TW201522642A - Cold-adapted temperature sensitive strains of enterovirus 71 and processes of developing cold-adapted temperature sensitive virus strains - Google Patents

Abstract

The present invention relates to cold-adapted temperature sensitive Enterovirus 71 strains, particularly to the cold-adapted temperature sensitive Enterovirus 71 strains EV71:TLL[beta]P20 and EV71:TLL[alpha]P20. The present invention also relates to processes of developing the cold-adapted temperature sensitive virus strains.

Description

Temperature sensitive strain suitable for cold enterovirus 71 and method for developing cold temperature sensitive virus strain Sequence submission

This application is filed with the sequence listing of the electronic format. The sequence listing name is 2577224PCTSequenceListing.txt, which was produced on January 11, 2013 and has a size of 21 kb. The information in the sequence listing of the electronic format is incorporated herein by reference in its entirety.

The present invention relates to a temperature sensitive strain suitable for cold enterovirus 71, specifically for cold enterovirus 71 temperature sensitive strains EV71:TLLβP20 and EV71:TLLαP20. The invention also relates to methods for developing cold temperature sensitive virus strains, specifically RNA virus strains.

The publications and other materials used herein to clarify the background of the invention or to provide further details of the practice are incorporated by reference.

Hand, Foot and Mouth Disease (HFMD) is caused by a human enterovirus that is essentially a group of enterovirus species A. Among these enteroviruses, Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) are responsible for more than 90% of HFMD cases. In addition to causing HFMD, EV71 is known to cause severe neurological diseases as well as death (especially in young children).

Human Enterovirus 71 (EV71) is a small, non-enveloped virus of approximately 30 nm in size with a single-strand positive RNA genome of approximately 7,450 nucleotides. The virus is classified as a human enterovirus A species under the genus Enterovirus of the family Piporaviridae (Alexander et al., 1994; Melnick, 1996). Phylogenetic analysis based on the major capsid protein (VP1) gene of EV71 was divided into three major genomes (indicated as A, B, and C), and genomes B and C were further subdivided into subgenome B1 to B5 and C1 to C5 (Bible et al., 2008). EV71 has been associated with a number of clinical diseases including primary hand, foot and mouth disease (HFMD), aseptic meningitis, encephalitis, and polio 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 novel serotype of Enterovirus (Schmidt et al., 1974). In the years following its initial isolation, HFMD with complications due to the virus was reported worldwide (Blomberg et al., 1974; Kennett et al., 1974; Deibel et al., 1975; Hagiwara et al., 1978).

In recent years, after the contagious and sporadic outbreak of neurotoxic EV71, EV71 infection has become a major public health burden and problem in the Asia-Pacific region of the world. The neurotoxicity of EV71 first gained significant global attention during the outbreak in Bulgaria in 1975, which caused 705 cases of poliomyelitis, including 44 deaths (Chumakov et al., 1979; Shindarov et al., 1979). An outbreak of similar nature occurred in Hungary in 1978 and produced multiple cases of poliomyelitis and 47 deaths (Nagy et al., 1982). Subsequently, several milder infectious diseases of CNS diseases associated with EV71 have been reported in New York, Hong Kong, Australia, and Philadelphia (Chomnaitree et al., 1958; Samuda et al., 1987; Gilbert et al., 1988; Hayward et al. 1989). Two EV71 infectious diseases have emerged in Japan, most of which are characterized by a low incidence of HFMD and CNS diseases (Tagaya et al., 1981; Ishimaru et al., 1980; Hagiwara et al., 1983). In 1997, a large outbreak of HFMD due to highly neurotoxic EV71 occurred in Malaysia and caused 48 deaths. In 1998, there was a larger outbreak in Taiwan with more than 100,000 HFMD, 405 severe infections and 78 deaths (due to acute brainstem encephalomyelitis and neurological heart failure and pulmonary edema) (Lum et al., 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 HFMD cases were recorded in the mainland of China, including 126 deaths (Chinese Center, 2008). According to reports, the number of HFMD cases increased to 1,155,525 in 2009, of which 353 died (Yang et al., 2011). In 2010, the country experienced the largest outbreak, with more than 1.7 million HFMD cases, 27,000 patients with severe neurological complications, and 905 deaths. In all three outbreaks, almost all of the severe cases with neurological complications and death were due to EV71 (Zeng et al., 2012).

At present, the molecular determinants of the viral toxicity and pathogenesis of EV71 infection are still not fully understood. There are no antiviral drugs approved for clinical treatment of serious infections and related neurological complications, and there are no vaccines available for controlling and preventing recurrent outbreaks. In terms of control and preventive strategies, the development of cost-effective vaccines, especially for developing countries, is a top priority and a priority. Various types of vaccines against EV71 in investigation and development appear to trigger immune responses in rodents or monkeys (Wu et al., 2001; Arita et al., 2005; Chiu et al., 2006; Arita et al., 2007; Tung et al. People, 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. Despite the virion particle vaccine based on VP1 capsid And subunit peptide vaccines make feasible potential vaccine strategies still worthy of further research and development, but based on the development and use of inactivated injectable Salk vaccine and live attenuated oral Sabin polio in control and almost eradication of wild-type poliovirus infection A large number of past experiences with inflammatory vaccines, injectable inactivated and orally attenuated EV71 vaccines remain the most promising candidates (Zhang et al., 2010).

Currently, vaccines are not available on the market to protect children from infections due to EV71 and hand, foot and mouth disease. Based on recent information, two centers in the People's Republic of China, one in Singapore and one in Taiwan are developing injectable inactivated EV71 vaccines (press release and personal communication). National Institute of Dr. H. Shimizu Enterovirus Unit in Infectious Diseases, Tokyo, Japan has been conducting the research on the pathogenesis of EV71 in monkeys since 1980 and the temperature-sensitive strain of EV71 as a potential candidate for live attenuated EV71 vaccine (Arita et al., 2005; Arita et al. , 2007). Although the degree of neurological invasion and histopathological pathology of CNS is lower than that of wild-type virus after intravenous inoculation, all potential vaccine candidates for temperature-sensitive strains of EV71 in monkey studies can still cause such lesions. .

Live attenuated vaccines represent one of the first successful methods of vaccination, dating back to the 18th century when British doctor Edward Jenner began using vaccinia virus to vaccinate children against devastating diseases. Live attenuated vaccines use live virus or microorganisms that have been attenuated so that they do not cause disease, but induce a protective immune response. Traditional, classical and genetic approaches have been successful to some extent in the use of viruses and microbial attenuation for live attenuated vaccines. ^44-48 Conventional methods use naturally occurring related organisms that are non-toxic in humans, such as the use of cowpox or vaccinia viruses. Classical methods involve multiple rounds of growth under conditions that attenuate virulent viruses or microorganisms, such as in tissue culture or harsh liquid media. Genetic methods utilize modern molecular biology techniques to manipulate the genome to reduce its toxicity (Huygelen, 1997; Robinson, 2008; Coleman et al, 2008; Lauring et al, 2010; Kenney et al, 2011). In a classical method of obtaining a temperature-sensitive phenotype virus as a marker for attenuation, vaccination of wild-type virus is performed by means of plaque assay techniques by culturing the virus in suitable cells grown at lower temperatures. The selected virus strain is then repeatedly subcultured at a lower target incubation temperature (Hagiwara et al., 1982; Hashimoto and Hagiwara, 1983; Richman and Murphy, 1997).

There is a need to develop viral strains that are useful in the treatment of viral diseases, maintain phenotype and genetic stability under specific in vitro cell culture conditions, and do not exhibit neurotoxicity in monkeys after intravenous inoculation. There is also a need to develop temperature-sensitive virus (including RNA virus) strains suitable for cold after successive passages in cell culture.

The present invention relates to a temperature sensitive strain suitable for cold enterovirus 71, specifically for cold enterovirus 71 temperature sensitive strains EV71:TLLβP20 and EV71:TLLαP20. The invention also relates to methods for developing cold temperature sensitive virus strains, specifically RNA virus strains.

Thus, in one aspect, the invention provides a temperature sensitive strain suitable for cold enterovirus 71. In one embodiment, the temperature sensitive strain suitable for cold enterovirus 71 is EV71:TLLβP20 as described herein. In another embodiment, the temperature sensitive strain suitable for cold enterovirus 71 is EV71:TLLαP20 as described herein.

The enterovirus 71 virus strain of the present invention is prepared by a method of using temperature sensitivity as a phenotypic marker to attenuate enterovirus 71. The method is an in vitro laboratory process that alters the biological growth characteristics of the virus to accommodate optimal replication at incubation temperatures below 30 °C. The adaptation process (described in detail below) is carried out in a stepwise manner in a system that incrementally reduces the incubation temperature for culturing the virus until the target temperature selected for optimal replication of the virus is achieved.

