JP2016511634A - Process for developing cold-adapted temperature-sensitive strains and cold-adapted temperature-sensitive virus strains of enterovirus 71 - Google Patents

Abstract

The present invention relates to cold-adapted temperature sensitive enterovirus 71 strains, in particular to cold-adapted temperature sensitive enterovirus 71 strains EV71: TLLβP20 and EV71: TLLαP20. The present invention also relates to a process for developing cold-adapted temperature sensitive virus strains.

Description

Sequence submission
[0001] This application has been filed with an electronic sequence listing. The sequence listing is 2577224PCTSsequenceListing. entitled txt, created on 11 January 2013, and is 21 kb in size. The information in electronic form of the sequence listing is incorporated herein in its entirety.

  [0002] The present invention relates to cold-adapted temperature sensitive enterovirus 71 strains, in particular to cold-adapted temperature sensitive enterovirus 71 strains EV71: TLLβP20 and EV71: TLLαP20. The invention also relates to the process of developing cold adapted temperature sensitive virus strains, particularly RNA virus strains.

  [0003] To clarify the background of the invention or to provide further details regarding its implementation, the publications and other materials used herein are hereby incorporated by reference and, for convenience, references, respectively. Grouped into a list.

  [0004] Hand-foot-and-mouth disease (HFMD) is caused by a group of human enteroviruses that essentially belong to the Enterovirus A species. Of these, enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are responsible for over 90% of HFMD cases. In addition to causing HFMD, EV71 has been known to cause severe neurological disease with death, especially in young children.

  [0005] Human enterovirus 71 (EV71) is a small non-enveloped virus approximately 30 nm in size and has a single-stranded plus-strand RNA genome of approximately 7,450 nucleotides. The virus is classified as a human enterovirus A species under the Enterovirus genus within the Picornaviridae family (Alexander et al., 1994; Melnick, 1996). EV71 is divided into three major genogroups (denoted A, B, and C), and genogroups B and C are further subclassified based on phylogenetic analysis of the major capsid protein (VP1) gene. Subdivided into genogroups B1-B5 and C1-C5 (Bible et al., 2008). EV71 has been associated with a range of clinical disorders, mainly in hand and foot children (HFMD), aseptic meningitis, encephalitis and polio-like paralysis, mainly in infants and young children (Alexander et al., 1994; Melnick). 1996). The virus was first isolated from a child with aseptic meningitis in California, USA and subsequently characterized as a new serotype of the Enterovirus genus (Schmidt et al., 1974). Several years after initial isolation, complications of complications of HFMD have been reported in various regions of the world (Bromberg et al., 1974; Kennett et al., 1974; Deibel et al., 1975; Hagiwara et al., 1978).

  [0006] In recent years, EV71 infection has become a major public health burden and concern worldwide, especially in the Asia Pacific region, following the outbreak of neurotoxic EV71 epidemics and sporadic outbreaks. The neurotoxicity of EV71 first attracted great global attention during the 1975 Bulgarian pandemic, causing 705 polio-like diseases and resulting in 44 deaths (Chumakov et al., 1979; Shindarov) Et al., 1979). A pandemic of similar nature occurred in Hungary in 1978 and resulted in many cases of polio-like disease and 47 deaths (Nagy et al., 1982). Subsequently, several milder CNS disease epidemics associated with EV71 have been reported in New York, Hong Kong, Austria and Philadelphia (Chomnairite et al., 1958; Samuda et al., 1987; Gilbert et al., 1988; Hayward Et al., 1989). Two epidemics of EV71 occurred in Japan, most cases were characterized by HFMD, and the incidence of CNS disease was low (Tagaya et al., 1981; Ishimaru et al., 1980; Hagiwara et al., 1983). In 1997, a very large outbreak of HFMD due to EV71, a highly neurotoxic in Malaysia, caused 48 deaths. A larger pandemic occurred in Taiwan in 1998, with over 100,000 deaths of HFMD, 405 severe infections, and 78 deaths from acute brainstem encephalomyelitis with neurogenic heart failure and pulmonary edema (Lum 1998a; Lum et al., 1998b; Chang et al., 1998; Yan et al., 200; Liu et al., 2000; Wang et al., 2000). In 2008, a total of 488,955 cases of HFMD with 126 deaths were recorded in the mainland of the People's Republic of China (Chinese center, 2008). In 2009, the number of HFMD cases increased to 1,155,525 and was reported to be accompanied by 353 deaths (Yang et al., 2011). In 2010, China experienced the largest epidemic ever, resulting in over 1.7 million HFMD cases, 27,000 patients with severe neurological complications, and 905 deaths. In all three outbreaks, almost all severe cases with neurological complications and death were due to EV71 (Zeng et al., 2012).

  [0007] Currently, the molecular determinants of virulence and development of EV71 infection are still not fully understood. There are no approved antiviral drugs for clinical treatment of severe infections and associated neurological complications, and no vaccines are available for use to control and prevent recurrent pandemics. With regard to control and prevention strategies, the development of cost-effective vaccines, especially for use in developing countries, is paramount and very urgent. Various types of vaccines against EV71, under research and development, appear to elicit an immune response in rodents or monkeys (Wu et al., 2001; Arita et al., 2005; Chiu et al., 2006; Arita et al., 2007). Tung et al., 2007; Chung et al., 2008; Chen et al., 2008; Ong et al., 2010; Chen et al., 2011; Lee and Chang, 2010; Xhang et al., 2010). Virus-like particle vaccines and subunit peptide vaccines based on VP1 capsids continue to be viable potential vaccine strategies that are worth further research and development, but very extensive past experience in development and Injectable inactivated and oral attenuated EV71 vaccines are the most promising, based on the fact that wild-type poliovirus infection is controlled and approaching eradication using activated injectable soak and live attenuated oral Sabin poliovirus vaccine (Zhang et al., 2010).

  [0008] Currently, there are no vaccines available on the market to protect children against infection with EV71 and hand-foot-and-mouth disease. Based on recent information, there are two facilities in the People's Republic of China, one in Singapore and one in Taiwan in the process of developing an injectable inactivated EV71 vaccine (press release and personal communication). H. The enterovirus division of the National Institute of Infectious Diseases, Tokyo, led by Dr. Shimizu, has been conducting thorough research on the development of EV71 in monkeys and a temperature-sensitive strain of EV71 as a potential candidate oral live attenuated EV71 vaccine since the 1980s. (Arita et al., 2005; Arita et al., 2007). All potential vaccine candidates for temperature-sensitive strains of EV71 in that monkey study are still to a lesser extent compared to wild-type virus after intravenous inoculation, but still with neuroinvasiveness and histopathological lesions of the CNS Was possible to cause.

[0009] The live attenuated vaccine is the first vaccination dating back to the 18th century when British doctor Edward Jenner started vaccinating children with cowpox virus against the destructive disease of smallpox Corresponds to one of the successful methods. Live attenuated vaccines use live viruses or microorganisms that are attenuated to prevent disease but still induce a protective immune response. Traditional, classical, and genetic methods have had some success in attenuating viruses and microorganisms for use as live attenuated vaccines ^44-48 . Traditional methods use naturally associated organisms that are non-pathogenic in humans, such as cowpox or vaccinia virus. Classical methods involve growing several cycles of pathogenic viruses or microorganisms under attenuated conditions, such as tissue culture or harsh liquid media. Genetic methods utilize modern molecular biotechnology to manipulate the genome and reduce virulence (Huygelen, 1997; Robinson, 2008; Coleman et al., 2008; Lauring et al., 2010; Kenney et al., 2011). In the classical method of obtaining a temperature sensitive phenotype virus as a marker of attenuation, wild-type virus is plaque selected through plaque assay techniques by culturing the virus in appropriate cells incubated at lower temperatures. Selected virus strains are subsequently passaged repeatedly at lower incubation temperatures that are targeted (Hagiwara et al., 1982; Hashimoto and Hagiwara, 1983; Richman and Murphy, 1997).

  [0010] Viral strains that can be used to treat viral diseases that retain phenotypic and genetic stability in certain in vitro cell culture conditions and are not neurotoxic in monkeys after intravenous inoculation It is desirable to develop It is also desirable to develop cold adapted temperature sensitive strains of viruses, including RNA viruses, obtained in cell culture after successive passages.

  [0011] The present invention relates to cold-adapted temperature sensitive enterovirus 71 strains, and in particular to cold-adapted temperature sensitive enterovirus 71 strains EV71: TLLβP20 and EV71: TLLαP20. The invention also relates to the process of developing cold adapted temperature sensitive virus strains, particularly RNA virus strains.

  [0012] Accordingly, in one aspect, the invention provides a cold adapted temperature sensitive enterovirus 71 strain. In one embodiment, the cold-adapted temperature sensitive enterovirus 71 strain is EV71: TLLβP20 as described herein. In another embodiment, the cold-adapted temperature sensitive enterovirus 71 strain is EV71: TLLαP20 as described herein.

  [0013] The enterovirus 71 strain of the present invention is prepared by a method of attenuating enterovirus 71 using temperature sensitivity as a phenotypic marker. The method is an in vitro laboratory process that alters the biological growth characteristics of the virus and adapts for optimal replication at incubation temperatures below 30 ° C. The adaptation process described in detail below is performed in a systematic and stepwise fashion that incrementally reduces the incubation temperature at which the virus is cultured until the target temperature selected for optimal replication of the virus is achieved.

