthesized. Recombinant human albt min 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 mer ibers of the family (e.g. a-fetoprotein, vitamin D binding protein, or afamin) may substitute for albumin in compositions and methods contemplated herein. Those skilled in the ar t will also recognize that modifications can be made to albumin by any means known in the a: t, for example, by recombinant DNA technology, by post-translational modification, by ] iroteolytic cleavage and/or by chemical means. Those substitutions and alterations to albumir that provide essentially equivalent stabilizing function to serum albumin without substil utions and alterations are contemplated herein.
In certain embodiments, compositions having a high molecular weig ht 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 c an include, but are not limited to, monosaccharides, (e.g. glucose, galactose, ribose, muinose,
rhamno.se, talose, xylose or allose arabinose), disaccharides (e.g. trehalose, sucrose, maltose, isomaltose, cellibiose, gentiobiose, laminaribose, xylobiose, mannobiose, lactose, cr 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 chi :in). Polyols can include, but are not limited to, mannitol, sorbitol, arabitol, erythritol, rx altitol, xylitol, glycitol, glycol, polyglycitol, polyethylene glycol, polypropylene glycol, ar d 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 pharmaco ogically acceptable buffer. In certain embodiments, trehalose can be present at concentratic ns 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 nari ire to protect organisms from stress. Anhydrobiotic organisms that can tolerate low wate r conditions contain large amounts of trehalose. Trehalose has been shown to prevent both membrane fusion events and phase transitions that can cause membrane destabiliza tion during drying. Structural analysis suggests that trehalose fits well between the poh r head groups in lipid bylayers. Trehalose also prevents denaturation of labile proteins duing drying. It is thought that trehalose stabilizes proteins by hydrogen bonding with pc lar 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 educing power and is thus essentially non-rcactivc 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 ECK 'O 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 vi ais stability.
In certain embodiments, compositions can be described that typicall y include a physiologically acceptable buffer. Those skilled in the art recognize that a variety i )f 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 pai n 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 equivalen t 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-bi iffered saline (PBS).
Some live, attenuated "viral vaccine compositions herein concern cor ipositions that increase stability and/or reduce deterioration of live, attenuated virus in addition to having reduced immunogenicity or are non-immunogenic. In accordance with thes<: embodiments compositions can include one or more protein agents st ccharides polyols and high molecular weight surfactants wherein the composition decreases inactivation of live attenuated virus. therefore certain contemplated herein have reduced adverse reaction when administered to a subject. in some exemplary surfactant agent consists eo-po block copolymers are selected from group consisting lactalbumin serum albumin a-fetoprotein vitamin d binding afamin derived vertebrate species carbohydrate is saccharide anc polyol. trehalose sucrose chitosan sorbitol> mannitol. In certain more particular embodiments, in order to reduce immune reac:ion 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 embodirr ents, the carbohydrate agent is trehalose. In certain embodiments, at least one surfactant agent is the EO-PO block copolymer Pluonic F127. In some live, attenuated viral vaccine com] tositions at least one carbohydrate agent is trehalose. In certain live, attenuated viral vaccine compositions include, the EO-PO block copolymer Pluronic F127 where the conce ltration 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 ac ministered in which any toxic effects are outweighed by the therapeutic effects of the ac live 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 achiev<: a desired result. for example therapeutically active amount of compound may vary cording to factors such as the disease state age sex and weight individual ability formulations elicit response in individual. dosage regima be idjusted provide optimum therapeutic response.> In some embodiments, composition (e.g. pharmaceutical chemical, protein, peptide of an embodiment) may be administered in a convenient manne:- 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 en tbodiment, 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 aqueaus buffer