In a second aspect, the invention provides a composition comprising a temperature sensitive strain suitable for cold enterovirus 71 as 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 another embodiment, the composition is a vaccine. Vaccines containing a temperature sensitive strain suitable for cold enterovirus 71 as described herein are prepared using techniques well known to those skilled in the art. Such vaccines are suitable for providing immunity against a parental strain by administering a vaccine to an individual, such as a human subject, using techniques well known to those skilled in the art.

In a third aspect, the invention provides a method of eliciting a protective immune response in an individual, such as a human subject, comprising administering to the individual a prophylactic or therapeutically or immunologically effective amount as described herein. Temperature sensitive strain of cold enterovirus 71. In one embodiment, the protective immune response protects the individual from 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 another embodiment, the disease is encephalitis. In another embodiment, the disease is The symptoms of poliomyelitis are paralyzed. In one embodiment, the cold enterovirus 71 temperature sensitive strain described herein is administered as a vaccine. In another embodiment, the individual has been exposed to wild-type enterovirus 71. In another embodiment, a temperature sensitive strain suitable for cold enterovirus 71 as described herein is administered to prevent an individual, such as a human subject, from suffering from a disease associated with enterovirus 71. In another embodiment, the individual has been exposed to wild-type enterovirus 71. In another embodiment, administration of a temperature sensitive strain suitable for cold enterovirus 71 as described herein delays the onset of or slows the progression of a disease associated with enterovirus 71 in an individual infected with a virus, such as a human subject.

In a fourth aspect, the invention provides a method of using temperature sensitivity as a phenotypic marker to attenuate a virus. According to this aspect, the method develops a temperature sensitive virus strain suitable for cold. The method of the present invention is an in vitro laboratory process that alters the biological growth characteristics of the virus to accommodate optimal replication at incubation temperatures below 30 °C. The adaptation process is carried out in a stepwise manner to incrementally reduce the incubation temperature for culturing the virus until the target temperature selected for optimal replication of the virus is achieved. Thus, in accordance with the present invention, the method comprises the steps of: (i) preparing a reference stock of the parental wild type virus, (ii) using a lower infection rate for each sub-derived complete cytopathic effect (CPE) The inoculum of (MOI) and the shorter incubation period is incubated at a higher MOI and a culture temperature of about 34 ° C to about 36 ° C, preferably about 34 ° C, to culture the cell culture infected with the reference stock of the parental wild type virus. Sub- or more than five subcultures until full CPE is obtained in each subculture, (iii) use of lower infection rate (MOI) and shorter incubation period for each sub-derived complete cytopathic effect (CPE) The inoculum is incubated at a higher MOI and at a incubation temperature lower than about 1 ° C to about 3 ° C in the preceding step, and the resulting virus-infected cell culture is subjected to five or more subcultures until each subsequent step. The complete CPE is obtained on the generation, and (iv) the step (iii) is repeated in a stepwise manner in a system that incrementally lowers the incubation temperature until the target temperature selected for optimal replication of the virus 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 is incrementally lowered to a temperature that is reduced by from about 1 °C to about 2 °C.

In one embodiment, the culture is carried out at a temperature of from about 36 ° C to about 38 ° C, preferably about 37 ° C. The cell culture of the wild-type virus-infected virus is bred one or two times until a complete cytopathic effect (CPE) is obtained to prepare a reference stock of the parental wild-type virus. An aliquot of the culture containing the resulting virus is placed in a vial or other suitable storage device. This culture was used as a reference stock for the parental wild type virus. The reference parental wild type virus is used in the subsequent attenuating process. In another embodiment, an aliquot of the reference stock of the parental wild type virus is stored at a suitable temperature, such as -80 °C.

In one embodiment, the virus is any virus. In another embodiment, the virus is an RNA virus. In another embodiment, the RNA virus is a positive strand RNA virus. In another embodiment, the virus is a member of the picornavirus family. In another embodiment, the virus is a member of the genus Enterovirus. In one embodiment, the virus is Enterovirus 71 (EV71). In another embodiment, the virus is Coxsackievirus A16 (CA16). The methods of the present invention are suitable for use in producing cold sensitive temperature sensitive strains of any of these viruses, including, but not limited to, cold temperature sensitive strains of EV71 and CA16.

In one embodiment, the cell to be infected by the virus is any cell that allows the virus to grow. In a preferred embodiment, the cells are Vero cells (ATCC CCL-81). In one embodiment, the cells are maintained by conventional passage in a medium suitable for cell growth. In an embodiment where the cells are Vero cells, Vero cells are cultured in Dulbecco's modified Eagles's medium (DMEM) supplemented with 10% fetal bovine serum (FCS). In one embodiment, the Vero cell line maintained in DMEM supplemented with 1% FCS is used to produce a parental wild type virus, virus culture, attenuation, titration, and assessment of a temperature sensitive phenotype. In another embodiment, the DMEM is supplemented with 1% FCS to adapt the virus strain to replication in successively lowered incubation temperatures.

The invention also relates to a temperature virus strain suitable for cold production by the methods described herein. The temperature virus suitable for cold generated by the methods described herein is suitable for use in the production of vaccines using techniques well known to those skilled in the art. Such vaccines are suitable for providing immunity against a parental strain of the vaccine by administering the vaccine to the individual using techniques well known to those skilled in the art.

Figure 1a shows peripheral blood mononuclear cells (arrows) derived from the blood of monkeys on day 4 after administration of an intravenous dose of enterovirus 71 (EV71:TLLβP20) using commercial monoclonal antibodies specific for the virus. Indirect immunofluorescence staining was positive.

Figure 1b shows a photograph of an electrophoresis agarose gel stained with GelRed reagent showing the use of an oligonucleotide primer pair specific for the detection of enterovirus 71 from the 4th day after immunization (2202F, 2891F) One-step RT-PCR amplification of the tissue. The expected size of the RT-PCR amplified product was 427 bp. The channels in the two gels are as follows. Gel 1 : channel 1: 100 bp DNA ladder; channel 2: 2202F-heart; channel 3: 2202F-spleen; channel 4: 2202F-lymph node; channel 5: 2202F-kid; channel 6: 2202F-liver; channel 7: 2891F - heart; channel 8: 2891F-spleen; channel 9: 2891F-lymph node; channel 10: 2891F-kid; channel 11: 2891F-liver; channel 12: 2202F-brain (brain bridge); channel 13: 2202F-brain ( Medulla); channel 14: 2202F-cortex (brain back); channel 15: 2202F-spinal (cervical spine); channel 16: 2202F-spinal (lumbar spine); channel 17: 2202F-spinal (thoracic); channel 18: 2891F-brain Stem (medulla); channel 19: 2891F-brain (pons); channel 20: 2891F-cortex (left cerebellum). Gel 2 : channel 21: 100 bp DNA ladder; channel 22: 2891F-cortex (right cerebellum); channel 23: 2891F-spinal (cervical vertebra); channel 24: 2891F-spinal (lumbar spine); channel 25: 2891F-spinal (thoracic) ) channel 26: no template control; channel 27: 100 bp DNA ladder.

The present invention relates to a temperature sensitive strain suitable for cold enterovirus 71, specifically for cold enterovirus 71 temperature sensitive strains EV71:TLLβP20 and EV71:TLLαP20. The invention also relates to methods for developing cold temperature sensitive virus strains, specifically RNA virus strains.

Thus, in one aspect, the invention provides temperature sensitivity that is suitable for cold. In one embodiment, the temperature sensitive strain suitable for cold enterovirus 71 is EV71:TLLβP20 as described herein. In another embodiment, the temperature sensitive strain suitable for cold enterovirus 71 is EV71: TLLαP20 as described herein. EV71: TLLβP20 was deposited on October 25, 2012 under the terms of the Budapest Treaty on the American Type Culture Collection at 10801 University Boulevard, Manassas, Virginia 20110, USA and the designated registration number is PTA. -13285. EV71: TLLαP20 was deposited with the American Type Culture Collection on October 25, 2012 under the terms of the Budapest Treaty, and the designated registration number is PTA-13284.