  [0014] In a second aspect, the present invention provides a composition comprising the cold-adapted temperature sensitive enterovirus 71 strain described herein. In one embodiment, the composition comprises an effective amount of a virus strain described herein. In another embodiment, the composition comprises one or more physiologically or pharmaceutically acceptable carriers. In a further embodiment, the composition is a vaccine. Using techniques well known to those skilled in the art, a vaccine containing the cold-adapted temperature-sensitive enterovirus 71 strain described herein is prepared. Such vaccines are useful for providing immunity against a parental virus strain by administering the vaccine to a subject, eg, a human subject, using techniques well known to those skilled in the art.

  [0015] In a third aspect, the present invention provides a method for inducing a protective immune response in a subject, eg, a human subject, comprising a prophylactically or therapeutically or immunologically effective amount of the present specification. The method is provided comprising administering to the subject a cold-adapted temperature sensitive enterovirus 71 as described. In one embodiment, the protective immune response protects the subject against diseases caused by enterovirus 71. In one embodiment, the disease is hand-foot-and-mouth disease. In another embodiment, the disease is aseptic meningitis. In a further embodiment, the disease is encephalitis. In a further embodiment, the disease is polio-like paralysis. In one embodiment, the cold-adapted temperature sensitive enterovirus 71 strain described herein is administered as a vaccine. In a further embodiment, the subject is exposed to wild type enterovirus 71. In another embodiment, administration of a cold-adapted temperature sensitive enterovirus 71 strain described herein prevents a subject, eg, a human subject, from suffering from an enterovirus 71 related disease. In a further embodiment, the subject is exposed to wild type enterovirus 71 strain. In further embodiments, administration of the cold-adapted temperature sensitive enterovirus 71 strain described herein delays the onset of or slows the rate of progression of enterovirus 71 related disease in a virally infected subject, eg, a human subject.

  [0016] In a fourth aspect, the present invention provides a method for attenuating a virus using temperature sensitivity as a phenotypic marker. According to this aspect, the method develops a cold-adapted temperature sensitive virus strain. The method of the invention is an in vitro laboratory process that alters the biological growth characteristics of the virus and adapts for optimal replication at incubation temperatures below 30 ° C. The adaptation process is performed in a systematic, stepwise manner that incrementally reduces the incubation temperature at which the virus is cultured until the target temperature selected for optimal replication of the virus is achieved. Thus, according to the present invention, the method comprises the following steps: (i) preparing a parental wild type virus reference stock and (ii) a culture of cells infected with the parental wild type virus reference stock, Each to obtain a lower MOI inoculum and complete CPE for 5 or more passages at a higher multiplicity of infection (MOI) and incubation temperature of about 34 ° C. to about 36 ° C., preferably about 34 ° C. Incubate until a complete cytopathic effect (CPE) is obtained at each passage using a shorter incubation period of passage, (iii) a culture of cells infected with the virus resulting from the previous step, Lower MOI inoculum for 5 or more passages at higher MOI and incubation temperatures about 1 ° C to about 3 ° C lower than the previous step Incubate until a complete cytopathic effect (CPE) is obtained at each passage with a shorter incubation period of each passage to obtain complete CPE, and (iv) be selected for optimal replication of the virus Step (iii) is repeated in a systematic stepwise fashion that gradually decreases the incubation temperature until a target temperature is achieved. In one embodiment, the target temperature is from about 26 ° C to about 29 ° C, preferably about 28 ° C. In one embodiment, the incubation temperature that decreases incrementally is a temperature that decreases by about 1 ° C. to about 2 ° C.

  [0017] In one embodiment, the parental wild-type virus reference stock has a complete cytopathic effect (CPE) for one or two passages at a temperature of about 36 ° C to about 38 ° C, preferably about 37 ° C. ) Is obtained by incubating a culture of cells infected with the wild type virus. An aliquot of the culture supernatant containing the produced virus is placed in a vial or other suitable storage device. This culture supernatant serves as a reference stock for the parental wild type virus. The reference parent wild type virus is used for the subsequent attenuation process. In another embodiment, an aliquot of the parental wild type virus reference stock is stored at a suitable temperature, eg, -80 ° C.

  [0018] In one embodiment, the virus is any virus. In another embodiment, the virus is an RNA virus. In a further embodiment, the RNA virus is a positive strand RNA virus. In a further embodiment, the virus is a member of the Picornaviridae family. In another embodiment, the virus is a member of the Enterovirus genus. In one embodiment, the virus is enterovirus 71 (EV71). In another embodiment, the virus is Coxsackievirus A16 (CA16). The methods of the invention are useful in producing cold-adapted temperature sensitive virus strains of any of these viruses, including but not limited to EV71 and CA16 cold-adapted temperature sensitive strains.

  [0019] In one embodiment, the cell infected with the virus is any cell that allows the virus to grow. In a preferred embodiment, the cell is a Vero cell (ATCC CCL-81). In one embodiment, the cells are maintained by normal passage in a medium suitable for cell growth. In embodiments where the cells are Vero cells, the Vero cells are cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS). In one embodiment, Vero cells maintained in DMEM supplemented with 1% FCS are used for parental wild type virus production, virus culture, attenuation, titer determination, and evaluation of temperature sensitive phenotype. In another embodiment, DMEM is supplemented with 1% FCS to adapt the virus strain for replication as the incubation temperature is continuously reduced.

  [0020] The present invention also relates to cold-adapted temperature sensitive strains of viruses produced by the methods described herein. Cold-adapted temperature sensitive viruses produced by the methods described herein are useful for the production of vaccines using techniques well known to those skilled in the art. Such vaccines are useful for providing immunity to the parental virus strain by administering the vaccine to a subject using techniques well known to those skilled in the art.

[0021] FIG. 1a is an indirect immunofluorescence assay using a commercial monoclonal antibody obtained from monkey blood 4 days after administration of an intravenous dose of enterovirus 71 (EV71: TLLβP20) and specific for the virus. Shows positively stained peripheral blood mononuclear cells (arrows).

  [0022] FIG. 1b is a one-step RT-PCR amplification product of tissue obtained from monkeys (2202F, 2891F) 4 days after immunization using a specific oligonucleotide primer pair for the detection of Enterovirus 71 strain. The expected size of the RT-PCR amplification product is 427 bp. The lanes in the two gels are as follows. Gel 1: Lane 1: 100 bp DNA ladder; Lane 2: 2202F-heart; Lane 3: 2202F-spleen; Lane 4: 2202F-lymph node; Lane 5: 2202F-kidney; Lane 6: 2202F-liver; Lane 7: 2891F -Heart; Lane 8: 2891F-Spleen; Lane 9: 2891F-Lymph node; Lane 10: 2891F-Kidney; Lane 11: 2891F-Liver; Lane 12: 2202F-Brainstem (bridge); Lane 13: 2202F-Brainstem (medullary medulla Lane 14: 2202F—cortex (cerebral gyrus); Lane 15: 2202F—spinal cord (cervical); Lane 16: 2202F—spinal cord (lumbar); Lane 17: 2202F—spinal cord (chest); Lane 18: 2891F—brain stem. (Medullary medulla); lane 19: 2891F—brain stem (bridge); lane 20: 2891F—cortex ( Cerebellum). Gel 2: Lane 21: 100 bp DNA ladder; Lane 22: 2891F—cortex (right cerebellum); Lane 23: 2891F—spinal cord (neck); Lane 24: 2891F—spinal cord (lumbar); Lane 25: 2891F—spinal cord (chest) ); Lane 26: Template-free control; Lane 27: 100 bp DNA ladder.

  [0023] The present invention relates to cold-adapted temperature sensitive enterovirus 71 strains, in particular cold-adapted temperature sensitive enterovirus 71 strains EV71: TLLβP20 and EV71: TLLαP20. The invention also relates to the process of developing cold adapted temperature sensitive virus strains, particularly RNA virus strains.

  [0024] Accordingly, in one aspect, the invention provides a cold-adapted temperature sensitive strain. In one embodiment, the cold-adapted temperature sensitive enterovirus 71 strain is EV71: TLLβP20 as described herein. In another embodiment, the cold-adapted temperature sensitive enterovirus 71 strain is EV71: TLLαP20 as described herein. EV71: TLLβP20 has been deposited with the American Type Culture Collection located at 10801 University Boulevard, Manassas, Virginia 201110, USA, under the provisions of the Budapest Treaty, and assigned the deposit number PTA-13285 It was. EV71: TLLαP20 was deposited with the American Type Culture Collection on 25 October 2012 under the terms of the Budapest Treaty and assigned deposit number PTA-13284.

  [0025] The enterovirus 71 strain of the present invention is prepared by a method of attenuating enterovirus 71 using temperature sensitivity as a phenotypic marker. The method is an in vitro laboratory process that changes the biological growth characteristics of the virus and is adapted for optimal replication at incubation temperatures below 30 ° C. The adaptation process is performed in a systematic, stepwise manner that incrementally reduces the incubation temperature at which the virus is cultured until the target temperature selected for optimal replication of the virus is achieved. As disclosed herein, a culture of cells infected with (i) a parental wild-type virus reference stock and (ii) a parental wild-type virus reference stock is treated with a higher multiplicity of infection (MOI). ) And 5 or more passages at an incubation temperature of about 34 ° C., complete cell degeneration at each passage, using a lower MOI inoculum and a shorter incubation period for each passage to obtain complete CPE. Incubate until effect (CPE) is obtained, and (iii) Incubate cell cultures infected with the virus resulting from the previous step at a higher MOI and about 1 ° C. to about 3 ° C. lower than the previous step 5 or more passages at temperature, shorter MOI inoculation and shorter incubation period for each passage to obtain complete CPE Incubate at each passage until a complete cytopathic effect (CPE) is obtained, and (iv) decrease the incubation temperature in increments until the target temperature selected for optimal replication of the virus is achieved Repeat step (iii) in a systematic stepwise manner. In one embodiment, the target temperature is from about 26 ° C to about 29 ° C, preferably about 28 ° C.