solutions. The active agent may also be administered parenterally or intrap ;ritoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, ani mixtures thereof and in oils. Under ordinary conditions of storage and use, these prepar itions may contain a preservative to prevent the growth of microorganisms.
Pharmaceutical compositions suitable for injectable use may be ac ministered 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 md can be fluid to the extent that easy syringability exists. It may further be preserved iigainst the contaminating action of microorganisms such as bacteria and fungi. The pharm aceutically acceptable carrier can be a solvent or dispersion medium containing, for example, water, cthanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene 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 ngredients 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 fo rmulations 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 mici ©particles, 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 c an include about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to ] .0 or even about 1 to 10 gram per dose. Single dose or multiple doses can also be administ ;red 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 inhala nts 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 miy 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 Vo 2%.
Oral formulations include such normally employed excipients as, fcr example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. In certain embodiments, oral phaimaceutical compositions can include an inert diluent or assimilable edible carrier, or may be i snclosed in hard or soft shell gelatin capsule, or may be compressed into tablets, or may be ir corporated 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, bu xal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compo jitions 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 1 >e 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 tie 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 on s 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, :he 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.
EXAMPLES
The following examples are included to demonstrate certain embodiments presented herein. It should be appreciated by those of skill in the art that the techniques disclosed in the Examples which follow represent techniques discovered to function well in the practices disclosed herein, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are Example 1
Base stability of DEN-2 PDK 53 flavivirus in liquid phase
In one illustrative method, the thermal stability for flaviviruses in lie uid phase was investigated. In accordance with this method, the base stability of the DEN-2 IDK 53 parental vaccine vector, stored in phosphate buffered saline (PBS), at different temperatures was determined (Table 1). In one example, lxlO4 pfu of DEN-2 PDK 53 virus in a total volume of 0.5ml PBS was incubated, in 2ml screw capped vials at either 4° C, room temperature (~ 21° C) or 37° C. After 24 hours of incubation viral titer and activity was determined by a Neutral Red agarose overlay plaque titration assay in Vero cells. A s illustrated in Table 1, incubation of DEN-2 PDK 53 in PBS at 4° C results in an awrage fourfold decrease in viral titer and complete loss in viral activity when incubated at 37° C for the same period. These results demonstrate the relatively poor stability of the DEN-2 PDK 53 flavivirus in the absence of stabilizing excipients.
Table 1 Stability of Den-2 PDK53 vims stored for 24 hours at different temperatures.
(Table Removed)
Exampile 2
Stabiliz ng Effects of Compositions
In certain exemplary compositions, pharmaceutically acceptable exc ipients contemplated herein that aid in thermal stability of live viral vaccines are known in the art. In one exemplary method, PBS was used as a base composition to assess the stabilizing effects of different excipients. In these examples, a stock solution of each excipient was mide in PBS and the pH adjusted to approximately 7.1 with NaOH, except for chitosan whore the pH of the stock solution was adjusted to approximately 6.8. Excipients were diluted in PBS to the final concentrations indicated (w/v) (Table 2). In accordance with this method, lxl d4 pfu of DEN-2 PDK 53 virus, in serum-free medium, was added to 0.5ml of each composiiion and stored a: 37° C for 24 hours. Following incubation, viral activity and titer was deteimined by plaque titration in Vero cells, as described above. As illustrated in Table 2, the stabilizing effects of compositions including a single excipient, at various concentrations comparable to previous experimental examples, was minimal. However, some excipients for example, trehalose and recombinant human serum albumin (rHSA), were more effective thai others at stabilizing DEN-2 PDK 53 virus at 37° C. Results of the study represented in Table 2 also revealec. that increased stabilizing effects of several excipients, including rHSA and trehalose, can be obtained within certain ranges of concentrations of these excipien :s. In this particular example, trehalose was more effective at concentrations above 15% (w/v) and F127 at concentrations between 0.5 and 3%.
