TWI705140B - 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:TLLβP20 and EV71:TLLαP20. The present invention also relates to processes of developing the cold-adapted temperature sensitive virus strains.

Description

A temperature-sensitive strain of Enterovirus 71 suitable for cold and a method for developing a temperature-sensitive virus strain suitable for cold Sequence submission

This application is applied together with the sequence table in electronic format. The sequence listing name is 2577224PCTSequenceListing.txt, which was generated on January 11, 2013 and has a size of 21 kb. The information in the sequence table in electronic format is incorporated into this article by reference in its entirety.

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

Publications and other materials used herein to clarify the background of the present invention or provide other details about the practice are incorporated by reference, and are separately collected in references for convenience.

Hand, foot and mouth disease (HFMD) is caused by human enteroviruses, which are basically a group of enterovirus species. Among these enteroviruses, enterovirus 71 (EV71) and Coxsackie virus A16 (CA16) are responsible for more than 90% of HFMD cases. In addition to causing HFMD, EV71 is known to cause severe neurological diseases and death (especially in young children).

Human Enterovirus 71 (EV71) is a small non-enveloped virus with a size of about 30 nm, which has a single-stranded positive RNA genome of about 7,450 nucleotides. The virus is classified as a species of human enterovirus A under the Picornaviridae endovirus genus (Alexander et al., 1994; Melnick, 1996). Based on the phylogenetic analysis of the major capsid protein (VP1) gene of EV71, it is divided into three major genomes (indicated as A, B, and C), and genomes B and C are further divided into subgenomes B1 to B5 and C1 to C5 (Bible et al., 2008). EV71 has been associated with a number of clinical diseases including hand, foot and mouth disease (HFMD), aseptic meningitis, encephalitis and polio symptom 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 then characterized as a novel serotype of Enterovirus (Schmidt et al., 1974). In the years after its initial isolation, outbreaks of HFMD with complications due to the virus have been reported worldwide (Blomberg et al., 1974; Kennett et al., 1974; Deibel et al., 1975; Hagiwara et al., 1978).

In recent years, after the emergence of neurotoxic EV71 infectivity and sporadic outbreaks, EV71 infection has become a major public health burden and problem in the entire world, specifically in the Asia-Pacific region. The neurotoxicity of EV71 first received significant global attention during the outbreak in Bulgaria in 1975, which caused 705 cases of polio-symptomatic diseases, including 44 deaths (Chumakov et al., 1979; Shindarov et al., 1979). An outbreak of similar nature occurred in Hungary in 1978 and resulted in multiple polio-symptomatic diseases and 47 deaths (Nagy et al., 1982). Subsequently, several milder infectious diseases related to EV71-related CNS diseases 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). There are two EV71 infectious diseases in Japan, most of the cases are characterized by the low incidence of HFMD and CNS disease (Tagaya et al., 1981; Ishimaru et al., 1980; Hagiwara et al., 1983). In 1997, a major outbreak of HFMD due to highly neurotoxic EV71 occurred in Malaysia and caused 48 deaths. A larger outbreak occurred in Taiwan in 1998, with more than 100,000 cases of HFMD, 405 severe infections, and 78 deaths (due to acute brainstem encephalomyelitis, 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 cases of HFMD were recorded in the mainland of the People's Republic of China, including 126 deaths (Chinese Center, 2008). It is reported that the number of HFMD cases increased to 1,155,525 in 2009, with 353 deaths (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 severe cases with neurological complications and death were due to EV71 (Zeng et al., 2012).

Currently, the molecular determinants of viral toxicity and pathogenesis of EV71 infection are still not fully understood. There are neither antiviral drugs approved for clinical treatment of severe infections and related neurological complications, nor any vaccines available to control and prevent recurrent outbreaks. As far as control and preventive strategies are concerned, the development of cost-effective vaccines, especially for developing countries, is the first and most urgent task. Various types of vaccines against EV71 under investigation and development seem to trigger 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. Human, 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. Although virus-like particle vaccine based on VP1 capsid Potential vaccine strategies that make it feasible with subunit peptide vaccines are 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 the control and almost eradication of wild-type poliovirus infections A lot of past experience of inflammatory virus vaccine, injectable inactivated and oral attenuated EV71 vaccine is still the most promising candidate (Zhang et al., 2010).