The enterovirus 71 virus strain of the present invention is prepared by a method of using temperature sensitivity as a phenotypic marker to attenuate enterovirus 71. The method is an in vitro laboratory process that alters the biological growth characteristics of the virus to accommodate optimal replication at incubation temperatures below 30 °C. The adaptation process is carried out in a stepwise manner to incrementally reduce the incubation temperature for culturing the virus until the target temperature selected for optimal replication of the virus is achieved. As disclosed herein, (i) preparing a reference stock of the parental wild-type virus, (ii) using a lower infection rate (MOI) and shorter incubation for each sub-derived complete cytopathic effect (CPE) The inoculum of the time period is incubated at a higher MOI and a incubation temperature of about 34 ° C for five or more subcultures of the cell culture infected with the reference stock of the parental wild type virus until complete CPE is obtained in each subculture. (iii) using a lower infection multiplication rate (MOI) for each passage to obtain complete cytopathic effect (CPE) and a shorter incubation period, the inoculum is at a higher MOI and is about 1 ° C lower than the previous step. Incubating the virus-infected cell culture obtained by the foregoing steps five or more times at a incubation temperature of about 3 ° C until a complete CPE is obtained in each subculture, and (iv) a system for incrementally lowering the incubation temperature Step (iii) is repeated in a stepwise manner until the target temperature selected for optimal replication of the virus 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 wild-type virus-infected cell culture is incubated one or two times at a temperature of from about 36 ° C to about 38 ° C, preferably about 37 ° C until complete cytopathic effect (CPE) is obtained. A reference stock for the parental wild type virus is prepared. Place an aliquot of the culture containing the resulting virus in a vial or other suitable storage device in. This culture was used as a reference stock for the parental wild type virus. The reference parental wild type virus is used in the subsequent attenuating process. In another embodiment, an aliquot of the reference stock of the parental wild type virus is stored at a suitable temperature, such as -80 °C.

In one embodiment, the cell to be infected by 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 conventional passage in a medium suitable for cell growth. In an embodiment where the cells are Vero cells, Vero cells are cultured in Dulbecco's modified Eagles's medium (DMEM) supplemented with 10% fetal bovine serum (FCS). In one embodiment, Vero cells maintained in DMEM supplemented with 1% FCS are used to generate a parental wild type virus, virus culture, attenuate, titrate, and assess a temperature sensitive phenotype. In another embodiment, the DMEM is supplemented with 1% FCS to adapt the strain to replicate in a continuously reduced incubation temperature.

In one embodiment, once the virus has achieved complete cytopathic effect (CPE), each successive fresh fresh culture is obtained by obtaining a clear culture containing the virus and delivering the supernatant to the cells (eg, Vero cells). The virus is subcultured in the various steps of the process. In another embodiment, the virus-containing culture supernatant is delivered at a higher infection magnification (MOI) of 20 at the beginning of each successive change to a lower incubation temperature. After noting that the virus was able to cause complete CPE in the Vero cells within 3 days after inoculation, the new culture flask containing fresh confluent monolayers of Vero cells was inoculated at least three times by the same MOI virus, and then reduced to 5 To a lower MOI of 10. Once it is noted that the virus is capable of causing complete CPE within 3 days after inoculation with a lower MOI of 5 to 10, after at least three subcultures at an MOI of 5 to 10, the attenuating process then proceeds to a lower continuous incubation temperature. stage. The number of days required for each passage depends on the rate at which the virus adapts at various stages of incrementally lowering the incubation temperature. Those skilled in the art will readily know when to reach full CPE. Further details of methods of preparing the temperature-sensitive strains of cold enterovirus 71 of the present invention are described below.

In a second aspect, the invention provides a method suitable for cold enteropathy as described herein A composition of a toxic 71 temperature sensitive strain. 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 another embodiment, the composition is a vaccine. Vaccines containing a temperature sensitive strain suitable for cold enterovirus 71 as described herein are prepared using techniques well known to those skilled in the art. Such vaccines are suitable for providing immunity against a parental strain by administering a vaccine to an individual, such as a human subject, using techniques well known to those skilled in the art.

It will be appreciated that the temperature sensitive strains described herein are suitable for inducing a protective immune response in an individual or preventing an individual from suffering from a virus-related disease or delaying the onset or slowing down of a virus-related disease. At the rate of progression, the individual is administered as a composition additionally 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 of the following: 0.01 M-0.1 M and preferably 0.05 M phosphate buffer, 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, physiological 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 oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and similar vehicles. The solid composition may comprise 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 the form of an aerosol, such as transpulmonary and/or intranasal delivery, preferably by non-toxic surfactants, for example, esters or partial esters of C6 to C22 fatty acids or natural glycerides and propellant formulations or combination. Other carriers such as lecithin may be included to facilitate intranasal delivery. The pharmaceutically acceptable carrier may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives and other additives such as antibacterial agents, antioxidants And chelating agents which increase the shelf life and/or effectiveness of the active ingredient. As is well known in the art, the compositions of the present invention can be formulated to provide rapid, sustained or delayed release of the active ingredient after administration to an individual.

In a third aspect, the invention provides a method of inducing a protective immune response in an individual, such as a human subject, comprising administering to the individual a prophylactic or therapeutically or immunologically effective amount as described herein. Temperature sensitive strain of cold enterovirus 71. In one embodiment, the protective immune response protects the individual from 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 another embodiment, the disease is encephalitis. In another embodiment, the disease is paralyzed by poliomyelitis symptoms. In one embodiment, the cold enterovirus 71 temperature sensitive strain described herein is administered as a vaccine. In another embodiment, the individual has been exposed to wild-type enterovirus 71. "Exposure" to enterovirus 71 means contact with enterovirus 71 to cause infection. In another embodiment, administration of a temperature sensitive strain suitable for cold enterovirus 71 as described herein prevents an individual, such as a human subject, from suffering from a disease associated with enterovirus 71. In another embodiment, the individual has been exposed to wild-type enterovirus 71. In another embodiment, administration of a temperature sensitive strain suitable for cold enterovirus 71 as described herein delays the onset of or slows the progression of a disease associated with enterovirus 71 in an individual infected with a virus, such as a human subject.

As used herein, "administering" means delivery using any of a variety of methods and delivery systems known to those skilled in the art. Can be, for example, intraperitoneal, intracranial, intravenous, oral, transmucosal, subcutaneous, transdermal, intradermal, intramuscular, topical, parenteral, via implantation, intrathecal, intralymphatic, intralesional, pericardium Or epidural administration. The agent or composition can also be administered in the form of an aerosol, such as by pulmonary and/or intranasal delivery. The vote can be made, for example, one time, multiple times, and/or over one or more extended periods.

The protective immune response in an individual can be elicited, for example, by administering a primary dose of the vaccine to the individual, followed by one or more subsequent administrations of the vaccine after a suitable period of time. Suitable periods of time between administration of the vaccine can be readily determined by those skilled in the art and are typically on the order of several weeks. Several months. However, the invention is not limited by any particular method, route or frequency of administration.

"Prophylactically effective dose" or "immunologically effective dose" is the induction of protection of an individual from infection or afflicted with an individual in the case of an individual who is predisposed to a viral infection or has a tendency to develop a disease associated with the virus. The amount of vaccine for the immune response. "Protecting" an individual means reducing the likelihood of an individual being infected with the virus or reducing the likelihood of the onset of the condition in the individual by at least two times, preferably at least ten times. For example, if an individual has a 1% chance of infecting a virus, twice the likelihood of an individual being infected with the virus will cause the individual to have a 0.5% chance of contracting the virus. Optimally, a "prophylactically effective dose" induces an immune response in an individual that completely prevents the individual from contracting the virus or completely preventing the onset of the condition in the individual.

Certain embodiments of any of the methods of immunization and treatment of the invention may further comprise administering to the individual one less adjuvant. "Adjuvant" shall mean any agent suitable for enhancing the immunogenicity of an antigen and enhancing the immune response in an individual. Many adjuvants, including particulate adjuvants, are suitable for use with vaccines based on proteins and nucleic acids, and methods for combining adjuvants with antigens are well known to those skilled in the art. Adjuvants suitable for protein immunization include, but are not limited to, sputum, Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), sputum adjuvant, saponin-based adjuvant (such as Quil A) And QS-21) and similar adjuvants.

The invention also provides a method of using temperature sensitivity as a phenotypic marker to attenuate a virus. According to this aspect, the method develops a temperature sensitive virus strain suitable for cold. The method of the present invention is an in vitro laboratory process that alters the biological growth characteristics of the virus to accommodate optimal replication at incubation temperatures below 30 °C. The adaptation process is carried out in a stepwise manner to incrementally reduce the incubation temperature for culturing the virus until the target temperature selected for optimal replication of the virus is achieved.