Table 2 Effects of different excipients; on DEN-2 PDK53 stability when stored at 37° C for
24 hours
(Table Removed)
Example 3
Stabilizing effects of compositions including specific combinations of excipients
In the following illustrative procedure, compositions including mult, pie excipients in differing combinations and concentrations were tested for stabilizing a pluronic co-polymer non-ionic surfactant and a protein were optimal at improving DEN-2 PDK 53 stability at 37° C. Formulations including trehalose, F127 and rHSA had tie greatest stabilizing effects. Unexpectedly, the combined stabilizing effect of these three excipients was much greater than the sum of that observed with each individual component suggesting synergism between the components. Improved thermal stability of the DEN-2 PDK 53 flavi vims was obtained through the synergistic activities of the combination of trehalose, F127 and rHSA could not have been anticipated based on prior art examples. Figs '. and 2 also illustrate that the stabilizing effect of the trehalose/F127/rHSA mixture was fuither enhanced by the addition of 0.05% chitosan. Fig. 2 shows that the rate of viral inacivation when stored over a 48 hour period at 37° C is significantly reduced by composition! containing trehalose, F127 and rHSA. Examples in the art suggest that the stability of flaviviruses can be enhanced by formulations containing Ca 2+ and Mg 2+ divalent cations. However, as represented in Figs. 1 and 2, the addition of Ca 2+ (0.0009M) and Mg '+ (0.0005M) to a formulation confers no additional stabilizing benefits. The results fiom Fig. 2 suggest that addition of divalent cations may have a negative impact to long term li :mid phase viral stability in the context of particular embodiments.
In one exemplary method, a composition including trehalose, F127 rnd rHSA was assessed for its stabilizing properties with multiple flaviviruses. The stability o f chimeric DEN-2 flaviviruses expressing the membrane and envelope proteins from either W;st Nile (DEN-2/WN), Dengue 1 (DEN-2/D1), Dengue 3 (DEN-2/D3, or Dengue 4 (DEN-2 /D4) viruses was determined as described for Example 1. Illustrative results in Table 3 rsveal greatly improved liquid phase stability of all the chimeric flaviviruses when stored n a composition including trehalose, F127 and rHSA. The different chimeras express d fferent envelope and membrane proteins from five serologically distinct flaviviruses. In ac dition, West Nile virus and the dengue viruses are significantly divergent. This result suggests that compositions herein may be useful for liquid phase stabilization of diverse member j of the family of Flaviviradae as well as other virus families. The ability to stabilize flaviviruses at room temperature (~21° C) and at 4° C was examined by representative procedures as outlined for Example 1. The exemplary results, illustrated in Table 4, reveal that a composition including trehalose, F127 and rHSA effectively preserves viral activity for 7 days at 21 ° C and for 48 days at 4° C.
Table 3. Stability of different chimeric flaviviruses stored at 37° C for 21 hours in 5BS or a composition (Fl) including 15% trehalose, 2% F127 and 1% rHSA.
(Table Removed)
Table 4. Stability of flaviviruses stored at different temperatures for 7 or 48 days in PBS or a compos ition (F1) including 15% trehalose, 2% F127 and 1 % rHSA.
(Table Removed)
Examplle 4
Use of alternate components
Another exemplary method was used to compare the stabilizing effe cts of bovine serum albumin (BSA) and, gelatin, to that of rHSA and of different pluroni F127. Thus, while proteins other than rHSA may be used in combination with treru lose and Fl 27 to aid in stabilization of flaviviral vaccines, the use of a serum albumin or closely related proteins is more suitable in accordance with this exemplary method. In addition, Fig. 3 illustrates that, as isolated excipients, the polymer Pluronic PI23 is comparable to Pluronic Fl27 in its ability to stabilitze the DEN-2 PDK-53 virus. However, in this exemph ry method. PI23 does not appear to be an effective subsititute for F127 in compositions also containing trehalose and serum albumin. As exemplified in Fig. 4, compositions containing trehalose, rHSA and other commonly used pharmaceutical surfactants such as Poly sorbate 20 (Tween 20), instead of a pluronic co-polymer, are not effective in stabilizing DEN-2 PDK 53 relative to formulations containing a pluronic co-polymer. These exemplary methc ds suggest better stabilizing efficiencies of formulations containing distinct high molecular we ight pluronic: co-polymer surfactants.