  [0026] In one embodiment, the parental wild type virus reference stock is infected with the wild type virus at a temperature of about 36 ° C. to about 38 ° C., preferably at about 37 ° C., for one or two passages. The cell culture is prepared by incubating until a complete cytopathic effect (CPE) is obtained. An aliquot of the culture supernatant containing the produced virus is placed in a vial or other suitable storage device. This culture supernatant serves as a reference stock for the parental wild type virus. The reference parent wild type virus is used for the subsequent attenuation process. In another embodiment, an aliquot of the parental wild type virus reference stock is stored at a suitable temperature, eg, -80 ° C.

  [0027] In one embodiment, the cell infected with the virus is any cell that allows the virus to grow. In a preferred embodiment, the cell is a Vero cell (ATCC CCL-81). In one embodiment, the cells are maintained by normal passage in a medium suitable for cell growth. In embodiments where the cells are Vero cells, the Vero cells are cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS). In one embodiment, Vero cells maintained in DMEM supplemented with 1% FCS are used for parental wild type virus production, virus culture, attenuation, titer determination, and evaluation of temperature sensitive phenotype. In another embodiment, DMEM is supplemented with 1% FCS to adapt the virus strain for replication as the incubation temperature is continuously reduced.

  [0028] In one embodiment, as soon as the virus has obtained a complete cytopathic effect (CPE), a clarified culture supernatant containing the virus is obtained and a continuous fresh new culture of cells, eg Vero cells. The virus is passaged at each step of the process by passing this supernatant into each of the flasks. In another embodiment, the culture supernatant containing the virus is passed at a higher multiplicity of infection (MOI) of 20, at the beginning of each successive change to a lower incubation temperature. Within 3 days after inoculation, fresh confluence after it was recognized in Vero cells that it was possible to cause full CPE, and subsequently reduced to a lower 5-10 MOI. A new culture flask containing monolayer Vero cells is inoculated with the virus at the same MOI for at least 3 more passages. If it was recognized that it was possible to cause complete CPE within 3 days after inoculation at a lower MOI of 5-10, then at least 3 more passages at 5-10 MOI and then attenuated The process then moves on to the next phase with a lower incubation temperature. The number of days required for each passage depends on how quickly the virus adapts to each phase of incubation temperature where it decreases incrementally. Those skilled in the art will readily know when full CPE has been reached. Further details of the method for preparing the cold-adapted temperature-sensitive enterovirus 71 strain of the present invention are described below.

  [0029] In a second aspect, the present invention provides a composition comprising the cold-adapted temperature sensitive enterovirus 71 strain described herein. In one embodiment, the composition comprises an effective amount of a virus strain described herein. In another embodiment, the composition comprises one or more physiologically or pharmaceutically acceptable carriers. In a further embodiment, the composition is a vaccine. Using techniques well known to those skilled in the art, a vaccine containing the cold-adapted temperature-sensitive enterovirus 71 strain described herein is prepared. Such vaccines are useful for providing immunity against a parental virus strain by administering the vaccine to a subject, eg, a human subject, using techniques well known to those skilled in the art.

  [0030] The cold-adapted temperature-sensitive enterovirus 71 strain described herein induces a protective immune response in a subject or prevents the subject from suffering from a virus-related disease, or initiates an enterovirus-related disease Is used to delay or slow its rate of progression, it is administered to a subject in the form of a composition further comprising one or more physiologically or pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, one or more 0.01M-0.1M and preferably 0.05M phosphate buffer. Solution, phosphate buffered saline (PBS), or 0.9% saline. Such carriers also include aqueous or non-aqueous solutions, suspensions, and emulsions. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, saline and buffered media. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, and fixed oil. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. The solid composition can also include a non-toxic solid carrier such as glucose, sucrose, mannitol, sorbitol, lactose, starch, magnesium stearate, cellulose or cellulose derivatives, sodium carbonate and magnesium carbonate. For administration in aerosols, for example for pulmonary and / or intranasal delivery, it is preferably formulated with non-toxic surfactants such as esters or partial esters of C6-C22 fatty acids or natural glycerides and propellants. Additional carriers such as lecithin may be included to facilitate intranasal delivery. Pharmaceutically acceptable carriers can also contain minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives and other additives such as antimicrobial agents, oxidation, which enhance the shelf life and / or effectiveness of the active ingredient. An inhibitor and a chelating agent may be included. The composition can be formulated to provide rapid, sustained or delayed release of the active ingredient after administration to a subject, as is well known in the art.

  [0031] In a third aspect, the present invention provides a method for eliciting a protective immune response in a subject, eg, a human subject, comprising a prophylactic or therapeutically or immunologically effective amount of the present specification. The method is provided comprising the step of administering the described cold adapted temperature sensitive enterovirus 71 strain to a subject. Accordingly, the present invention also provides a composition comprising a cold adapted temperature sensitive enterovirus 71 strain or a cold adapted temperature sensitive enterovirus 71 strain for use in inducing a protective immune response in a subject. The present invention also provides the use of a cold adapted temperature sensitive enterovirus 71 strain or a composition comprising a cold adapted temperature sensitive enterovirus 71 strain for the manufacture of a medicament for inducing a protective immune response in a subject. In one embodiment, the protective immune response protects the subject against diseases caused by enterovirus 71. In one embodiment, the disease is hand-foot-and-mouth disease. In another embodiment, the disease is aseptic meningitis. In a further embodiment, the disease is encephalitis. In a further embodiment, the disease is polio-like paralysis. In one embodiment, the cold-adapted temperature sensitive enterovirus 71 strain described herein is administered as a vaccine. In a further embodiment, the subject is exposed to wild type enterovirus 71. “Exposed” to enterovirus 71 means contact with enterovirus 71 such that an infection can occur. In another embodiment, administration of a cold-adapted temperature sensitive enterovirus 71 strain described herein prevents a subject, eg, a human subject, from suffering from an enterovirus 71 related disease. Accordingly, the present invention also provides a cold-adapted temperature-sensitive enterovirus 71 strain or a cold-adapted temperature-sensitive enterovirus 71 strain for use in preventing a subject, eg, a human subject, from suffering from an enterovirus 71-related disease. Compositions comprising are also provided. The present invention also includes a cold-adapted temperature sensitive enterovirus 71 strain or a cold-adapted temperature sensitive enterovirus 71 strain for the manufacture of a medicament for preventing a subject, eg, a human subject, from suffering from an enterovirus 71-related disease. Compositions are also provided. In a further embodiment, the subject is exposed to wild type enterovirus 71 strain. In further embodiments, administration of the cold-adapted temperature sensitive enterovirus 71 strain described herein delays the onset of or slows the rate of progression of enterovirus 71 related disease in a virally infected subject, eg, a human subject. Thus, the present invention also provides a cold adapted temperature sensitive enterovirus 71 strain or cold strain for use in delaying the onset of or slowing the rate of progression of enterovirus 71 related disease in a virally infected subject, eg, a human subject. Also provided is a composition comprising an adaptive temperature sensitive enterovirus 71 strain. The present invention also provides a cold-adapted temperature-sensitive enterovirus 71 strain or cold-adapted for the manufacture of a medicament for delaying the onset of or slowing the onset of enterovirus 71-related disease in a virus-infected subject, eg, a human subject. Compositions comprising temperature sensitive enterovirus 71 are also provided.

  [0032] As used herein, "administering" means delivering using any of a variety of methods and delivery systems known to those skilled in the art. Administration is, for example, intraperitoneal, intracerebral, intravenous, oral, transmucosal, subcutaneous, transdermal, intradermal, intramuscular, topical, parenteral, through transplantation, intrathecal, intralymphatic, intralesional, It can be performed around the heart or epidurally. The agent or composition can also be administered in an aerosol, eg for pulmonary and / or intranasal delivery. Administration can be performed, for example, once, multiple times, and / or for one or more extended periods.

  [0033] Induction of a protective immune response in a subject can be achieved, for example, by administering a primary dose of vaccine to the subject and continuing with one or more subsequent doses of vaccine after an appropriate period of time. An appropriate period between administrations of the vaccine can be readily determined by one skilled in the art and is usually from about several weeks to several months. The present invention, however, is not limited to any particular method, route or frequency of administration.

  [0034] A "prophylactically effective dose" or "immunologically effective dose" is defined by a subject when administered to a subject that is prone to viral infection or is susceptible to a virus-related disorder. Any amount of vaccine that induces in a subject an immune response that protects against infection with a virus or suffering from a disorder. “Protecting” a subject means reducing the likelihood of the subject becoming infected with the virus, or reducing the likelihood of initiating a disorder in the subject, at least 2-fold, preferably at least 10-fold. . For example, if a subject has a 1% chance of being infected with a virus, a two-fold reduction in the likelihood that the subject will be infected with a virus will result in a subject having a 0.5% chance of being infected with the virus. Will occur. Most preferably, a “prophylactically effective dose” induces an immune response in the subject that completely prevents the subject from being infected with the virus or completely prevents the onset of the disorder in the subject.