Thus, in accordance with the present invention, the method comprises the steps of: (i) preparing a reference stock of the parental wild-type virus, and (ii) using a lower infection rate for each sub-derived complete cytopathic effect (CPE) ( The inoculum of MOI) and the shorter incubation period is incubated with the parental wild-type virus at a higher MOI and a incubation temperature of about 34 ° C to about 36 ° C, preferably about 34 ° C. Test the cell culture of the infected material for five or more passages until the complete CPE is obtained in each subculture, (iii) use a lower infection rate for each sub-derived complete cytopathic effect (CPE) The inoculum of (MOI) and the shorter incubation period is incubated at a higher MOI and a culture temperature lower than the previous step by about 1 ° C to about 3 ° C for five or more cell cultures obtained by the aforementioned steps. Subsequent until the complete CPE is obtained in each subculture, and (iv) the step (iii) is repeated in a stepwise manner with a system that incrementally lowers the incubation temperature until the target temperature selected for optimal replication of the virus 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 is incrementally lowered to a temperature that is reduced by from about 1 °C to about 2 °C.

In one embodiment, the wild-type virus-infected cell culture is incubated one or two times at a temperature of from about 36 ° C to about 38 ° C, preferably about 37 ° C until complete cytopathic effect (CPE) is obtained. A reference stock for the parental wild type virus is prepared. An aliquot of the culture containing the resulting virus is placed in a vial or other suitable storage device. This culture was used as a reference stock for the parental wild type virus. The reference parental wild type virus is used in the subsequent attenuating process. In another embodiment, an aliquot of the reference stock of the parental wild type virus is stored at a suitable temperature, such as -80 °C.

In one embodiment, once the virus has achieved complete cytopathic effect (CPE), each successive fresh fresh culture is obtained by obtaining a clear culture containing the virus and delivering the supernatant to the cells (eg, Vero cells). The virus is subcultured in the various steps of the process. In another embodiment, the virus-containing culture supernatant is delivered at a higher infection magnification (MOI) of 20 at the beginning of each successive change to a lower incubation temperature. After noting that the virus was able to cause complete CPE in the Vero cells within 3 days after inoculation, the new culture flask containing fresh confluent monolayers of Vero cells was inoculated at least three times by the same MOI virus, and then reduced to 5 To a lower MOI of 10. Once it is noted that the virus is capable of causing complete CPE within 3 days after inoculation with a lower MOI of 5 to 10, after at least three subcultures at an MOI of 5 to 10, the attenuating process then proceeds to a lower continuous incubation temperature. stage. The day of each generation The number depends on the speed at which the virus adapts at various stages of incrementally lowering the incubation temperature. Those skilled in the art will readily know when to reach full CPE.

In one embodiment, the virus is any virus. In another embodiment, the virus is an RNA virus. In another embodiment, the RNA virus is a positive strand RNA virus. In another embodiment, the virus is a member of the picornavirus family. In another embodiment, the virus is a member of the genus Enterovirus. In one embodiment, the virus is Enterovirus 71 (EV71). In another embodiment, the virus is Coxsackievirus A16 (CA16). The methods of the present invention are suitable for use in producing cold sensitive temperature sensitive strains of any of these viruses, including, but not limited to, cold temperature sensitive strains of EV71 and CA16.

In one embodiment, the cells to be infected by the virus are any cells that allow 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 conventional passage in a medium suitable for cell growth. In the example where the cells are Vero cells, Vero cells are cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FCS). In one embodiment, Vero cells maintained in DMEM supplemented with 1% FCS are used to generate a parental wild type virus, virus culture, attenuate, titrate, and assess a temperature sensitive phenotype. In another embodiment, the DMEM is supplemented with 1% FCS to adapt the virus strain to replication in successively lowered incubation temperatures.

The invention also relates to a temperature virus strain suitable for cold production by the methods described herein. The temperature virus suitable for cold generated by the methods described herein is suitable for use in the production of vaccines using techniques well known to those skilled in the art. Such vaccines are suitable for providing immunity against a parental strain of the vaccine by administering the vaccine to the individual using techniques well known to those skilled in the art.

The method of the invention is suitable for the production of cold temperature sensitive viruses of the picornavirus family and the enterovirus genus. This applicability has been demonstrated herein by producing temperature sensitive strains EV71 (TLLα) and EV71 (TLLβ) suitable for cold, which are obtained after successive passages in cell culture at incrementally lower incubation temperatures. . EV71 (TLLβ) virus strain Phenotypic and genetic stability were maintained under specific in vitro cell culture conditions and did not exhibit neurotoxicity in monkeys following intravenous inoculation. This applicability has also been demonstrated herein by generating a temperature sensitive CA16 suitable for cold, which is obtained after successive passages in cell culture at incrementally lower incubation temperatures.

The invention also provides a kit for immunizing an individual by a temperature sensitive strain suitable for cold enterovirus 71 as described herein. The kit comprises a temperature sensitive strain suitable for cold enterovirus 71, a pharmaceutically acceptable carrier, an applicator, and instructions for use thereof as described herein. The invention includes other embodiments that are familiar to those skilled in the art. Such instructions may provide any information suitable for directing administration of a temperature sensitive strain suitable for cold enterovirus 71 as described herein.

Unless otherwise indicated, the practice of the present invention employs conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture, and genetic engineering, where such techniques are employed. Within the skills of the technology. See, for example, Maniatis et al , 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York); Sambrook et al , 1989, Molecular Cloning , 2nd Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) ); Sambrook and Russell, 2001, Molecular Cloning , 3rd edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York); Green and Sambrook, 2012, Molecular Cloning , 4th edition (Cold Spring Harbor Laboratory Press, Cold Spring) Harbor, New York); Ausubel et al ., 1992, Current Protocols in Molecular Biology (John Wiley & Sons, including regular updates); Glover, 1985, DNA Cloning (IRL Press, Oxford); Russell, 1984, Molecular biology of plants: a laboratory course manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); Anand, Techniques 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 Pr Ess, Cold Spring Harbor, New York); Nucleic Acid Hybridization (BD Hames & SJ Higgins, 1984); Transcription And Translation (BD Hames & SJ Higgins, 1984); Culture Of Animal Cells (RI Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); Theory In Enzymology (Academic Press, Inc., NY); Methods In Enzymology , Volumes 154 and 155 (Wu Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, ed., Academic Press, London, 1987); Handbook Of Experimental Immunology , Volumes I-IV (DM Weir and CC Blackwell, eds., 1986); Riott, Essential Immunology , Sixth Edition, Blackwell Scientific Publications, Oxford, 1988; Fire et al, RNA Interference Technology: From Basic Science to Drug 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.

Instance

The invention is described with reference to the following examples, which are not intended to limit the invention in any way. Standard techniques well known in the art or techniques specifically described below are used.

Example 1 Materials and methods for developing cold-sensitive human enterovirus 71 (EV71) temperature sensitive strains

Cell, Virus, and Cold Adaptation Process : Vera Cells (ATCC CCL-81) maintained by conventional subculture in Dulbec's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FCS) Used for virus culture, attenuation, titration and evaluation of temperature sensitive phenotypes. Three EV71 isolates belonging to genotypes C1, B3 and B4 isolated in Vero cells from individual oral secretions, stool and brain stem specimens of patients presenting hand, foot and mouth disease (HFMD) according to the previously described technique Spot purification was performed once (Dougherty, 1964). After plaque purification, virus stocks indicated as individual wild-type virus strains were prepared after two passages in Vero cells at 37 °C. All virus stocks and each successive virus strain were stored in a minus 80 °C freezer.

Fresh confluent young monolayer Vero cell lines in DMEM containing 1% FCS were used to adapt virus strains to replicate at successively lower incubation temperatures starting at an initial low incubation temperature of 34 °C. Once the virus has achieved complete cytopathic effect (CPE), the clarified culture containing the virus is passed to each successive fresh, fresh flask of Vero cells. The virus-containing culture supernatant was delivered at a higher infection multiplication rate (MOI) of 20 at the beginning of each successive change to a lower incubation temperature. After noting that the virus was able to cause complete CPE in the Vero cells within 3 days after inoculation, the new culture flask containing fresh confluent monolayers of Vero cells was inoculated at least three times by the same MOI virus, and then reduced to 5 To a lower MOI of 10. Once the virus is noted to be capable of causing complete CPE within 3 days after inoculation with a lower MOI of 5 to 10, after at least three subsequent passages, the attenuating process is then advanced to the next consecutive lower incubation temperature. The lower incubation temperatures used in this example to develop subsequent increments for cold enterovirus 71 temperature sensitive strains were 34 °C, 32 °C, 30 °C, 29 °C, and 28 °C.