Exemplary data is further illustrated in Fig 4. Fig. 4. represents stability of the DEN-2 PDK 53 virus in compositions containing different surfactants. DEN-2 PD C 53 was stored at 37° C for 23 hours in each formulation. Surfactants evaluated in this example include n-octyl-p-D-glucopyranoside (P -OG), Polysorbate 20 (P 20), Polysorbate i 10 (P 80 ) and F127 (F). Other formulation components include trehalose (T) and rHSA (A). Values are expressed as a percentage of the viral titer remaining after incubation relative to the input titer.
Examplle 5
Comparison of the stabilizing effects of different compositions
The stabilizing properties of one exemplary composition were compared to that of compositions known in the art. A stabilizing composition for live flaviviral vaccines, disclosed in the art (U.S. Pat. No. 4,500,512), includes 4% lactose, 2 % sorbitol, 0. g/L CaCl2, 0.076 MgS04 and amino acids on the order of 0.0005M to 0.05M in PBS. Another composition reported by Adebayo et al (1998) consists of 10% sucrose, 5% lactalbumin, 0. lg/L CaCb, and 0.076 g/L MgSO Adebayo, et al. lxlO4 pfu of DEN-2 PDK 53 vaccine virus were incubated at 37° C in 0.5ml of each composition for 23 hours, after which viral activity and titer was assayed as described in Example 1. As exemplified in Fig. 5, some embodiments, for example formulation Fl, represents a significant improvement over those previously described compositions. In the example shown, virtually no viral activity was recovered after storage in the formu ations known in the art (formulations F3 and F4), whereas upwards of 50% of the initial viral titer was recovered after storage in a composition disclosed herein. These results reveal that previous formulations are ineffective at promoting live viral vaccine stability durin % liquid phase storage.
Examplle 6
Preservation of viral activity after multiple freeze-thaws
In one exemplary method, the ability of select compositions to preserve viral activity after freeze-thaw cycles was demonstrated. lxlO4 pfu of DEN-2 PDK 53 vuccine virus was suspended in 0.5 ml of each composition in screw cap vials. For the first freeze-thaw cycle vials were frozen at -80° C for 24 hours and thawed rapidly at 37° C. This was immediately followed by a second freeze-thaw cycle where the vials were frozen al -80° C for 1 hour and thawed rapidly at 37° C. Viral titer and activity was then assessed by a plaque titration assay as described in Example 1. As illustrated in Fig. 6, particular compof itions that include trehalose, F127 and rHSA effectively preserved full viral activity through two freeze-thaw cycles. Additionally, compositions including these three excipients were more effective than those containing just a single excipient. The results of this particular illustrative experiment suggest the compositions and methods disclosed herein are an effective cryoprotectant for flaviviral vaccines and may facilitate viral preservation during fr;eze-drying, spray-drying, or other dehydration techniques.
Example 7
Stabilization of other live, attenuated viruses.
Examples illustrated previously reveal effective liquid phase stabiliz ition of several live, attenuated flaviviruses in compositions including trehalose, F127 and iHSA. It is anticipated that embodiments disclosed herein may also be effective at stabilizing o ther live, attenuated viruses. For example, a formulation including trehalose, F127 and rHSA may be
used to stabilize live attenuated measles virus, an attenuated sindbis virus, an attenuated influenza virus, a recombinant, attenuated adenovirus or a recombinant, attenuated vaccinia virus. In one exemplary method, these non-flaviviral viruses can be suspended and maintained in liquid phase, in a composition including trehalose, F127, and rHSA directly after hai-vesting from cell culture. In another illustrative method, non-flaviviral vin ses can be suspended in a composition prior to, or subsequent to, freeze or spray-drying. Statistically improved viral stability may demonstrate that the formulation of this embodiment is applicable to other attenuated viral vaccines outside of the Flavivirus family. Those skilled in the art recognize that application may then be extended to other live, attenuated vir ises.
Example 8
Safety and in vivo immunogenicity.
Molecular interactions between excipients and molecular or cellular components may serve, not only to enhance stability of viral vaccines, but also to ciuse increased cell or tissue damage in vivo. Formulations may decrease the immunogenicity of these viral vaccines in live animals. In this example, it is demonstrated that exemp ary compositions are safe after subcutaneous injection and are essentially immunologically inert. Four different exemplary compositions were selected for testing in mice as follow:.
Formulation 1: 15% Trehalose, 2% F-127, 1 % rHSA
Formulation 2: 15% Trehalose, 2% F-127, 1% rHSA, ImM CaCl2/ 0.5mM MgS04
Formulation 3: 15% Trehalose, 2% F-127, 1% rHSA, 0.5% chitosan
Formulation 4: 22.5% Trehalose, 3% F-127, 1.5% rHSA
Formulation 5: PBS