Currently, there is no vaccine available in the market to protect children from infections attributed to EV71 and hand, foot and mouth disease. Based on recent information, two centers in the People's Republic of China, one center in Singapore and one center in Taiwan are in the process of developing injectable inactivated EV71 vaccine (press release and personal communication). National Institute of headed by Dr. H. Shimizu The Enterovirus Unit in Infectious Diseases, Tokyo, Japan has been conducting in-depth research on the pathogenesis of monkey EV71 and the temperature-sensitive strain of EV71 as a potential candidate for oral live attenuated EV71 vaccine since the 1980s (Arita et al., 2005; Arita et al. , 2007). Although compared with the wild-type virus after intravenous vaccination, the degree of neural invasion and histopathological changes in the CNS is lower, but all potential vaccine candidates of the temperature-sensitive strain of EV71 in the monkey study can still cause these changes .

Live attenuated vaccines represent one of the first successful methods of vaccination, dating back to the 18th century, when British doctor Edward Jenner began to use vaccinia virus to vaccinate children against the destructive disease smallpox. Live attenuated vaccines use weakened live viruses or microorganisms so that they cannot cause disease, but induce a protective immune response. Traditional, classical and genetic methods have been successfully used as live attenuated vaccines to attenuate viruses and microorganisms to a certain extent. ^44-48 Traditional methods use related organisms that are non-toxic in the human body, such as cowpox (vaccinia) viruses. The classic method involves multiple rounds of growth under conditions that attenuate virulent viruses or microorganisms, such as in tissue culture or harsh liquid media. Genetic methods use modern molecular biotechnology 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 the classic method of obtaining a temperature-sensitive phenotype virus as a marker of attenuation, plaque selection of wild-type viruses is performed by culturing the virus in suitable cells cultivated at a lower temperature by means of plaque analysis technology. The selected virus strains are then repeatedly subcultured at the target lower cultivation temperature (Hagiwara et al., 1982; Hashimoto and Hagiwara, 1983; Richman and Murphy, 1997).

There is a need to develop virus strains that can be used to treat 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 strains of temperature-sensitive viruses (including RNA viruses) suitable for cold that are obtained after successive generations in cell culture.

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

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

The enterovirus 71 virus strain of the present invention is prepared by using temperature sensitivity as a phenotypic marker to attenuate enterovirus 71. This method is an in vitro laboratory process, which changes the biological growth characteristics of the virus to adapt to the optimal replication under the incubation temperature below 30°C. The adaptation process (which is described in detail below) is carried out in a systematic and stepwise manner that gradually reduces the incubation temperature for culturing the virus until the selected target temperature for optimal replication of the virus is reached.

In a second aspect, the present invention provides a composition comprising the temperature sensitive strain of Enterovirus 71 suitable for cold as described herein. In one embodiment, the composition contains an effective amount of the virus strain described herein. In another embodiment, the composition includes one or more physiologically or pharmaceutically acceptable carriers. In another embodiment, the composition is a vaccine. A vaccine containing the temperature sensitive strain of Enterovirus 71 suitable for cold as described herein is prepared using techniques well known to those skilled in the art. These vaccines are suitable for administering vaccines to individuals, such as human individuals, to provide immunity against parental virus strains by using techniques well known to those skilled in the art.

In a third aspect, the present invention provides a method for eliciting a protective immune response in an individual such as a human individual, which comprises administering to the individual a prophylactic or therapeutically or immunologically effective amount of the appropriate amount described herein. Enterovirus 71 is a temperature-sensitive strain of cold. 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 Symptoms of polio paralysis. In one example, the temperature-sensitive strain of Enterovirus 71 described herein suitable for cold is administered as a vaccine. In another example, the individual has been exposed to wild-type enterovirus 71. In another embodiment, the administration of the enterovirus 71 temperature-sensitive strain suitable for cold described herein prevents individuals, such as human individuals, from suffering from enterovirus 71-related diseases. In another example, the individual has been exposed to wild-type enterovirus 71. In another embodiment, the administration of the enterovirus 71 temperature-sensitive strain suitable for cold as described herein delays the onset or slows the progression rate of enterovirus 71-related diseases in a virus-infected individual such as a human individual.