Viral titration : slightly modified according to the method described in the Polio Laboratory Manual 2004 of the World Health Organization. The virus titration concentration was determined by microtiter analysis in Vero cells and the virus titration concentration was calculated per Reed and Muench method (1938). 50 ml cell culture infection dose (CCID ₅₀ ). Briefly, after treatment with equal amounts of chloroform to disperse viral aggregates, a 10-fold serial dilution of the clarified viral supernatant was prepared in DMEM containing 1% FCS. The Vero cell monolayer (10 ⁴ cells per well) in a 96-well flat-bottom tissue culture plate was inoculated with 100 μl of serially diluted virus stocks and at each incubation temperature in a 5% CO ₂ atmosphere. The cultivation was carried out for 5 days, and then the presence of CPE was observed.

Temperature sensitivity analysis : Two methods were used to evaluate the growth characteristics of virus strains in Vero cells at incubation temperatures of 28 ° C, 37 ° C and 39.5 ° C. The first method assesses the number of days (viral kinetics) used by the virus strain to cause complete CPE in infected cells and the second method assesses the titer concentration of the virus strain in cells cultured at each particular test temperature. Briefly, in the first method, three growth media containing T-25 tissue culture flasks of confluent single-layered Vero cells of similar age were replaced by maintenance medium (DMEM containing 1% FCS). The medium in each flask was then equilibrated to the specific temperature to be tested by placing in a separate incubator for 1 hour and then inoculated with a virus strain at a dose of 10 infection magnification (MOI). If the CPE was not noticed at the end of the 10 day culture, the supernatant was transferred to a new single-layered Vero cell flask and similarly incubated for another 10 days. If the CPE is not noticed after the second pass, it is considered to be virus free. In the second method, a Vero cell suspension having a density of 10 ⁴ cells per 100 μl is inoculated into each well of three 96-well cell culture dishes and cultured at 37 ° C in a 5% CO ₂ atmosphere. . After 10 hours of incubation, each cell culture plate was then equilibrated to the specific temperature to be tested by placing in a separate incubator for 1 hour. The cells in each well were then inoculated by 10-fold serial dilutions of 100 μl of virus strain, which were then transferred to incubation chambers at respective temperatures and incubated for 5 days, after which the presence of CPE was observed.

Genetic stability and temperature sensitivity analysis : To assess the genetic stability of virus strains cultured in a specific culture environment and cell type, the virus strain was subcultured 20 times in cell culture under the 5 MOI virus inoculum. Incubation was carried out at a incubation temperature of 28 °C. At the end of 20 passages, the temperature sensitive phenotypic characteristics of the virus strains were assessed by culturing at incubation temperatures of 28 ° C, 37 ° C and 39.5 ° C as described above. The entire nucleotide sequence of each individual genome is then sequenced and analyzed relative to the genome of its respective parental wild type virus.

The reversal of temperature sensitivity analysis was performed on stable temperature sensitive virus strains suitable for cold. The selected virus strain was subcultured 5 times in a single layer of Vero cells incubated at 37 ° C in a 5% CO ₂ atmosphere. In each subculture, a virus of 10 MOI was used to inoculate the body. The growth characteristics of each successively obtained virus strain in Vero cells were evaluated by similar methods for temperature sensitivity analysis as described above at incubation temperatures of 28 ° C, 37 ° C and 39.5 ° C. The whole genome of each successive passage of virus at a culture temperature of 37 ° C was sequenced and analyzed.

RNA extraction, RT-PCR and sequencing : Viral genomic RNA was extracted from the culture medium of infected cells under complete CPE using a commercially available Viral RNA Extraction Kit (Qiagen, Germany). First strand synthesis was performed by EV71-specific primers using Superscript II RNA polymerase (Invitrogen, USA), and subsequent PCR was performed by 18 degenerate primer pairs using GoTaq Green PCR mix (Promega, USA). The resulting fragments were sequenced using a BigDye Terminator sequencing kit (Applied Biosystems, USA). 5' RACE was performed to ligate 5'-GAA GAG AAG GTG GAA ATG GCG TTT TTG G- by 5'-GAA GAG AAG GTG GAA by using standard T4 DNA ligase (Fermentas, USA) Cordycepin-3'; SEQ ID NO: 1) ligated to the 5' end of the EV71 cDNA to determine the 5'-UTR viral sequence, and then used a primer complementary to DT88 (5'-CCA AAA CGC CAT TTC CAC CTT Standard PCR was performed for CTC TTC3'; SEQ ID NO: 2) and EV71-specific primer (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 the oligo-dT primer (Li et al., 2005).

Molecular colonization and plastid purification : Fragments with ambiguous sequences were cloned into pZero-2 plastids (Invitrogen, USA) and transformed into TOP10 E. coli cells (Invitrogen, USA). The plastid containing the EV71 fragment was extracted from at least 10 colonies from each of the transformants using a commercially available plastid Miniprep Kit (Qiagen, Germany) and the sequence of the cloning fragment was subsequently determined.

The obtained fragment sequences were combined using the European Molecular Biology Open Software Suite (EMBOSS; http://mobyle.pasteur.fr/cgi-bin/portal.py?#forms::merger) (Rice et al. 2000). The pooled sequences were aligned with the reference sequence of EV71 strain 3799-SIN-98 (GenBank Accession No. DQ341354.1) using the program BioEdit Sequence Alignment Editor version 7.0.9.0 (Hall, 1997).

Monkey Study : The monkey study on the safety and immunogenicity of the selected EV71 temperature sensitive strain (EV71:TLLβP20) was contracted to the Research Facility of DUKE-NUS, Singapore. 7 macaca fascicularis (3 females (2202F, 2207F, 2891F) and 4 males (0791M, 2247M, 2889M, 2890M) with no Mycobacterium tuberculosis and simian immunodeficiency virus The average body weight of 3.23 Kg (range 2.44 to 4.11, SD = 0.7) was used to study the safety and immunogenicity of a stable temperature sensitive EV71:TLLβP20. All 7 monkeys were pre-screened for the absence of binding to EV71 (indirect immunofluorescence) and neutralizing antibodies. The study and all animal procedures were approved by the Committee for Biosafety and Animal Handling and the Ethical Committee of DUKE-NUS, Singapore. Viral vaccination and observation, animal care and necropsy according to the guidelines of the committee.

A 1 ml virus inoculum was intravenously inoculated into the right saphenous vein under light anesthesia with ketamine. Three monkeys (2889M, 0791M and 2891F) were given an intravenous dose of EV71:TLLβP20 at 10 ⁷ CCID ₅₀ per monkey, and 3 monkeys (2890M, 2202F and 2247M) were given 10 ⁸ CCID ₅₀ per monkey and 7th The monkey served as a negative control. The clinical disease of the monkey was observed twice daily, especially for neurological manifestations and its body temperature was recorded by an implanted temperature sensor. Stool from each monkey was collected daily and stored in a minus 80 °C freezer for virus isolation in the future. Two monkeys (one inoculum from different viral doses) were killed under deep anesthesia on day 4 post-inoculation (PI). At the time of necropsy, various parts of the central nervous system (CNS), non-neural tissue, and blood were collected for histopathology and virological analysis. On the 8th day after inoculation, two monkeys of another similar group were killed and the same type of tissue and blood were collected for histopathology and virology studies at necropsy. The remaining two monkeys were given respective equivalent booster doses of EV71:TLLβP20 on the 14th day after inoculation after collecting blood samples for evaluation of anti-EV71 antibodies. On the 16th day after receiving the booster dose, it was painlessly killed and the CNS tissue was collected for histopathological study at necropsy.

Histology and immunohistochemistry : CNS tissue specimens (brain, cerebellum, basal ganglia, brainstem and spinal cord) and non-neural tissues (lymph nodes, spleen, liver, kidney, lung and heart) were fixed in phosphate buffered saline (PBS) In 10% of formalin and embedded in paraffin after fixation. The spinal cord was sliced horizontally 10 times, 8 times, and 10 times at the cervical, thoracic, and lumbar levels, respectively. Paraffin section with a thickness of 6 μm by hematoxylin and eosin (H&E) and by Luxol-fast blue/cresyl violet (Kluver-Barrera method) after removing the paraffin and rehydration process Dyeing.