In certain methods described herein, groups of 8 or 9 NIH Swiss mice were immunized by subcutaneous injection with 1x10s pfu of a formulated DEN-2 PDK- 53/WN recombinant flavivirus vaccine at day 0 (d0), were boosted with the same formulate d vaccine at d29 and were then challenged with 103 pfu on a pathogenic West Nile strain (N"V 99) on d45. Control mice (four groups of 8) received formulations 1—4 alone with no vir is. No adverse events after administration in any of the immunized mice were observed. Thus, in this example, no apparent adverse events are caused by the exemplary formulations with or without vaccine virus. Sera were collected prior to immunization at dO, prior to boost at d28, prior to challenge at d44 and post-challenge at d75. West Nile neutralizing antibod y titers in
the sera were determined by plaque reduction neutralization test (PRNT). The results of the study are represented in Table 5.
Table 5: Neutralizing antibody and protection induced by formulated DEN2/\^N
vaccines
1 GMT = geometric mean titer; titers of 10.
A majority of the animals receiving the DEN-2/WN vaccine sero-co nverted
after the first dose regardless of whether no formulation (Formulation 5) or one of the
exemplary formulations (Formulations 1 - 4) was used. In addition, all of the vacc nated
animals sero-converted after the booster administration. Geometric mean PRNT titers
(GMT) demonstrate few differences between the vaccine groups. Titers were low * iter the
primary immunization, increased 3-10 fold after the boost and then showed a drariatic
anamnestic response upon challenge. 100% of all the vaccinated animals survived challenge,
again independent of vaccine formulation. Only 22% of the control animals survived; those
that did survive showed evidence of potent neutralizing antibody responses after challenge.
One advantage is that this example demonstrates that the exemplary formulations d 3 not
reduce the ability of an exemplary recombinant DEN-2/WN vaccine to prevent Wert Nile
disease in a mice.
Example 9
In another example, liquid compositions were used containing trehalose, rHSA and F127 to stabilize a West Nile chemeric flavivirus stored for various periods at e ither 25°C or 4°C. IxlO4 pfu of chimeric DEN-2/WN vaccine virus were incubated at each ten perature and viral activity was assessed at one or two week intervals as described in Exampl; 1. As illustrated in Figs.7 and 8, formulations containing trehalose, rHSA and F127 signiiicantly improved the thermal stability of the DEN-2/WN vaccine virus during storage at 2*°C and
4°C, respectively. At 25°C loss of viral activity was less than one log over 7 days. \t 4°C viral inactivation was negligible for periods up to 12 weeks when stored in exempl try formulations including trehalose, F127 and rHSA. Example 10
In another exemplary method, stabilizing effects of compositions were demonstrated including trehalose, rHSA and a pluronic co-polymer with dehydrated DEN-2 PDK 53 vaccines, lxl 04 pfu of DEN-2 PDK 53 vaccine virus formulated in accordance with procedures disclosed herein. Formulated vaccines were placed in serum vials and s lbjected to conventional lyophilzation procedures. Dried vaccines were stoppered under vacuum, stored at either 37°C or 4°C for 14 days followed by reconstitution of the vaccine to its original liquid volume by addition of sterile water. Viral activity of the reconstituted vaccina was assessed as outlined earlier. At 37°C, in the presence of compositions containing trehalose, rHSA and a pluronic co-polymer formulated in phosphate buffered saline, an average viral titer loss of 1 log was observed (Fig. 9). No loss in viral activity was observed for formulated dehydrated DEN-2 PDK 53 viral vaccines stored at 4°C for 14 days. These results demonstrate effective preservation of a dehydrated viral vaccine utilizing compositions disclosed herein.
Fig. 9. represents stability of lyophilized DEN-2 PDK 53 at differen; temperatures. Log titer loss of formulated lyophilized DEN-2 PDK 53 vaccine virus following incubation at 37°C or 4°C for 2 weeks as indicated. Formulations Fl (15% trehalose, 2% F127, 1% rHSA) and F2 (15% trehalose, 2% F127, 0.01% rHSA) were formulated in phosphate buffered saline. Formulation F3 (15% trehalose, 2% F127. 0.01% rHSA) was formulated in 10 mM Tris base.
All of the COMPOSITIONS and METHODS disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods have been described in terms of preferred embodiments, it is apparent to those of skill in the art that variations maybe applied to the COMPOSITIONS and METHODS and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope herein. More specifically, cen ain agents that are both chemically and physiologically related may be substituted for the agei ts described herein while the same or similar results would be achieved. All such similar
substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept as defined by the appended claims.
All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety.