  [0035] Certain embodiments of any of the present immunization methods and therapies can further comprise administering to the subject at least one adjuvant. “Adjuvant” shall mean any agent suitable for enhancing the immunogenicity of an antigen and boosting an immune response in a subject. Many adjuvants, including certain adjuvants, and methods for combining antigens and adjuvants, well known to those skilled in the art, are suitable for use in both protein and nucleic acid based vaccines. Adjuvants suitable for use in protein immunization include, but are not limited to, alum, Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), alum adjuvant, saponin based adjuvants such as Quil A and QS-21 are included.

  [0036] The present invention also provides a method of attenuating viruses using temperature sensitivity as a phenotypic marker. According to this aspect, the method develops a cold adapted temperature sensitive virus strain. The method of the invention is an in vitro laboratory process that alters the biological growth characteristics of the virus and adapts for optimal replication at incubation temperatures below 30 ° C. The adaptation process is performed in a systematic, stepwise manner that incrementally reduces the incubation temperature at which the virus is cultured until the target temperature selected for optimal replication of the virus is achieved.

  [0037] Thus, according to the present invention, the method comprises the following steps: (i) preparing a reference stock of the parent wild type virus and (ii) culturing cells infected with the reference stock of the parent wild type virus To obtain lower MOI inoculum and complete CPE for 5 or more passages at higher multiplicity of infection (MOI) and incubation temperature of about 34 ° C. to about 36 ° C., preferably about 34 ° C. Incubate until a complete cytopathic effect (CPE) is obtained at each passage using a shorter incubation period for each passage, and (iii) cell cultures infected with the virus resulting from the previous step. Lower MOI inoculum for 5 or more passages at higher MOI and incubation temperatures about 1 ° C. to about 3 ° C. lower than the previous step. Incubate until a complete cytopathic effect (CPE) is obtained at each passage with a shorter incubation period of each passage to obtain complete CPE, and (iv) be selected for optimal replication of the virus Step (iii) is repeated in a systematic stepwise fashion that gradually decreases the incubation temperature until a target temperature is achieved. In one embodiment, the target temperature is from about 26 ° C to about 29 ° C, preferably about 28 ° C. In one embodiment, the incubation temperature that decreases incrementally is a temperature that decreases by about 1 ° C. to about 2 ° C.

  [0038] In one embodiment, the parental wild type virus reference stock is infected with the wild type virus at a temperature of about 36 ° C. to about 38 ° C., preferably at about 37 ° C., for one or two passages. The cell culture is prepared by incubating until a complete cytopathic effect (CPE) is obtained. An aliquot of the culture supernatant containing the produced virus is placed in a vial or other suitable storage device. This culture supernatant serves as a reference stock for the parental wild type virus. The reference parent wild type virus is used for the subsequent attenuation process. In another embodiment, an aliquot of the parental wild type virus reference stock is stored at a suitable temperature, eg, -80 ° C.

  [0039] In one embodiment, as soon as the virus has obtained a complete cytopathic effect (CPE), a clarified culture supernatant containing the virus is obtained and a continuous fresh new culture of cells, eg, Vero cells. The virus is passaged at each step of the process by passing this supernatant into each of the flasks. In another embodiment, the culture supernatant containing the virus is passed at a higher multiplicity of infection (MOI) of 20, at the beginning of each successive change to a lower temperature incubation. Within 3 days after inoculation, after recognizing that it is possible to cause complete CPE in Vero cells, the fresh confluent monolayer Vero is then subsequently reduced to a lower 5-10 MOI. A new culture flask containing cells is inoculated with the same MOI virus for at least three more passages. If it was recognized that it was possible to cause complete CPE within 3 days after inoculation at a lower MOI of 5-10, then at least 3 more passages at 5-10 MOI and then attenuated The process then moves on to the next phase with a lower incubation temperature. The number of days required for each passage depends on how quickly the virus adapts to each phase of incubation temperature that decreases incrementally. Those skilled in the art will readily know when full CPE has been reached.

  [0040] In one embodiment, the virus is any virus. In another embodiment, the virus is an RNA virus. In a further embodiment, the RNA virus is a positive strand RNA virus. In a further embodiment, the virus is a member of the Picornaviridae family. In another embodiment, the virus is a member of the Enterovirus genus. In one embodiment, the virus is enterovirus 71 (EV71). In another embodiment, the virus is Coxsackievirus A16 (CA16). The methods of the invention are useful in producing cold-adapted temperature sensitive virus strains of any of these viruses, including but not limited to EV71 and CA16 cold-adapted temperature sensitive strains.

  [0041] In one embodiment, the cell infected with the virus is any cell that allows the virus to grow. In a preferred embodiment, the cell is a Vero cell (ATCC CCL-81). In one embodiment, the cells are maintained by normal passage in a medium suitable for cell growth. In embodiments where the cells are Vero cells, the Vero cells are cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS). In one embodiment, Vero cells maintained in DMEM supplemented with 1% FCS are used for parental wild type virus production, virus culture, attenuation, titer determination, and evaluation of temperature sensitive phenotype. In another embodiment, DMEM is supplemented with 1% FCS to adapt the virus strain for replication as the incubation temperature is continuously reduced.

  [0042] The present invention also relates to cold-adapted temperature-sensitive strains of viruses produced by the methods described herein. Cold-adapted temperature sensitive viruses produced by the methods described herein are useful for the production of vaccines using techniques well known to those skilled in the art. Such vaccines are useful for providing immunity to the parental virus strain by administering the vaccine to a subject using techniques well known to those skilled in the art.

  [0043] The methods of the invention are applicable to the production of cold-adapted temperature-sensitive viruses of the Picornaviridae and Enterovirus genus. Applicability has been demonstrated herein by the production of cold-adapted temperature-sensitive strains of EV71 (TLLα) and EV71 (TLLβ) obtained after successive passages in cell culture at incrementally decreasing incubation temperatures. ing. The EV71 (TLLβ) strain retains phenotypic and genetic stability in certain in vitro cell culture conditions and is not neurotoxic in monkeys after intravenous inoculation. Applicability has also been demonstrated by the production of cold-adapted temperature-sensitive CA16 obtained after successive passages in cell culture at incrementally decreasing incubation temperatures.

  [0044] The present invention also provides kits for immunizing a subject with the cold-adapted temperature sensitive enterovirus 71 strain described herein. The kit includes a cold-adapted temperature sensitive enterovirus 71 strain described herein, a pharmaceutically acceptable carrier, an applicator, and instructions for use. The invention also includes other embodiments of kits known to those skilled in the art. The instructions can provide any information useful for directing administration of the cold-adapted temperature sensitive enterovirus 71 strain described herein.

  [0045] The practice of the present invention, unless otherwise indicated, is within the skill of the art, including chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture and Conventional techniques of transgenic biology are used. For example, Maniatis et al., 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); Sambrook et al., 1989, Molecular Cloning, New York (Cold Spring Harbor Co .; Cold Spring Harbor Co., Ltd.). Russell, 2001, Molecular Cloning, 3rd edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); Green and Sambrook, 2012, Molecular Cl oning, 4th edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); Ausubel et al., 1992, Current Protocols in Molecular Biology (including John Wiley & Sons, L Press, Oxford); Russell, 1984, Molecular biology of plants: a laboratory course manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); ues for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Guthrie and Fink, Guide to Yeast Genetics and Molecular Biology (Academic Press, New York, 1991); Harlow and Lane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); Nucleic Acid Hybridization (B. D. Hames and S.H. J. et al. Transgition And Translation (BD D Hames and S. J. Higgins 1984); Culture Of Animal Cells (R. I. Fredney, C. R. I. Fredney, 1994); (IRL Press, 1986); Perbal, A Practical Guide To Molecular Cloning (1984); Papers, Methods In Enzymology (Academic Press, Inc., NY); Methods In Enzymology. 154 and 155 (supervised by Wu et al.), Immunochemical Methods In Cell And Molecular Biology (supervised by Mayer and Walker, Academic Press, London, 1987); Handbook Of. Ex. Supervised by Blackwell, 1986); Riot, Essential Immunology, 6th edition, Blackwell Scientific Publications, Oxford, 1988; Fire et al., RNA Interference Technology: Fc. rug Development, Cambridge University Press, Cambridge, 2005; Schepers, RNA Interference in Practice, Wiley-VCH, 2005; Engelke, RNA Interference (RNAi): The Nuts & Bolts of siRNA Technology, DNA Press, 2003; Gott, RNA Interference, Editing, and Modification: Methods and Protocols (Methods in Molecular Biology), Human Press, Totowa, NJ, 2004; Sohail , Gene Silencing by RNA Interference: Technology and Application, CRC, 2004.

  [0046] The invention will be described by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described below were utilized.

Example 1
Materials and methods for developing cold-adapted temperature-sensitive strains of human enterovirus 71 (EV71)
[0047] Cells, viruses and cold adaptation process: Vero cells (ATCC CCL-81) maintained by periodic passage in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). , Virus culture, attenuation, titer determination and temperature sensitive phenotype evaluation. Three EV71 isolates belonging to genotypes C1, B3 and B4 isolated in Vero cells from oral secretions, stool and brain stem specimens, respectively, of patients presenting with hand, foot and mouth disease (HFMD) are described above The plaque was purified once (Doughherty, 1964) according to the technique described. After plaque purification, the Vero cells were subcultured twice at 37 ° C., and then virus stocks called parent wild type strains were prepared. All virus stocks and their respective passages were stored in a −80 ° C. freezer.