In the fourth aspect, the present invention provides a method of using temperature sensitivity as a phenotypic marker to attenuate viruses. According to this aspect, the method was developed for cold temperature-sensitive virus strains. The method of the present invention is an in vitro laboratory process, which changes the biological growth characteristics of the virus to adapt to the optimal replication at an incubation temperature below 30°C. The adaptation process is carried out in a stepwise manner with a system that gradually reduces the incubation temperature used for culturing the virus until the selected target temperature for the best replication of the virus is reached. Therefore, according to the present invention, the method includes the following steps: (i) preparing a reference stock of the parental wild-type virus, (ii) using a lower infection rate in terms of obtaining a complete cytopathic effect (CPE) for each successive generation (MOI) and the inoculum with a shorter incubation period are cultivated at a higher MOI and an incubation temperature of about 34°C to about 36°C, preferably about 34°C. Cell cultures infected with the reference stock of the parental wild-type virus One or more subcultures until complete CPE is obtained in each subculture, (iii) Use lower infection rate (MOI) and shorter incubation period in terms of obtaining complete cytopathic effect (CPE) for each subculture The inoculum is cultured at a higher MOI and at an incubation temperature of about 1°C to about 3°C lower than the previous step. The virus-infected cell culture obtained by the previous step is subcultured five or more times until each incubation Generation of complete CPE is obtained, and (iv) Step (iii) is repeated in a stepwise manner with a system of incrementally lowering the incubation temperature until the selected target temperature for optimal replication of the virus is reached. In one embodiment, the target temperature is about 26°C to about 29°C, preferably about 28°C. In one embodiment, reducing the incubation temperature incrementally is a temperature reduction of about 1°C to about 2°C.

In one embodiment, by culturing at a temperature of about 36°C to about 38°C, preferably about 37°C Breed cell cultures infected with wild-type virus once or twice until the complete cytopathic effect (CPE) is obtained to prepare the reference stock of the parental wild-type virus. Place an aliquot of the culture supernatant containing the generated virus in a vial or other suitable storage device. The supernatant of this culture serves as a reference stock for the parental wild-type virus. The reference parent wild-type virus line is used for the subsequent attenuation 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 Picornaviridae 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 Coxsackie virus A16 (CA16). The method of the present invention is suitable for producing cold-suitable temperature-sensitive virus strains of any of these viruses, including but not limited to the cold-suitable 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 cell is a Vero cell (ATCC CCL-81). In one embodiment, the cells are maintained by routine subculture in a medium suitable for cell growth. In the example where the cells are Vero cells, the Vero cells are cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). In one embodiment, the Vero cell line maintained in DMEM supplemented with 1% FCS is used to generate parental wild-type virus, virus culture, attenuate, titrate, and evaluate temperature-sensitive phenotypes. In another embodiment, DMEM is supplemented with 1% FCS to adapt the virus strain to replicate in successively lowering incubation temperatures.

The present invention also relates to cold temperature virus strains produced by the methods described herein. The cold temperature virus produced by the method described herein is suitable for vaccine production using techniques well known to those skilled in the art. These vaccines are suitable for administering vaccines to individuals to provide immunity against parental virus strains by using techniques well known to those skilled in the art.

Figure 1a shows peripheral blood mononuclear cells (arrows) derived from monkey blood on the 4th day after intravenous dose of Enterovirus 71 (EV71: TLLβP20), using commercial monoclonal antibodies specific to the virus. Indirect immunofluorescence analysis was positive.

Figure 1b shows a photograph of an electrophoresis agarose gel stained with GelRed reagent, which shows the use of oligonucleotide primers specific for the detection of enterovirus 71 from monkeys (2202F, 2891F) on day 4 after immunization One-step RT-PCR amplification product of the tissue. The expected size of the RT-PCR amplified product is 427bp. The channels in the two gels are as follows. Gel 1 : Channel 1: 100bp DNA ladder; Channel 2: 2202F-heart; Channel 3: 2202F-spleen; Channel 4: 2202F-lymph nodes; Channel 5: 2202F-kidney; Channel 6: 2202F-liver; Channel 7: 2891F -Heart; Channel 8: 2891F-Spleen; Channel 9: 2891F-Lymph nodes; Channel 10: 2891F-Kidney; Channel 11: 2891F-Liver; Channel 12: 2202F-Brainstem (pons); Channel 13: 2202F-Brainstem ( Medulla oblongata); passage 14: 2202F-cortex (cerebral gyrus); passage 15: 2202F- spinal cord (cervical spine); passage 16: 2202F- spinal cord (lumbar spine); passage 17: 2202F- spinal cord (thoracic); passage 18: 2891F-brain Stem (medulla oblongata); passage 19: 2891F-brain stem (pons); passage 20: 2891F-cortex (left cerebellum). Gel 2 : Passage 21: 100bp DNA ladder; Passage 22: 2891F-cortex (right cerebellum); Passage 23: 2891F-spine (cervical spine); Passage 24: 2891F-spine (lumbar spine); Passage 25: 2891F-spine (thoracic) ); Lane 26: No template control; Lane 27: 100bp DNA ladder.