We claim:
1. A live attenuated virus composition comprising:
one or more live, attenuated viruses;
one or more high molecular weight surfactant agents wherein the high molecular weight surfactant agents have a molecular weight of 1500 or greater;
one or more proteins agents, the one or more protein agents are selected from the group consisting of lactalbumin, serum albumin, α-fetoprotein, vitamin D binding protein, afamin derived from a vertebrate species and a combination thereof, and
one or more carbohydrate agents, the one or more carbohydrate agent are selected from the group consisting of trehalose, sucrose, chitosan, sorbitol, mannitol and a combination thereof,
wherein the composition is capable of reducing the inactivation of the live attenuated virus.
2. The virus composition of claim 1, wherein the live, attenuated viruses are selected from the group consisting of Flavivirus, Togavirus, Coronavirus, Rhabdovirus, Filovirus, Paramyxovirus, Orthomyxovirus, Bunyavirus, Arenavirus, Retrovirus, Hepadnavirus, Herpesvirus, Poxvirus families and combinations thereof.
3. The virus composition of claim 1, wherein the live, attenuated viruses are Flaviviruses.
4. The virus composition of claim 1, wherein the composition is in aqueous form.
5. The virus composition of claim 1, wherein the composition is partially or wholly dehydrated.
6. The virus composition of claim 1, wherein the one or more high molecular weight surfactant agents further comprises one or more co-polymers wherein the molecular weight of the high molecular weight surfactant agent is 3000 or greater.
7. The virus composition of claim 1, wherein the surfactant agent(s) consists of one or more EO-PO block copolymers.
8. The virus composition of claim 1, wherein the protein agent(s) is serum albumin derived from a vertebrate species.
9. The virus composition of claim 1, wherein one of the one or more carbohydrate agent is trehalose.
10. The virus composition of claim 1, wherein at least one high molecular weight surfactant agent is an EO-PO block copolymer, at least one protein agent is serum albumin, and at least one carbohydrate agent is trehalose.
11. The virus composition of claim 10, wherein the EO-PO block copolymer concentration is less than from 4% (w/v), wherein the one or more protein concentration is from 0.001 to 3% (w/v) and the one or more carbohydrate concentration is from 5 to 50% (w/v).
12. A method for decreasing inactivation of a live, attenuated virus composition comprising, combining one or more live attenuated viruses with a composition comprising one or more high molecular weight surfactant agents wherein the high molecular weight surfactant agents have a molecular weight of 1500 or greater, one or more protein agents, the one or more protein agents are selected from the group consisting of lactalbumin, serum albumin, α-fetoprotein, vitamin D binding protein, afamin derived from a vertebrate species and a combination thereof, and one or more carbohydrates, the one or more carbohydrate agent are selected from the group consisting of trehalose, sucrose, chitosan, sorbitol, mannitol and a combination thereof, wherein the composition is capable of reducing inactivation of the live, attenuated virus.
13. The method of claim 12, wherein the live, attenuated viruses are selected from the group consisting of Flavivirus, Togavirus, Coronavirus, Rhabdovirus, Filovirus, Paramyxovirus, Orthomyxovirus, Bunyavirus, Arenavirus, Retrovirus, Hepadnavirus, Herpesvirus, Poxvirus families and combinations thereof.
14. The method of claim 12, further comprising partially or wholly dehydrating the combination.
15. The method of claim 14, further comprising partially or wholly re-hydrating the composition prior to administration.