  [0048] Using young fresh confluent monolayer Vero cells in DMEM containing 1% FCS, starting with an initial low incubation temperature of 34 ° C., the virus strains were replicated for replication during a continuously decreasing incubation temperature. Adapted. As soon as the virus achieved a complete cytopathic effect (CPE), the clarified culture supernatant containing the virus was passed into each successive fresh culture flask of Vero cells. The culture supernatant containing the virus was passed at a higher multiplicity of infection (MOI) of 20, at the beginning of each successive change to a lower temperature incubation. Within 3 days after inoculation, after recognizing that it is possible to cause complete CPE in Vero cells, the fresh confluent monolayer Vero is then subsequently reduced to a lower 5-10 MOI. A new culture flask containing the cells was inoculated with the virus at the same MOI for three additional passages. If it was recognized that it was possible to cause complete CPE within 3 days after inoculation at a lower MOI of 5-10, after at least 3 additional passages, the attenuation process was then continued Moved to a lower incubation temperature. In this example, the progressively decreasing incubation temperatures for developing cold-adapted temperature-sensitive strains of enterovirus 71 were 34 ° C, 32 ° C, 30 ° C, 29 ° C, and 28 ° C.

[0049] Viral titration: With minor modifications, virus titer is determined by microtiter assay in Vero cells according to the method described in World Health Organization Polio Lab Manual 2004 and Reed And according to the method of Muench (1938), virus titers were calculated as 50% cell culture infectious dose (CCID ₅₀ ) per milliliter. Briefly, a 10-fold serial dilution of the clarified virus supernatant was made in DMEM containing 1% FCS after treatment with an equal volume of chloroform to disperse virus aggregates. Vero cell monolayers (10 ⁴ cells per well) in 96-well flat bottom tissue culture plates are inoculated with 100 μl of serial dilutions of each virus stock and at each respective incubation temperature in an ambient environment of 5% CO _2. After 5 days incubation, the presence of CPE was observed.

[0050] Temperature sensitivity assay: Two approaches were used to assess the growth characteristics of virus strains in Vero cells at incubation temperatures of 28 ° C, 37 ° C and 39.5 ° C. The first approach evaluates the number of days it takes for the virus strain to cause complete CPE in the infected cells (replication kinetics), and the second approach involves the virus in cells incubated at each specific test temperature. Stock titers were evaluated. Briefly, in the first approach, the growth medium of three T-25 tissue culture flasks containing similar day-old confluent monolayer Vero cells was replaced with maintenance medium (DMEM with 1% FCS). The medium in each flask was then equilibrated to the specified temperature to be tested by placing in each incubator for 1 hour and subsequently inoculated with a multiplicity of infection (MOI) dose of the virus strain. . If CPE was not observed at the end of 10 days of culture, the supernatant was passed into a new flask of monolayer Vero cells and incubated for another 10 days as well. If no CPE was observed after the second passage, it was interpreted as no virus replication. In the second approach, a Vero cell suspension at a density of 10 ⁴ cells per 100 μl was seeded into each well of three 96-well cell culture plates and incubated at 37 ° C. in an ambient environment of 5% CO ₂ . . Each cell culture plate was then equilibrated to the specified temperature to be tested by incubating in each incubator for 1 hour after 10 hours of incubation. Subsequently, cells in each well were inoculated with 100 μl of a 10-fold serial dilution of the virus strain, then transferred to the respective temperature incubator and incubated for 5 days before observing for the presence of CPE.

  [0051] Genetic Stability and Temperature Sensitivity Assay: To assess the genetic stability of virus strains cultured in the specified culture environment and cell type, the virus strains were cultured in cell culture at 5 MOI virus inoculum. Passage 20 more times and incubation at an incubation temperature of 28 ° C. At the end of 20 passages, the virus strains were evaluated for temperature sensitive phenotypic characteristics by culturing at incubation temperatures of 28 ° C., 37 ° C. and 39.5 ° C. as described above. Subsequently, the complete nucleotide sequence of each genome was sequenced and analyzed for the complete genome of each parental wild type virus.

[0052] The temperature sensitivity assay was reinstated for a stable cold-adapted temperature sensitive virus strain. Selected strains were passaged 5 times in monolayer Vero cells in an ambient environment of 5% CO ₂ at a temperature of 37 ° C. At each passage, a 10 MOI virus inoculum was used. Virus strains obtained at each passage were evaluated for growth characteristics in Vero cells at incubation temperatures of 28 ° C., 37 ° C. and 39.5 ° C. by methods similar to those described above for the temperature sensitivity assay. The complete genome of the virus strain was sequenced and analyzed at each successive passage at a culture temperature of 37 ° C.

  [0053] RNA extraction, RT-PCR and sequencing: Viral genomic RNA was extracted from cultures of intact CPE infected cells using a commercially available viral RNA extraction kit (Qiagen, Germany). First-strand synthesis was performed with EV71 specific primers using Superscript II RNA polymerase (Invitrogen, USA) and subsequently using 18 degenerate primer pairs using GoTaq Green PCR mix (Promega, USA). PCR was performed. The generated fragment was sequenced using the BigDye Terminator sequencing kit (Applied Biosystems, USA). Using standard T4 DNA ligase (Fermentas, USA), 5′-cordycepin blocking adapter DT88 (5′-GAA GAG AAG GTG GAA ATG GCG TTT TTG G-cordycepin-3 ′; SEQ ID NO: 1) was converted to EV71. By ligating to the 5 ′ end of the cDNA, 5 ′ RACE is performed to determine the 5′-UTR viral sequence and then the DT88 complementary primer (5′-CCA AAA CGC CAT TTC CAC CTT CTC TTC 3 ′ Standard PCR was performed using SEQ ID NO: 2) and EV71 specific primers (5′-ATT CAG GGG CCG GAG GAC TAC-3 ′; SEQ ID NO: 3). 3'-RACE was also performed to determine the 3'-UTR viral sequence using oligo dT primers (Li et al., 2005).

  [0054] Molecular cloning and plasmid purification: Fragments with ambiguous sequences were cloned into the pZero-2 plasmid (Invitrogen, USA) and transformed into TOP10 E. coli cells (Invitrogen, USA). Using a commercially available plasmid miniprep kit (Qiagen, Germany), a plasmid containing the cloned EV71 fragment was extracted from at least 10 colonies of each transformant and subsequently cloned fragments The sequence of was determined.

  [0055] European Molecular Biology Open Software Suite (EMBOSS; http://mobile.pasture.fr/cgi-bin/portal.py?#forms:merger) (Rice et al. 2000) Merged. Program, BioEdit Sequence Alignment Editor v. 7.0.9.0 (Hall, 1997) was used to align the merged sequence with the reference sequence of EV71 strain 3799-SIN-98 (GenBank accession number DQ341354.1).

  [0056] Monkey studies: Monkey studies on the safety and immunogenicity of selected cold-adapted temperature-sensitive strains of EV71 (EV71: TLLβP20) were undertaken by researchers based on animal facilities in DUKE-NUS, Singapore (Contracted). Mycobacterium tuberculosis and 7 cynomolgus monkeys (Macaca fascicularis), 3 females (2202F, 2207F, 2891F) and 4 males (0791M, 2289F, 4789M, 4789M, 4789M, 4789F) 2890M), an average body weight of 3.23 kg (range 2.44-4.11, SD = 0.7) was used to study the safety and immunogenicity of stable cold-adaptive temperature-sensitive EV71: TLLβP20. All seven monkeys were prescreened for binding to EV71 (indirect immunofluorescence) and the absence of neutralizing antibodies. The study and all animal procedures were approved by DUKE-NUS, Singapore Biosafety and Animal Handling and Ethics Committee. Viral inoculation and observation, animal breeding and necropsy were performed according to committee guidelines.

[0057] Under light anesthesia with ketamine, 1 ml of the virus inoculum was inoculated intravenously into the right saphenous vein. Three monkeys (2889M, 0791M and 2891F) received an intravenous dose of EV71: TLLβP20 at 10 ⁷ CCID ₅₀ per monkey, and another 3 (2890M, 2202F and 2247M) each with 10 ⁸ CCID ₅₀ And the seventh monkey served as a negative control. Monkeys were observed twice daily for clinical illness, particularly neurological signs, and body temperature was recorded by an implant temperature sensor. Feces from each monkey were collected daily and stored in a minus 80 ° C. freezer for later virus isolation. Two monkeys, one from each inoculum at different viral doses, were sacrificed under deep anesthesia 4 days after inoculation (PI). At necropsy, various parts of the central nervous system (CNS), non-neural tissues and blood were collected for histopathological and virological analysis. On day 8 PI, another similar set of two monkeys were sacrificed and the same type of tissue and blood was collected for histopathological and virological analysis at necropsy. After collecting blood samples for evaluation of 14-day PI, anti-EV71 antibody, the remaining two monkeys were each administered an equivalent booster dose of EV71: TLLβP20. Sixteen days after the booster dose was administered, they were euthanized and CNS tissues were collected for histopathological studies at necropsy.

  [0058] Histology and immunohistochemistry: Phosphate buffered saline for CNS tissue specimens (cerebrum, cerebellum, basal ganglia, brainstem and spinal cord) and non-neural tissues (lymph node, spleen, liver, kidney, lung and heart) Fixing with 10% formalin in (PBS) and embedding in paraffin after fixation. The spinal cord was sliced horizontally, 10 times in the neck, 8 times in the chest, and 10 times in the lumbar region. Paraffin sections, 6 μm thick, were stained with hematoxylin and eosin (H & E) and Luxol-fast blue / cresyl violet (Cluver-Verrara method) after the paraffin removal and rehydration process.