Antigen detection and isolation of viruses from monkeys : Blood samples were collected in non-additive tubes and heparinized tubes. Using ^{Ficoll-Paque TM PLUS (GE Healthcare} , Sweden) of blood was collected from heparinized peripheral blood mononuclear cells (PBMC). After washing twice with sterile PBS, an aliquot of the PBMC suspension was inoculated onto wells coated with Teflon slides to use commercially available monoclonal antibodies (Catalog No. 3360, Light) Diagnostics, USA) detects EV71 antigen by indirect immunofluorescence assay. Virus isolation was performed by inoculating the PBMC suspension into wells of a 24-well cell culture dish containing a single layer of Vero cells. Each serum sample was subjected to virus isolation by inoculating 50 μl and 100 μl of serum into respective wells of a 24-well culture plate containing single-layered Vero cells. Monkey tissue was slightly washed with sterile PBS exchanged twice and homogenized by grinding with a mortar and pestle in a Class II biosafety cabinet. The tissue homogenate (10%, w/v) prepared in DMEM was clarified by centrifugation at 1000 g for 10 minutes. The supernatant was filtered through a 0.22 micron syringe filter and virus isolation was performed by inoculating 100 μl and 200 μl of filtrate. A 10% stool suspension was prepared in PBS and clarified by centrifugation at 1000 g for 10 minutes. After filtration through a 0.22 micron syringe filter, virus isolation was performed by inoculating 100 μl and 200 μl of stool filter. Monkey virus samples were subjected to all virus isolation work in duplicate, a set of seeded cell cultures were incubated at 28 °C and the other group was incubated at 37 °C.

Molecular Detection and Whole Genome Sequencing : Commercial viral RNA extraction and purification kits (Qiagen, Germany) are used to extract viral genomic RNA from serum, PBMC and clarified tissue homogenates. A commercial one-step RT-PCR kit (Qiagen, Germany) and a common oligonucleotide primer pair that amplifies the proximal third of the VP1 gene of all EV71 genotypes (sense: 5'-CACCCTTGTGATACCATGGATCAG-3' (SEQ ID NO: 4); antisense: 5'-GTGAATTAAGAACRCAYCGTGTYT-3' (SEQ ID NO: 5)) is used for molecular amplification and detection of EV71-specific viral RNA after self-tissue extraction. 18 pairs of sequence-specific primers based on the whole genome sequence EV71:TLLβ were used to amplify and sequence the EV71 genome in the serum of the two monkeys obtained on the 4th and 8th day after immunization. Any PCR amplified fragment that failed to give a good sequence read by direct sequencing was cloned into pZero-2 (Invitrogen, USA) and transformed into TOP10 E. coli. At least ten colonies were selected from each of the transformants and sequenced on the purified plasmid carrying the insert.

Serum Binding and Neutralizing Antibody Assay : EV71-infected Vero cells genotype B3 were collected under almost complete CPE and washed five times with sterile PBS. After the last wash, the suspension of infected cells was mixed with the washed uninfected Vero cell suspension at a ratio of about 4 infected cells to 1 uninfected cells. Ten microliters of the mixed cell suspension containing 250 cells were carefully layered onto each well of a 12-well slide coated with Teflon and allowed to dry on a warm dish. The dried slides were fixed in cold acetone for 10 minutes and used as an EV71 binding antibody assay by indirect immunofluorescence analysis starting at 1:10 for IgM and an initial dilution of 1:20 for IgG. Internal antigen. In an analysis of anti-EV71 IgM titration concentrations, monkey serum was treated with an appropriate concentration of Protein A (Invitrogen, USA) to remove IgG that was previously serially diluted 2-fold by sterile PBS.

Slightly modified according to the method described in the Polio Laboratory Manual 2004 of the World Health Organization, the concentration of the neutralizing antibody in the monkey was determined by micro-neutralization analysis using Vero cells. The concentration of each genotype used for neutralization of EV71 was 100 CCID ₅₀ per 100 μl. Virus neutralization assays were performed using 96-well flat bottom plates. Serial 2-fold dilutions of each serum sample were prepared in duplicate in the amount of 100 μl DMEM (1% FCS) starting at 1:10 dilution. An equal amount (100 μl) of the virus working stock in DMEM (1% FCS) containing 100 CCID ₅₀ EV71 was added to each well of the diluted serum and incubated at 37 ° C for two hours. After the incubation, 100 μl of a Vero cell suspension in DMEM (10% FCS) containing 250 cells was added to each well. 96-well plates were carefully sealed and incubated at 37 ° C in a 5% CO ₂ atmosphere. The discs were read daily for up to 8 days for the presence of CPE affecting Vero cells in each well. The titer concentration of the neutralizing antibody in each serum sample was determined by not showing the highest dilution of the CPE.

Example 2 Results for phenotypic and genotypic characteristics of cold-tested enterovirus 71 temperature-sensitive strains

(EV71: TLLα, EV71: TLLαP20, EV71: TLLβ, EV71: TLLβP20, and EV71: TLLβP40)

The three original parental EV71 viruses used to obtain a cold virus strain caused complete CPE in the Vero cells within 3 days after inoculation at 10 MOI of the virus inoculum and at the incubation temperature of 37.5 °C. In the same virus inoculum, the original parental EV71 virus caused complete CPE in the Vero cells within 5 days at a incubation temperature of 39.5 °C. However, all three of the original parental EV71 viruses derived from the first two passages of the Vero cells cultured at 37.5 °C were not induced in the Vero cells at the 10 MOI virus inoculum and at the incubation temperature of 28 °C. CPE. CPE was also not noted after blind passage at the end of the 10th day at 28 °C. The absence of viral replication in the vaccinated Vero cells was further supported by indirect immunofluorescence analysis using a commercially available monoclonal antibody against EV71 at the end of the 10 day culture in the presence of negatively stained suspension cells in the culture supernatant.

After more than 90 passages at successively reduced incubation temperatures, all three subcultured EV71 strains were able to 5 MOI virus inoculum and 3 days of inoculation at 28 ° C incubation temperature caused complete CPE in Vero cells. The virus strain was further subcultured at the incubation temperature of 28 °C until the 100th passage. In the 100th passage, the virus strains of the original EV71 derived from the oral fluid, brain stem tissue, and stool specimens of the patient were indicated as EV71:TLL, EV71:TLLα, and EV71:TLLβ, respectively. In the temperature sensitivity analysis, all three virus inoculants suitable for cold virus strains using 10 MOI virus inoculum caused complete CPE in Vero cells within 2 days at incubation temperature of 28 °C, but required at incubation temperature of 37 °C 4 days to reach full CPE. At the incubation temperature of 39.5 ° C, all three strains did not notice CPE after 10 days of culture. However, EV71:TLL gave 2+ CPE on day 10 of culture after blind subculture to a new flask of Vero cells grown at 39.5 °C. Even after another two blind subcultures to a new flask of Vero cells grown at 39.5 °C, EV71:TLLα and EV71:TLLβ did not notice CPE. Suspension cells collected from cultures inoculated with EV71:TLLα or EV71:TLLβ at the end of each 10-day culture (including cells derived from blind passages) were not detected by EV71 Positive immunofluorescence staining. Approximately 1% of the suspension cells collected from the EV71:TLL-inoculated culture supernatant gave positive staining, however, CPE was not noted after 10 days of culture.

Incubation temperatures of 28 ° C and 37 ° C were used to analyze the viral replication titration concentrations of three strains suitable for cold virus and the analysis was repeated at least 4 times. When the titrated culture was incubated at 28 ° C and 37 ° C, respectively, the titration concentration of EV71:TLL was 1 × 10 ⁸ CCID ₅₀ /ml and 2 to 3 × 10 ⁷ CCID ₅₀ /ml. EV71: TLLα gave a virus titration concentration of 1 × 10 ⁸ CCID ₅₀ /ml at a incubation temperature of 28 ° C and a titration concentration of 1 to 2 × 10 ^5.5-6 CCID ₅₀ /ml at 37 °C. EV71: TLLβ gives a titration concentration of 2 to 5 × 10 ⁸ CCID ₅₀ /ml at a incubation temperature of 28 ° C and a titration concentration of 1 to 1 × 10 ⁷ CCID ₅₀ /ml at 37 ° C.

The cold stability of the two strains was evaluated by substituting EV71:TLLα and EV71:TLLβ in the Vero cells at a incubation temperature of 28 ° C and a virus inoculum of 10 MOI. After another 20 passages, EV71:TLLα caused complete CPE in cells cultured at 28 °C within 2 days after inoculation but did not cause CPE in cells cultured at 37 °C, even after 2 blind passages. Its ability to maintain a virus titration concentration of 1 x 10 ⁸ CCID ₅₀ /ml at a incubation temperature of 28 °C. EV71: TLLβ was maintained at 28 ° C and 37 ° C incubation temperature after the other 20 passages to maintain the same cold phenotype in terms of growth kinetics and virus titration concentration. The stability of cold adaptation of EV71:TLLβ was further evaluated by subculture for another 20 passages under the same culture conditions (from the other 40 passages of the 100th passage) and found to remain similar to the temperature sensitive phenotype suitable for cold.