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

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

The enterovirus 71 virus strain of the present invention is prepared by using temperature sensitivity as a phenotypic marker to attenuate enterovirus 71. This method is an in vitro laboratory process, which changes the biological growth characteristics of the virus to adapt to the optimal replication under the incubation temperature below 30°C. The adaptation process is carried out in a stepwise manner with a system that gradually reduces the incubation temperature used for culturing the virus until the selected target temperature for the best replication of the virus is reached. As disclosed herein, (i) prepare a reference stock of the parental wild-type virus, (ii) use a lower infection rate (MOI) and shorter incubation in terms of obtaining a complete cytopathic effect (CPE) for each passage The inoculum of the time period is cultivated at a higher MOI and an incubation temperature of about 34°C. The cell culture infected with the reference stock of the parental wild-type virus is subcultured five or more times until a complete CPE is obtained in each subculture. , (Iii) Use the inoculum with a lower infection rate (MOI) and a shorter incubation period in terms of obtaining a complete cytopathic effect (CPE) for each successive generation at a higher MOI and about 1°C lower than the previous step Cultivate the virus-infected cell culture obtained by the aforementioned steps for five or more subcultures at an incubation temperature of about 3°C until a complete CPE is obtained at each subculture, and (iv) a system that reduces the incubation temperature incrementally , Repeat step (iii) step by step until reaching the selected target temperature for optimal virus replication. In one embodiment, the target temperature is about 26°C to about 29°C, preferably about 28°C.

In one embodiment, the cell culture infected with the wild-type virus is subcultured once or twice at a temperature of about 36°C to about 38°C, preferably about 37°C, until a complete cytopathic effect (CPE) is obtained. Prepare the reference stock of the parental wild-type virus. Place an aliquot of the culture supernatant containing the generated virus in a vial or other suitable storage device in. The supernatant of this culture serves as a reference stock for the parental wild-type virus. The reference parent wild-type virus line is used for the subsequent attenuation 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 routine subculture in a medium suitable for cell growth. In the example where the cells are Vero cells, the Vero cells are cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). In one embodiment, Vero cells maintained in DMEM supplemented with 1% FCS are used to produce parental wild-type virus, virus culture, attenuation, titration, and assessment of temperature-sensitive phenotypes. In another embodiment, DMEM is supplemented with 1% FCS to adapt the virus strain to replicate in continuously decreasing incubation temperatures.

In one embodiment, once the virus has achieved a complete cytopathic effect (CPE), the clear culture supernatant containing the virus is obtained and the supernatant is transferred to each continuous fresh new culture of cells (such as Vero cells) The virus is subcultured in the bottle and in each step of the process. In another embodiment, the virus-containing culture supernatant is delivered at a higher infection rate (MOI) of 20 at the beginning of each successive change to a lower incubation temperature. After noticing that the virus can cause complete CPE in Vero cells within 3 days after inoculation, inoculate a new culture flask containing freshly confluent single-layer Vero cells with virus of the same MOI for at least three subcultures, and then reduce to 5 To the lower MOI of 10. Once it is noted that the virus can cause complete CPE within 3 days after inoculation with a lower MOI of 5 to 10, and after at least three subcultures at an MOI of 5 to 10, the attenuation process then proceeds to a continuous lower incubation temperature. stage. The number of days required for each successive generation depends on the speed at which the virus adapts to each stage of increasing the incubation temperature. Those who are familiar with this technique will easily know when to reach full CPE. Other details of the method for preparing the temperature sensitive strain of enterovirus 71 suitable for cold of the present invention are described below.

In the second aspect, the present invention provides a cold bowel disease Composition of Toxin 71 temperature-sensitive strain. In one embodiment, the composition contains an effective amount of the virus strain described herein. In another embodiment, the composition includes one or more physiologically or pharmaceutically acceptable carriers. In another embodiment, the composition is a vaccine. A vaccine containing the temperature sensitive strain of Enterovirus 71 suitable for cold as described herein is prepared using techniques well known to those skilled in the art. These vaccines are suitable for administering vaccines to individuals, such as human individuals, to provide immunity against parental virus strains by using techniques well known to those skilled in the art.