[0059] Antigen detection and virus isolation from monkeys: Blood specimens were collected in both simple and heparinized tubes. Peripheral blood mononuclear cells (PBMC) were collected from heparinized blood using Ficoll-Paque ^™ PLUS (GE Healthcare, Sweden). After washing twice with sterile PBS, PBMCs were detected on wells of Teflon-coated slides for detection of EV71 antigen by an indirect immunofluorescence assay using a commercial detection monoclonal antibody (Cat. No. 3360, Light Diagnostics, USA). An aliquot of the suspension was planted. Virus isolation was performed by inoculating a PBMC suspension into the wells of a 24-well cell culture plate containing monolayer Vero cells. Virus isolation on each serum specimen was performed by inoculating 50 μl and 100 μl of serum into each well of a 24-well culture plate containing monolayer Vero cells. The tissue was homogenized by changing the sterile PBS twice, gently washing the monkey tissue, and grinding with a mortar and pestle in a Class II biosafety cabinet. Tissue homogenate (10%, w / v) prepared in DMEM was clarified by centrifugation at 1000 g for 10 minutes. Virus isolation was performed by filtering the clarified supernatant through a 0.22 micron syringe filter and inoculating 100 μl and 200 μl of filtrate. A 10% fecal suspension was made in PBS and clarified by centrifugation at 1000 g for 10 minutes. Virus isolation was performed by inoculating 100 μl and 200 μl fecal filtrate after filtration through a 0.22 micron syringe filter. All virus isolation work on monkey samples was performed in duplicate, one set of inoculated cell cultures was incubated at 28 ° C and the other was incubated at 37 ° C.

  [0060] Molecular detection and complete genome sequencing: Viral genomic RNA was extracted from serum, PBMC and clarified tissue homogenates using a commercial viral RNA extraction and purification kit (Qiagen, Germany). A commercial one-step RT-PCR kit (Qiagen, Germany) and a consensus oligonucleotide primer pair (sense: 5'-CACCCTTTGATACCATGATCAG-3 '(SEQ ID NO: 3) that amplifies the proximal third of the VP1 gene of all EV71 genotypes 4); Antisense: 5′-GTGAATTAAGAAACCAYCGTGTYT-3 ′ (SEQ ID NO: 5)) was used for molecular amplification after extraction from tissues and EV71 specific viral RNA detection. EV71: The complete genome of EV71 present in the serum of two monkeys obtained at 4 and 8 days PI was amplified and sequenced using 18 pairs of sequence specific primers based on the complete genome sequence of EV71: TLLβ. Any PCR amplified fragment that failed to produce a superior sequence read by direct sequencing was cloned into pZero-2 (Invitrogen, USA) and transformed into TOP10 E. coli. At least 10 colonies were selected from each transformant and sequenced on a purified plasmid carrying the insert.

  [0061] Serum binding and neutralizing antibody assay: Vero cells infected with genotype B3 EV71 were harvested close to complete CPE and washed 5 times with sterile PBS. After the last wash, the infected cell suspension was mixed with the washed uninfected Vero cell suspension at a ratio of approximately 4 infected cells to 1 uninfected cells. A 10 microliter mixed cell suspension containing 250 cells was carefully layered onto each well of a 12-well Teflon-coated slide and dried on a warm plate. The dried slides were fixed in cold acetone for 10 minutes and used as in-house antigen, assayed for EV71 binding antibody by indirect immunofluorescence assay, starting with an initial dilution of 1:10 for IgM and 1:20 for IgG. did. In an assay for anti-EV71 IgM titers, monkey serum was treated with the appropriate concentration of protein A (Invitrogen, USA) to remove IgG before performing serial 2-fold dilutions with sterile PBS.

[0062] With minor modifications, monkey neutralizing antibody titers were determined by microneutralization assay using Vero cells according to the method described in the World Health Organization Polio Laboratory Manual 2004. The concentration of each genotype of EV71 used for neutralization was 100 CCID ₅₀ per 100 μl. Virus neutralization assays were performed using 96 well flat bottom culture plates. Starting from a 1:10 dilution, serial 2-fold dilutions of each serum sample were prepared in duplicate in a volume of 100 μl DMEM (1% FCS). An equal volume (100 μl) of virus working stock in DMEM (1% FCS) containing 100 CCID ₅₀ EV71 was added to each well of diluted serum and incubated at 37 ° C. for 2 hours. After incubation, 100 μl of Vero cell suspension in DMEM (10% FCS) containing 250 cells was added to each well. The 96-well culture plate was carefully sealed and incubated at 37 ° C. in an ambient environment of 5% CO ₂ . In each well, plates were read daily for up to 8 days for the presence of CPE affecting Vero cells. The titer of neutralizing antibody in each serum sample was determined by the highest dilution of wells showing no CPE.

Example 2
Results on phenotypic and genotypic characteristics of cold-adapted temperature-sensitive strains of enterovirus 71 (EV71: TLLα, EV71: TLLαP20, EV71: TLLβ, EV71: TLLβP20 and EV71: TLLβP40)
[0063] The original three parental EV71 viruses used to obtain cold-adapted strains were completely isolated in Vero cells within 3 days after inoculation at 10 MOI of virus inoculum and 37.5 ° C. incubation temperature. Caused CPE. With the same virus inoculum, the original parental EV71 virus caused complete CPE in Vero cells within 5 days at an incubation temperature of 39.5 ° C. However, all three original parental EV71 viruses obtained from the first two passages of Vero cells cultured at 37.5 ° C. were obtained at 10 MOI virus inoculum and 28 ° C. incubation temperature at Vero cells. Did not cause CPE. Moreover, CPE was not seen at all after blind passage at the end of 10 days of culture at 28 ° C. The absence of viral replication in the inoculated Vero cells was further confirmed by the presence of suspended cells in the culture supernatant at the end of 10-day culture with a monoclonal antibody commercially detected against EV71 by an indirect immunofluorescence assay. Further support by negative staining.

  [0064] After more than 90 passages in which the incubation temperature is continuously reduced, all three passage EV71 strains are within 3 days of inoculation with a viral inoculum of ≦ 5 MOI and an incubation temperature of 28 ° C. It was possible to cause complete CPE in Vero cells. The virus strain was further passaged at an incubation temperature of 28 ° C. up to 100 passages. At the 100th passage, the original EV71-derived virus strains isolated from the patient's oral fluid, brainstem tissue and stool specimens were designated EV71: TLL, EV71: TLLα and EV71: TLLβ, respectively. In the temperature sensitivity assay, all three cold-adapted strains caused complete CPE in Vero cells within 2 days at an incubation temperature of 28 ° C. using a 10 MOI virus inoculum, but incubation at 37 ° C. At temperature, it took 4 days to achieve full CPE. No CPE was seen for all three strains after 10 days in culture at an incubation temperature of 39.5 ° C. However, EV71: TLL produced 2+ CPE on day 10 of culture after blind passage into a fresh flask of Vero cells incubated at 39.5 ° C. Even after two additional blind passages in a fresh flask of Vero cells incubated at 39.5 ° C., no CPE was seen for EV71: TLLα and EV71: TLLβ. Suspension cells that were inoculated with either EV71: TLLα or EV71: TLLβ and collected from the culture supernatant, including those obtained from blind passage, were each at the end of 10 days of culture, respectively. Did not produce positive immunofluorescent staining with. Approximately 1% of the suspension cells collected from the culture supernatant inoculated with EV71: TLL produced positive staining, but no CPE was seen after 10 days in culture.

[0065] Virus replication titers of the three cold-adapted strains were assayed using incubation temperatures of 28 ° C and 37 ° C, and the assay was repeated at least four times. The titers of EV71: TLL were 1 × 10 ⁸ CCID ₅₀ / ml and 2-3 × 10 ⁷ CCID ₅₀ / ml when the cultures titered at 28 ° C. and 37 ° C., respectively, were incubated. EV71: TLLα produced a titer of 1 × 10 ⁸ CCID ₅₀ / ml at an incubation temperature of 28 ° C. and a titer of ^1-2 × 10 ^5.5-6 CCID ₅₀ / ml at 37 ° C. EV71: TLLβ produced a titer of 2-5 × 10 ⁸ CCID ₅₀ / ml at an incubation temperature of 28 ° C. and a titer of 1 × 10 ⁷ CCID ₅₀ / ml at 37 ° C.

[0066] The stability of the cold adaptation of EV71: TLLα and EV71: TLLβ was compared to both virus strains over a further 20 passages in Vero cells at 28 ° C incubation temperature and 10 MOI virus inoculum. Was evaluated by passage. After an additional 20 passages, EV71: TLLα caused complete CPE in cells incubated at 28 ° C. within 2 days PI, but at 37 ° C. even after 2 blind passages. In incubated cells, it failed to cause CPE. The ability to achieve a virus titer of 1 × 10 ⁸ CCID ₅₀ / ml was maintained at an incubation temperature of 28 ° C. EV71: TLLβ maintained the same cold-adapted phenotype for growth kinetics and virus titer at both 28 ° C. and 37 ° C. incubation temperatures after an additional 20 passages. By further 20 passages (100 passages to 40 passages) under the same culture conditions, the stability of cold adaptation of EV71: TLLβ was further evaluated and similar cold adaptation temperature sensitivity It was found to maintain the phenotype.