Evaluation from a reversal of a temperature-sensitive phenotype suitable for cold was performed on 6 consecutive passages of virus in Vero cells incubated at 37 °C under each complete CPE on EV71:TLLβP20. The growth characteristics of the virus derived from each of the individual cultures cultured at 37 ° C in terms of growth kinetics and virus titration concentrations at incubation temperatures of 28 ° C, 37 ° C and 39.5 ° C are shown in Table 1. The virus still cannot produce viable infectious particles (lack of positive immunofluorescent staining cells) in Vero cells at 39.5 ° C incubation temperature after 3 consecutive passages in cells cultured at 37 ° C and cannot be in the 6th time Repeated passages induced complete CPE in cell culture at incubation temperatures of 39.5 °C.

The growth characteristics and titration concentration of the virus at each subculture were cultured or titrated in Vero cells at incubation temperatures of 28 ° C, 37 ° C and 39.5 ° C.

P1: Succession 1

IFA: Indirect immunofluorescence analysis

The genomes of EV71:TLLα and EV71:TLLβ, their respective virus strains (EV71:TLLαP20 and EV71:TLLβP20) and the original parent wild type were sequenced and analyzed. The nucleotide sequence of EV71:TLLαP20 is set forth in SEQ ID NO: 6. The nucleotide sequence of EV71:TLLβP20 is set forth in SEQ ID NO:7. The nucleotide number change of each fragment of the gene between EV71:TLLα, EV71:TLLαP20 and the original parent wild type is shown in Table 2. The nucleotide number change between the original parent wild type, EV71:TLLβ, EV71:TLLβP20 and EV71:TLLβP40 (additional 40 generations) is shown in Table 3. The whole genome of the virus strains derived from the 6 consecutive successive temperature-sensitive reversal studies at the incubation temperature of 37 ° C was also sequenced and the change in nucleotide number relative to EV71:TLLβP20 is shown in Table 4.

Example 3 Results of monkey studies for cold human enterovirus 71 temperature-sensitive strains (EV71: TLLβP20)

Clinical findings : General observations of the clinical status of the monkeys studied and body temperature measurements were performed twice daily in the morning and evening. Throughout the process, it was noted that all monkeys were active and eating normally. None of the monkeys produced any subtle local nerve defects such as limb weakness, tremors or abnormal movements. None of the monkeys except the one given the intravenous dose of 1×10 ⁸ CCID ₅₀ /ml (2247M) produced a peak of low grade fever (39.3 ° C) on the 3rd day after inoculation. None of the monkeys had a weight loss when they were scaled when they were killed under deep anesthesia.

Autopsy and histology found that all necropsy monkeys did not notice the overall postmortem lesion. No abnormal histological findings were observed in all monkey tissues collected for histopathological examination.

Virological studies : Virus isolation of monkey serum, PBMC, stool samples and all necropsy tissues was performed using Vero cells at incubation temperatures of 28 ° C and 37 ° C. Although blind passage was performed after 10 days of culture, the virus was not isolated from any of the monkey samples. Another blind subdivision in Vero cells was performed on serum, PBMC samples and their tissue homogenates that were positive for EV71 by RT-PCR. Although the virus was not isolated, two monkeys 2891F (received 1×10 ⁷ CCID ₅₀ EV71:TLLβP20) collected on the 4th day after inoculation using indirect immunofluorescence analysis (Fig. 1a) using commercial monoclonal antibodies were used. EV71 antigen was detected in several PBMCs of heparinized blood of 2890 M (EV71: TLLβP20 receiving 1×10 ⁸ CCID ₅₀ ). No viral antigen was detected in the heparinized blood collected PBMC of monkeys collected on the 8th day after inoculation.

EV71-specific genomic sequences were detected by RT-PCR in serum samples from all monkeys collected on days 4 and 8 after inoculation. In non-neuronal tissues, the EV71 genomic sequence was detected only in the spleen homogenate of 2 monkeys (2202F and 2891F) (Fig. 1b). The EV71 genomic sequence was not detected in any of the neuronal tissue homogenates. Serum samples from two monkeys (2889M and 2890M) (collected on days 4 and 8 after inoculation) The genome of EV71:TLLβP20 was extracted and completely sequenced. The number of nucleotides (NT) and corresponding amino acid (AA) mutations or reversals present in each of the genomic fragments of the virus strain present in the serum compared to the genome of EV71:TLLβP20 is shown in the table. 5 in.

Monkey humoral immune response : The presence of bound antibodies (IgM and IgG) in the serum of monkeys administered intravenous EV71:TLLβP20 was analyzed by indirect immunofluorescence analysis using internally prepared infected Vero cells as antigen. Anti-EV71 IgM in the blood of two remaining monkeys (2889M and 2890M) collected on the 14th day after the inoculation (previously given the equivalent intravenous booster dose) and the 30th day after the inoculation (the 16th day after the boost) The titration concentrations of IgG and IgG are shown in Table 6.

Determination of EV71 genotype A (BrCr), B3, B4, B5, C1 and C5 in serum samples of two monkeys (2889M and 2890M) collected on the 14th and 30th day after inoculation by micro-neutralization analysis Neutralize the antibody titration concentration. The respective titrated concentrations of monkey serum neutralizing antibodies against each of the EV71 genotypes are shown in Table 7.

Example 4 Materials and methods for developing human coxsackievirus A16 suitable for cold temperature sensitive strains

MATERIALS AND METHODS : Conventional subcultured Vero cells (ATCC CCL-81) in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FCS) were used for virus culture. , attenuate, titrate and assess the temperature sensitivity of Coxsackievirus A16. The parental wild-type coxsackievirus A16 (CA16) used in the attenuating process is derived from oral secretions from patients with hand-foot-and-mouth disease (HFMD). The virus was plaque purified once according to the techniques described previously. After plaque purification, virus stocks indicated as individual wild-type virus strains were prepared after two passages in Vero cells at 37 °C. All virus stocks and each successive virus strain were stored in a minus 80 °C freezer.

A young, freshly confluent, single-layered Vero cell line in DMEM containing 1% FCS was used to adapt to the original wild-type CA16 for replication at successively lower incubation temperatures starting at an initial lower incubation temperature of 34 °C. The virus was subcultured into each successive new culture flask of Vero cells under complete cytopathic effect (CPE). The virus was subcultured at a higher infection multiplication rate (MOI) of 20 when it was changed to a continuous lower incubation temperature and was reduced to 5 to 10 after at least three passages after it was noted that it was able to cause complete CPE within 3 days after inoculation. The lower MOI. After noting that the virus was able to cause complete CPE within 3 days after inoculation with an MOI of 5 to 10, it was advanced to the next consecutive lower incubation temperature after at least three more subcultures. Each successively subcultured medium was stored in a minus 80 ° C freezer for later analysis.

Viral titration : slight modification was performed according to the method described in the Polio Laboratory Manual 2004 of the World Health Organization. The virus titration concentration was determined by microtiter analysis in Vero cells and the virus titration concentration was calculated per Reed & Muench method (1938). 50 ml cell culture infection dose (CCID ₅₀ ). Briefly, after treatment with equal amounts of chloroform to disperse viral aggregates, a 10-fold serial dilution of the clarified viral supernatant was prepared in DMEM containing 1% FCS. The Vero cell monolayer (10 ⁴ cells per well) in a 96-well flat-bottom tissue culture plate was inoculated with 100 μl of serially diluted virus stocks and at each incubation temperature in a 5% CO ₂ atmosphere. The cultivation was carried out for 5 days, and then the presence of CPE was observed.

Temperature sensitivity analysis : Two methods were used to evaluate the growth characteristics of a virus strain suitable for cold in Vero cells at incubation temperatures of 28 ° C, 37 ° C and 39.5 ° C. The first method assesses the number of days (viral kinetics) used by the virus strain to cause complete CPE in infected cells and the second method assesses the titer concentration of the virus strain in cells cultured at each particular test temperature. Briefly, in the first method, the growth medium of three T-25 tissue culture flasks containing confluent monolayer Vero cells of similar age was replaced by maintenance medium (DMEM containing 1% FCS). The medium in each flask was then equilibrated to the specific temperature to be tested by placing in a separate incubator for 1 hour and then inoculated with a virus strain at a dose of 10 infection magnification (MOI). If CPE was not noticed at the end of the 10 day culture, the supernatant was transferred to a new single-layered Vero cell flask and similarly incubated for another 10 days. If the CPE is not noticed after the second pass, it is considered to be virus free. In the second method, a Vero cell suspension having a density of 10 ⁴ cells per 100 μl is inoculated into each well of three 96-well cell culture dishes and cultured at 37 ° C in a 5% CO ₂ atmosphere. . After 10 hours of incubation, each cell culture plate was then equilibrated to the specific temperature to be tested by placing in a separate incubator for 1 hour. The cells in each well were then inoculated by 10-fold serial dilutions of 100 μl of virus strain, which were then transferred to incubation chambers at respective temperatures and incubated for 5 days, after which the presence of CPE was observed.