16. The method of claim 12, wherein the composition increases the shelf life of a virus composition when the composition is maintained at room temperature or lower compared to a control composition.
17. The method of claim 12, wherein the composition decreases inactivation of an aqueous live, attenuated virus for 24 hours or greater.
18. The method of claim 12, wherein the composition decreases inactivation of an aqueous live, attenuated virus during one or more freeze and thaw cycles compared to a control composition.
19. The method of claim 12, wherein at least one high molecular weight surfactants is an EO-PO block copolymer polyxamer 407, at least one of the protein agents is serum albumin, and at least one of the carbohydrate agents is trehalose.
20. The method of claim 12, wherein the virus composition reduces the onset of or prevents a health condition selected from the group consisting of West Nile infection, Dengue fever, Japanese encephalitis, Kyasanur Forest disease, Murray Valley encephalitis, St. Louis encephalitis, Tick-borne encephalitis, Yellow fever and Hepatitis C Virus Infection.
21. A kit for decreasing the inactivation of a live, attenuated virus composition comprising:
at least one container; and
a composition comprising one or more protein agents, the one or more protein agents are selected from the group consisting of lactalbumin, serum albumin, α-fetoprotein, vitamin D binding protein, afamin derived from a vertebrate species and a combination thereof, one or more carbohydrate agents, the one or more carbohydrate agents are selected from the group consisting of trehalose, sucrose, chitosan, sorbitol, mannitol and a combination thereof, and one or more high molecular weight
surfactants wherein the high molecular weight surfactant agents have a molecular weight of 1500 or greater, wherein the composition decreases inactivation of a live, attenuated virus.
22. The kit of claim 21, wherein at least one surfactant agent is an EO-PO block copolymer poloxamer 407, at least one protein agent is serum albumin, and at least one carbohydrate agent is trehalose.
23. The kit of claim 22, wherein the trehalose concentration is from 5 to
50% (w/v).
24. The kit of claim 22, wherein the EO-PO block copolymer poloxamer 407 concentration is from 0.1 to 4% (w/v).
25. The kit of claim 22, wherein the serum albumin concentration is from 0.001 to 3% (w/v).
26. The kit of claim 21, wherein the composition further comprises one or more live, attenuated viruses.
27. The kit of claim 26, wherein the live, attenuated viruses are selected from the group consisting of Flavivirus, Togavirus, Coronavirus, Rhabdovirus, Filovirus, Paramyxovirus, Orthomyxovirus, Bunyavirus, Arenavirus, Retrovirus, Hepadnavirus, Herpesvirus, Poxvirus families and combinations thereof.

Documents:

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Patent Number

279320

Indian Patent Application Number

7036/DELNP/2009

PG Journal Number

03/2017

Publication Date

20-Jan-2017

Grant Date

18-Jan-2017

Date of Filing

03-Nov-2009

Name of Patentee

Takeda Vaccines, Inc.

Applicant Address

One Takeda Parkway, Deerfield, IL 60015, USA

Inventors:

#	Inventor's Name	Inventor's Address
1	STINCHCOMB, DAN T.	8409 COUNTY ROAD 3, FORT COLLINS, COLORADO 80528, USA.
2	OSORIO, JORGE E.	560 HIGHWAY 78 SOUTH, MOUNT HOREB, WISCONSIN 53572, USA.
3	WIGGAN, O'NEIL	1600 WEST PLUM STREET, FORT COLLINS, COLORADO 80521, USA.

PCT International Classification Number

C12N 7/04

PCT International Application Number

PCT/US2008/059472

PCT International Filing date

2008-04-04

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	60/910,579	2007-04-06	U.S.A.