  [0067] Evaluation of reversion from the cold-adaptive temperature-sensitive phenotype was performed by passage of the virus six times in succession in Vero cells incubated at 37 ° C with each complete CPE. The growth characteristics of the virus obtained from each of the respective cultures incubated at 37 ° C. with respect to growth kinetics and virus titer at incubation temperatures of 28 ° C., 37 ° C. and 39.5 ° C. are shown in Table 1. The virus remains unable to produce viable infectious particles in Vero cells after 3 consecutive passages in cells incubated at 37 ° C. and at an incubation temperature of 39.5 ° C. (positive immunofluorescent staining). Lacking cells), and at the 6th repeated passage, it remained impossible to cause complete CPE in cell cultures at an incubation temperature of 39.5 ° C.

Table 1
Evaluation of EV71: TLLβP20 reversion from a cold-adapted temperature-sensitive phenotype after six consecutive passages of Vero cells incubated at 37 ° C

  Growth characteristics and virus titer at each passage were cultured or titered in Vero cells at incubation temperatures of 28 ° C., 37 ° C. and 39.5 ° C.

P1: Passage 1
IFA: Indirect immunofluorescence assay
[0068] EV71: TLLα and EV71: TLLβ, the respective strains after 20 additional passages (EV71: TLLαP20 and EV71: TLLβP20) and the original parental wild type complete genome were sequenced and analyzed. The nucleotide sequence of EV71: TLLαP20 is shown in SEQ ID NO: 6. The nucleotide sequence of EV71: TLLβP20 is shown in SEQ ID NO: 7. Table 2 shows the number of nucleotide changes in each gene segment between EV71: TLLα, EV71: TLLαP20 and the original parental wild type. The number of nucleotide changes between the original parental wild type, EV71: TLLβ, EV71: TLLβP20 and EV71: TLLβP40 (an additional 40 passages) is shown in Table 3. The complete genome of the virus strain obtained from a temperature-sensitive reversion study with 6 consecutive passages at an incubation temperature of 37 ° C. was also sequenced and the number of nucleotide changes compared to that of EV71: TLLβP20 is shown in Table 4 .

Table 2
The number of nucleotide (NT) and corresponding amino acid (AA) mutations that occur in each of the EV71: TLLα and EV71: TLLαP20 genomic segments compared to the complete genome of the original parental wild-type virus

Table 3
The number of nucleotide (NT) and corresponding amino acid (AA) mutations occurring in each of the EV71: TLLβ, EV71: TLLβP20 and EV71: TLLβP40 genomic segments compared to the original parental wild-type virus genome

Table 4
EV71: Number of nucleotide (NT) and corresponding amino acid (AA) mutations that occur in each genomic segment of a virus strain from a temperature-sensitive reversion study compared to the genome of TLLβP20

Example 3
Results on monkey study of cold-adapted temperature-sensitive strain of human enterovirus 71 (EV71: TLLβP20)
[0069] Clinical findings: General observations regarding the clinical status and temperature measurements of the monkeys studied were made twice daily in the morning and evening. Throughout, all monkeys were found to be active and eating normally. None of the monkeys developed any minor local neurological defects such as leg weakness, tremor or abnormal movement. These cases, except that one monkey (2247M) receiving an intravenous dose of 1 × 10 ⁸ CCID ₅₀ / ml showed a transient low grade fever (39.3 ° C.) 3 days after inoculation There was no. None of the monkeys lost weight during reweight measurements until the monkey was sacrificed under deep anesthesia.

  [0070] Autopsy and histological findings: No overall postmortem lesions were seen for all monkeys at necropsy. Abnormal histological findings were not found for all monkey tissues collected for histopathology.

[0071] Virological study: Virus isolation was performed on monkey serum, PBMC, stool samples and all autopsy tissues using Vero cells at incubation temperatures of 28 ° C and 37 ° C. After 10 days in culture, no virus was isolated from any of the monkey samples despite blind passage. One additional blind passage was performed in Vero cells for sera, PBMC samples and tissue homogenates that gave a positive test result for EV71 by RT-PCR. Two monkeys, 2891F (EV71: TLLβP20 1 × 10 ⁷ CCID ₅₀ ) collected at 4 days PI by indirect immunofluorescence assay using commercial monoclonal antibodies (FIG. 1a), although virus isolation failed. EV71 antigen was detected in several PBMCs obtained from heparinized blood at 2890M (administered) and 2890M (administered 1 × 10 ⁸ CCID ₅₀ of EV71: TLLβP20). No viral antigens were detected in PBMCs collected from monkey heparinized blood collected at 8 days PI.

  [0072] RT-PCR detected EV71-specific genomic sequences in all monkey serum samples collected on both the 4-day and 8-day PIs. Among non-neural tissues, the EV71 genomic sequence was detected only in spleen homogenates of two monkeys (2202F and 2891F) (FIG. 1b). No EV71 genomic sequence was detected in any of the neural tissue homogenates. The genome of EV71: TLLβP20 present in the serum samples of two monkeys (2889M and 2890M) collected on both the 4-day and 8-day PIs was extracted and fully sequenced. The number of nucleotide (NT) and corresponding amino acid (AA) mutations or reversions that occur in each genome segment of the virus strain present in serum compared to the genome of EV71: TLLβP20 is shown in Table 5.

Table 5
EV71: nucleotide (NT) and corresponding corresponding to each genomic segment of EV71 present in the serum samples of 2 monkeys (2889M and 2890M) collected at 4 and 8 days PI compared to the complete genome of TLLβP20 Number of amino acid (AA) mutations

  [0073] Monkey humoral immune response: an assay for the presence of bound antibodies (IgM and IgG) in monkey serum administered intravenous EV71: TLLβP20 by an indirect immunofluorescence assay using in-house prepared infected Vero cells as antigen Executed. Anti-EV71 IgM present in the blood of two remaining monkeys (2889M and 2890M) collected at 14-day PI and at 30-day PI (16 days after booster) before administering an equivalent intravenous booster dose And IgG titers are shown in Table 6.

Table 6
Titers of monkey-bound antibodies (IgM and IgG) after administration of intravenous EV71: TLLβP20 as determined by indirect immunofluorescence assay using in-house prepared infected Vero cells as antigen

  [0074] Neutralizing antibody titers in serum samples of 2 monkeys collected at 14 and 30 days PI against EV71 genotype A (BrCr), B3, B4, B5, C1 and C5 by microneutralization assay It was determined. The respective titers of monkey serum neutralizing antibodies against each of the EV71 genotypes are shown in Table 7.

Table 7
Medium to several genotypes of EV71 (A, B3, B4, B5, C1 and C5) in sera of two monkeys after administration of intravenous EV71: TLLβP20 as determined by microneutralization assay The titer of the Japanese antibody.

Example 4
Materials and methods for developing cold-adapted temperature-sensitive strains of human coxsackievirus A16
[0075] Materials and Methods: Vero cells (ATCC CCL-81) maintained by periodic passage in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) were obtained from Coxsackievirus A16. Used for virus culture, attenuation, titer determination and temperature sensitivity evaluation. Parental wild-type Coxsackievirus A16 (CA16) for the attenuation process is obtained from the oral secretions of patients with hand, foot and mouth disease (HFMD). The virus was plaque purified once according to the technique described above. After plaque purification, the Vero cells were subcultured twice at 37 ° C., and then virus stocks called parent wild type strains were prepared. All virus stocks and their respective passages were stored in a −80 ° C. freezer.

  [0076] Using young fresh confluent monolayer Vero cells in DMEM containing 1% FCS, starting with the first lower incubation temperature of 34 ° C, the original for replication Wild type CA16 was adapted. At the time of complete cytopathic effect (CPE), Vero cells were subcultured into continuous culture flasks. Recognizing that complete CPE can be caused by passage at 20 higher multiplicity of infection (MOI) and within 3 days after inoculation when continuously changing to lower incubation temperatures And then reduced to a lower 5-10 MOI after at least 3 additional passages. If it was recognized that it was possible to cause complete CPE within 3 days after inoculation with an MOI of 5-10, then at least 3 more passages were followed, followed by continuous lower incubation temperatures. The culture supernatant from each successive passage was stored in a minus 80 ° C. freezer for later analysis.

[0077] Viral titer determination: With minor modifications, virus titer is determined by microtiter assay in Vero cells according to the method described in the World Health Organization Polio Laboratory Manual 2004, and Reed And according to the method of Muench (1938), virus titers were calculated as 50% cell culture infectious dose (CCID ₅₀ ) per milliliter. Briefly, a 10-fold serial dilution of the clarified virus supernatant was made in DMEM containing 1% FCS after treatment with an equal volume of chloroform to disperse virus aggregates. Vero cell monolayers (10 ⁴ cells per well) in 96-well flat bottom tissue culture plates are inoculated with 100 μl of serial dilutions of each virus stock and at each respective incubation temperature in an ambient environment of 5% CO _2. After 5 days incubation, the presence of CPE was observed.