Example 5 Results of viral characteristics in cells suitable for cold human Coxsackievirus A16 temperature-sensitive strains

Under the 100th subculture of continuously decreasing incubation temperature, the CA16 strain suitable for cold can 5 MOI virus inoculum and 3 days of inoculation at 28 ° C incubation temperature caused complete CPE in the Vero cells, but it took 4 days to reach full CPE at the incubation temperature of 37 °C. At the incubation temperature of 39.5 ° C, no CPE was noted.

Incubation temperatures of 28 ° C and 37 ° C were used to analyze viral replication titration concentrations for cold CA16 temperature sensitive strains. When the titration plate was incubated at 28 ° C and 37 ° C, respectively, the titration concentration of the CA16 temperature sensitive strain suitable for cold was 1 × 10 ⁸ CCID ₅₀ /ml and 1 × 10 ⁷ CCID ₅₀ /ml.

The terms "a" and "the" are used in the context of the present invention (especially in the context of the following claims) unless otherwise indicated herein. And similar indicators should be construed as covering both singular and plural. Unless otherwise stated, the terms "including", "having", "including" and "including" are explained. It is an open term (meaning "including (but not limited to)"). The recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to the individual values of the range, and the individual values are incorporated in the specification as if individually recited herein. For example, if the range 10-15 is 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 clearly contradicted by the context. The use of any and all examples or exemplary language, such as "such as" The language in the specification should not be construed as indicating that any element not claimed is essential to the practice of the invention.

It will be appreciated that the methods and compositions of the present invention can be combined in a variety of embodiments, only a few of which are disclosed herein. The embodiments of the invention are described herein, including the best mode known to the inventors to carry out the invention. Variations of the embodiments may become apparent to those of ordinary skill in the art after reading the foregoing description. The inventors intend for the skilled artisan to employ such variations as appropriate, and the inventors intend to practice the invention in a manner other than as specifically described herein. Accordingly, to the extent permitted by applicable law, the invention includes all modifications and equivalents of the subject matter described in the claims. In addition, the present invention encompasses any combination of the above-described elements in all possible variations thereof unless otherwise indicated herein or otherwise clearly contradicted by the context.

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<120> Suitable for cold enterovirus 71 temperature sensitive strains and development for cold temperatures Sensitive virus strain method

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Claims (36)

A temperature sensitive strain suitable for cold enterovirus 71. The temperature-sensitive strain suitable for cold enterovirus 71 according to claim 1 is EV71:TLLβP20. The temperature-sensitive strain of cold enterovirus 71 is as claimed in claim 1, wherein the strain is EV71:TLLαP20. A composition comprising a cold enterovirus 71 temperature sensitive strain according to any one of claims 1 to 3. The composition of claim 4, which further comprises a pharmaceutically acceptable carrier. The composition of claim 4 or 5, further comprising an adjuvant. The composition of any one of claims 4 to 7, which is a vaccine. A composition of any one of claims 1 to 3, which is suitable for use in a cold-to-intestinal virus 71 temperature-sensitive strain, or a composition according to any one of claims 4 to 8, for use in eliciting a protective immune response in an individual. A composition suitable for cold enterovirus 71 temperature-sensitive strains, or a composition according to any one of claims 4 to 8, for use in preventing an individual from suffering from a disease associated with enterovirus 71, as claimed in any of claims 1 to 3. A composition suitable for cold enterovirus 71 temperature-sensitive strains, or a composition according to any one of claims 4 to 8, for delaying the intestinal tract in an individual infected with enterovirus 71, according to any one of claims 1 to 3. The onset of a virus 71-related disease or slows the rate of the disease. The composition of any one of claims 1 to 3, wherein the composition is a cold sensitive enterovirus 71 temperature sensitive strain or a composition according to any one of claims 4 to 8 for use in the manufacture of a medicament for eliciting a protective immune response in an individual. . A composition suitable for cold enterovirus 71 temperature-sensitive strains or a composition according to any one of claims 4 to 8 for use in the manufacture of a medicament for preventing an individual from suffering from intestinal diseases An agent for the disease associated with 71. A composition suitable for cold enterovirus 71 temperature-sensitive strains, or a composition according to any one of claims 4 to 8, for use in the manufacture of a delay in an individual infected with enterovirus 71, as claimed in any one of claims 1 to 3. An agent that causes the onset of a disease associated with enterovirus 71 or slows the rate of the disease. A method of eliciting a protective immune response in an individual comprising administering to the individual a prophylactically, therapeutically or immunologically effective amount of a temperature suitable for cold enterovirus 71 according to any one of claims 1 to 3 A sensitive strain or a composition according to any one of claims 4 to 8. The method of claim 14, wherein the individual has been exposed to wild-type enterovirus 71. A method for preventing an individual from suffering from a disease associated with enterovirus 71, comprising administering to the individual a prophylactically, therapeutically or immunologically effective amount of a cold enterovirus according to any one of claims 1 to 3 A temperature sensitive strain or a composition according to any one of claims 4 to 8. The method of claim 16, wherein the individual has been exposed to wild-type enterovirus 71. A method of delaying the onset of or slowing the onset of a disease associated with enterovirus 71 in an individual infected with enterovirus 71, comprising administering to the individual a prophylactic, therapeutically or immunologically effective amount as claimed The temperature-sensitive strain of the cold enterovirus 71 or the composition of any one of claims 4 to 8 of any one of items 1 to 3. The method of any one of claims 14 to 18, wherein the individual is a human individual. A kit for use in a single immunization of cold enterovirus 71 temperature sensitive strains, comprising a cold sensitive enterovirus 71 temperature sensitive strain according to any one of claims 1 to 3 or as claimed in claims 4 to 8 A composition, a pharmaceutically acceptable carrier, and instructions for use thereof. A kit of claim 20, further comprising an applicator. A method of producing a temperature sensitive virus suitable for cold, comprising the steps of: (i) preparing a reference stock of the parental wild-type virus; (ii) using a lower infection multiplication rate (MOI) for each sub-derived complete cytopathic effect (CPE) and a shorter incubation period of the inoculum Incubating the cell culture of the reference stock of the parental wild-type virus for five or more passages at a higher MOI and incubation temperature of about 34 ° C to about 36 ° C, preferably about 34 ° C until Sub-passives obtain complete CPE; (iii) use lower infectivity (MOI) and shorter incubation periods for inoculations with complete cytopathic effect (CPE) for each subdivision at higher MOI and above Incubating the cell cultures infected with the virus obtained by the foregoing steps for five or more passages at a medium to low incubation temperature of about 1 ° C to about 3 ° C until complete CPE is obtained in each subculture; and (iv) Step (iii) is repeated in a stepwise manner in a system that incrementally lowers the incubation temperature until the target temperature selected for optimal replication of the virus is achieved. The method of claim 22, wherein the wild-type virus-infected cell culture is incubated one or two times at a temperature of from about 36 ° C to about 38 ° C, preferably about 37 ° C until a complete cytopathic effect is obtained ( CPE) This reference stock for the preparation of the parental wild type virus. The method of claim 22 or 23, wherein the virus is a member of the picornavirus family, preferably an enterovirus genus. The method of claim 24, wherein the virus is enterovirus 71 or coxsackievirus A16. The method of any one of claims 22 to 25, wherein the target temperature is from about 26 ° C to about 29 ° C, preferably about 28 ° C. The method of any one of clauses 22 to 26, wherein the cytopathic effect (CPE) of the virus is obtained by obtaining a clarified culture containing the virus and delivering the supernatant to the cells. The new culture flask is fresh and the virus is subcultured in each step of the process. The method of any one of clauses 22 to 27, wherein the higher MOI is 20. The method of claim 28, wherein the new culture flask containing freshly confluent single-layered Vero cells is inoculated three or more times by virus under MOI of 20 after the virus causes complete CPE. The method of any one of clauses 22 to 29, wherein the lower MOI is 5 to 10. The method of claim 30, wherein the new culture flask containing the freshly confluent monolayer of Vero cells is inoculated at least three more times after the virus causes complete CPE by virusing at an MOI of 5 to 10. The method of claim 31, wherein the step (iii) is initiated or repeated after the at least three more subdivisions at an MOI of 5 to 10. The method of any one of claims 27 to 32, wherein the complete CPE occurs within 3 days after inoculation. The method of any one of claims 22 to 33, wherein the cells are Vero cells. The method of any one of claims 22 to 34, wherein the Vero cells are cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal bovine serum (FCS). The method of claim 35, wherein the medium is supplemented with 1% FCS.