[0078] Temperature Sensitive Assay: Two approaches were used to assess the growth characteristics of cold-adapted virus strains in Vero cells at incubation temperatures of 28 ° C, 37 ° C and 39.5 ° C. The first approach evaluates the number of days it takes for the virus strain to cause complete CPE in the infected cells (replication kinetics), and the second approach involves the virus in cells incubated at each specific test temperature. Stock titers were evaluated. Briefly, in the first approach, the growth medium of three T-25 tissue culture flasks containing similar day-old confluent monolayer Vero cells was replaced with maintenance medium (DMEM with 1% FCS). The medium in each flask was then equilibrated to the specified temperature to be tested by placing in each incubator for 1 hour and subsequently inoculated with a multiplicity of infection (MOI) dose of the virus strain. . If CPE was not observed at the end of 10 days of culture, the supernatant was passed into a new flask of monolayer Vero cells and incubated for another 10 days as well. If no CPE was observed after the second passage, it was interpreted as no virus replication. In the second approach, a Vero cell suspension at a density of 10 ⁴ cells per 100 μl was seeded into each well of three 96-well cell culture plates and incubated at 37 ° C. in an ambient environment of 5% CO ₂ . . Each cell culture plate was then equilibrated to the specified temperature to be tested by incubating in each incubator for 1 hour after 10 hours of incubation. Subsequently, cells in each well were inoculated with 100 μl of a 10-fold serial dilution of the virus strain, then transferred to the respective temperature incubator and incubated for 5 days before observing for the presence of CPE.

Example 5
Results on virus characteristics in cells of cold-adapted temperature-sensitive strain of human coxsackievirus A16
[0079] At 100 passages in which the incubation temperature is continuously reduced, cold-adapted CA16 strains were treated with complete CPE in Vero cells within 3 days of inoculation at a virus inoculum of ≦ 5 MOI and an incubation temperature of 28 ° C. However, at an incubation temperature of 37 ° C., it took 4 days to achieve full CPE. At the incubation temperature of 39.5 ° C., no CPE was seen.

[0080] Virus replication titers of cold-adapted temperature-sensitive strains of CA16 were assayed using incubation temperatures of 28 ° C and 37 ° C. The titer of the cold-adapted temperature sensitive strain of CA16 was 1 × 10 ⁸ CCID ₅₀ / ml and 1 × 10 ⁷ CCID ₅₀ / ml when the titer culture plates were incubated at 28 ° C. and 37 ° C., respectively.

  [0081] In the context of describing the present invention (especially in the context of the following claims), the use of the terms "a" and "an" and "the" and similar reference controls are indicated elsewhere herein. Unless otherwise clearly contradicted by context, it shall be considered to include both the singular and plural. The terms “including”, “having”, “included” and “containing” are to be considered open-ended terms (ie, including but not limited to) unless otherwise indicated. ”). The recitation of value ranges herein is intended to serve as a simplification for individually referring to each distinct value within the range, unless otherwise indicated herein, and each distinct value is It is incorporated within this specification as if it were individually mentioned in the specification. For example, if ranges 10-15 are disclosed, 11, 12, 13, and 14 are also disclosed. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by background. The use of any examples or exemplary language (eg “for example”) provided herein is merely intended to facilitate an understanding of the invention and, unless otherwise claimed, the scope of the invention No restrictions are imposed on No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

  [0082] It will be appreciated that the methods and compositions of the present invention can be incorporated in a variety of forms, only a few are disclosed herein. Aspects of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these aspects will be apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors contemplate that those skilled in the art will properly use such variations and intend to practice the invention differently from those specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

  List of references

Claims (36)

  Low temperature adaptive temperature sensitive enterovirus 71 strain.   The cold-adapted temperature-sensitive enterovirus 71 strain according to claim 1, which is EV71: TLLβP20.   The cold-adapted temperature-sensitive enterovirus 71 strain according to claim 1, which is EV71: TLLαP20.   A composition comprising the cold-adapted temperature-sensitive enterovirus 71 strain according to any one of claims 1 to 3.   The composition of claim 4 further comprising a pharmaceutically acceptable carrier.   The composition of claim 4 or 5, further comprising an adjuvant.   The composition according to any one of claims 4 to 7, which is a vaccine.   The cold-adapted temperature-sensitive enterovirus 71 strain according to any one of claims 1 to 3, or the composition according to any one of claims 4 to 8 for use in inducing a protective immune response in a subject.   The cold-adapted temperature-sensitive enterovirus 71 strain of any one of claims 1-3, or any one of claims 4-8 for use in preventing a subject from suffering from an enterovirus 71-related disease. Item composition.   The cold-adapted temperature-sensitive enterovirus 71 strain of any one of claims 1-3 for use in delaying or slowing the onset of enterovirus 71-related disease in a subject infected with enterovirus 71, or claim Item 9. The composition according to any one of Items 4 to 8.   A cold-adapted temperature sensitive enterovirus 71 strain according to any one of claims 1 to 3, or a composition according to any one of claims 4 to 8 for the manufacture of a medicament for inducing a protective immune response in a subject. Use of.   The cold-adapted temperature-sensitive enterovirus 71 strain according to any one of claims 1 to 3, or any one of claims 4 to 8 for the manufacture of a medicament for preventing a subject from suffering from an enterovirus 71-related disease. Use of a composition according to claim.   A cold-adapted temperature-sensitive enterovirus 71 strain according to any one of claims 1 to 3, for the manufacture of a medicament for delaying or slowing the onset of enterovirus 71-related disease in a subject infected with enterovirus 71, or Use of a composition according to any one of claims 4-8.   Prevention of a protective immune response in a subject, comprising the cold-adapted temperature-sensitive enterovirus 71 strain of any one of claims 1-3, or the composition of any one of claims 4-8. Administering a therapeutically, therapeutically or immunologically effective amount to the subject.   15. The method of claim 14, wherein the subject is exposed to wild type enterovirus 71.   A method for preventing a subject from suffering from an enterovirus 71-related disease, the cold-adapted temperature-sensitive enterovirus 71 strain according to any one of claims 1 to 3, or any one of claims 4 to 8. Administering a prophylactically, therapeutically or immunologically effective amount of the composition of claim to the subject.   17. The method of claim 16, wherein the subject is exposed to wild type enterovirus 71.   A method for delaying or slowing the onset of enterovirus 71-related disease in a subject infected with enterovirus 71, comprising the cold-adapted temperature sensitive enterovirus 71 strain of any one of claims 1 to 3, or claim 4 9. The method comprising administering to a subject a prophylactic, therapeutically or immunologically effective amount of the composition of any one of -8.   The method according to any one of claims 14 to 18, wherein the subject is a human subject.   A kit for immunizing a subject with a cold-adapted temperature-sensitive enterovirus 71 strain, the cold-adapted temperature-sensitive enterovirus 71 strain according to any one of claims 1 to 3, or any one of claims 4 to 8. The kit comprising a composition of: a pharmaceutically acceptable carrier, and instructions for its use.   21. The kit of claim 20, further comprising an applicator. A method for producing a cold-adapted temperature-sensitive virus comprising the following steps:
(I) preparing a reference stock of the parental wild type virus;
(Ii) a culture of cells infected with a reference stock of the parental wild-type virus, 5 times or more at a higher multiplicity of infection (MOI) and an incubation temperature of about 34 ° C to about 36 ° C, preferably about 34 ° C; Incubate until a complete cytopathic effect (CPE) is obtained at each passage, using a higher passage number, a lower MOI inoculum and a shorter incubation period of each passage to obtain complete CPE;
(Iii) Five or more passages of the culture of cells infected with the virus resulting from the previous step at a higher MOI and incubation temperature about 1 ° C. to about 3 ° C. lower than the previous step. Incubate with a lower MOI inoculum and a shorter incubation period at each passage to obtain complete CPE until a complete cytopathic effect (CPE) is obtained at each passage; and (iv) Repeating step (iii) in a systematic stepwise manner that incrementally reduces the incubation temperature until the target temperature selected for optimal replication is achieved;
Said method comprising the steps.   A culture of cells infected with wild type virus is obtained at a temperature of about 36 ° C. to about 38 ° C., preferably about 37 ° C., until a complete cytopathic effect (CPE) is obtained for one or two passages. 23. The method of claim 22, wherein the reference stock of the parental wild type virus is prepared by incubation.   24. The method of claim 22 or 23, wherein the virus is a member of the Picornaviridae family, preferably the Enterovirus genus.   25. The method of claim 24, wherein the virus is enterovirus 71 or coxsackievirus A16.   26. The method of any of claims 22-25, wherein the target temperature is from about 26C to about 29C, preferably about 28C.   As soon as the virus has a complete cytopathic effect (CPE), a clarified culture supernatant containing the virus is obtained and this supernatant is passed into each successive fresh culture flask of cells. 27. The method of any of claims 22-26, wherein the virus is passaged at each step of the process.   28. The method of any of claims 22-27, wherein the higher MOI is 20.   29. A new culture flask containing fresh confluent monolayer Vero cells is inoculated with 20 MOI viruses for 3 additional passages after the virus has caused full CPE. the method of.   30. The method of any of claims 22-29, wherein the lower MOI is 5-10.   31. A new culture flask containing fresh confluent monolayer Vero cells is inoculated with a virus at a MOI of 5-10 for at least 3 more passages after the virus has caused complete CPE. the method of.   32. The method of claim 31, wherein step (iii) is initiated or repeated at a MOI of 5-10 after said at least 3 further passages.   33. A method according to any one of claims 27 to 32, wherein complete CPE occurs within 3 days after inoculation.   34. The method of any of claims 22-33, wherein the cell is a Vero cell.   35. The method of any of claims 22-34, wherein Vero cells are cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal calf serum (FCS).   36. The method of claim 35, wherein the medium is supplemented with 1% FCS.

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