It should be understood that the temperature-sensitive strain of Enterovirus 71 suitable for cold as described herein can be used to induce a protective immune response in an individual or prevent the individual from suffering from a virus-related disease or delay the onset or slow down the onset of a virus-related disease At the rate of progression, it is administered to an individual in the form of a composition that additionally contains 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: 0.01M-0.1M and preferably 0.05M phosphate buffer, phosphate buffer Normal saline (PBS) or 0.9% normal saline. Such carriers also include aqueous or non-aqueous solutions, suspensions and emulsions. Aqueous vehicles include water, alcohol/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 contain non-toxic solid carriers such as glucose, sucrose, mannitol, sorbitol, lactose, starch, magnesium stearate, cellulose or cellulose derivatives, sodium carbonate, and magnesium carbonate. For administration in aerosol form, such as pulmonary and/or intranasal delivery, it is better to use non-toxic surfactants, (for example) esters or partial esters of C6 to C22 fatty acids or natural glycerides and propellant formulation reagents or combination. Other carriers such as lecithin may be included to facilitate intranasal delivery. The pharmaceutically acceptable carrier may further contain a small amount of auxiliary substances, such as wetting or emulsifying agents, preservatives and other additives, such as antibacterial agents, antioxidants Agents and chelating agents, which increase the shelf life and/or effectiveness of active ingredients. As is well known in the art, the composition 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 present invention provides a method for inducing a protective immune response in an individual such as a human individual, which comprises administering to the individual a prophylactic or therapeutically or immunologically effective amount of the appropriate amount described herein. Enterovirus 71 is a temperature-sensitive strain of cold. 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 polio symptom paralysis. In one example, the temperature-sensitive strain of Enterovirus 71 described herein suitable for cold is administered as a vaccine. In another example, the individual has been exposed to wild-type enterovirus 71. "Exposure" to enterovirus 71 means contact with enterovirus 71 so that infection can occur. In another embodiment, the administration of the enterovirus 71 temperature-sensitive strain suitable for cold as described herein prevents individuals such as human individuals from suffering from enterovirus 71-related diseases. In another example, the individual has been exposed to wild-type enterovirus 71. In another embodiment, the administration of the enterovirus 71 temperature-sensitive strain suitable for cold as described herein delays the onset or slows the progression rate of enterovirus 71-related diseases in a virus-infected individual such as a human individual.

As used herein, "administration" means delivery using any of various methods and delivery systems known to those skilled in the art. Can (e.g.) be intraperitoneal, intracranial, intravenous, oral, transmucosal, subcutaneous, percutaneous, intradermal, intramuscular, topical, parenteral, via implantation, intrathecal, intralymphatic, intralesional, pericardial Or administered epidurally. The agent or composition can also be administered in aerosol form, such as pulmonary and/or intranasal delivery. The investment may be made, for example, once, multiple times, and/or over one or more extended periods.

A protective immune response in the individual can be elicited, for example, by administering a main dose of the vaccine to the individual, followed by one or more subsequent administrations of the vaccine after a suitable period of time. The suitable period between the administration of the vaccine can be easily determined by those familiar with the technology, and is usually about several weeks to Several months. However, the present invention is not limited to any particular method, approach or frequency of administration.

"Prophylactically effective dose" or "immunologically effective dose" means that when administered to an individual who is prone to viral infection or has a tendency to suffer from a virus-related disease, it induces and protects the individual from infection or disease The amount of vaccine for the immune response. "Protecting" an individual means reducing the possibility of the individual being infected with a virus or reducing the probability of the onset of a disease in the individual at least twice, preferably at least ten times. For example, if an individual has a 1% chance of contracting the virus, a two-fold reduction in the possibility of the individual being infected with the virus will give the individual a 0.5% chance of contracting the virus. Optimally, the "prophylactically effective dose" induces an immune response in the individual that completely prevents the individual from being infected with the virus or completely prevents the onset of the disease in the individual.

Certain embodiments of any of the immunization and treatment methods of the present invention may further comprise administering to the individual at least one 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 of combining adjuvants and antigens are well known to those skilled in the art. Adjuvants suitable for 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) and similar adjuvants.

The present invention also provides a method of using temperature sensitivity as a phenotypic marker to attenuate viruses. According to this aspect, the method was developed for cold temperature-sensitive virus strains. The method of the present invention is an in vitro laboratory process, which changes the biological growth characteristics of the virus to adapt to the optimal replication at an incubation temperature below 30°C. The adaptation process is carried out in a stepwise manner with a system that gradually reduces the incubation temperature used for culturing the virus until the selected target temperature for the best replication of the virus is reached.

Therefore, according to the present invention, the method includes the following steps: (i) preparing a reference stock of the parental wild-type virus, (ii) using a lower infection rate in terms of obtaining a complete cytopathic effect (CPE) for each passage ( MOI) and the inoculum with a shorter incubation period is cultivated with the parental wild-type virus at a higher MOI and an incubation temperature of about 34°C to about 36°C, preferably about 34°C. Consider five or more subcultures of the cell culture infected with the stock material until a complete CPE is obtained at each subculture. (iii) Use a lower infection rate for each subculture to obtain a complete cytopathic effect (CPE) (MOI) and the inoculum with a shorter incubation period. Cultivate the virus-infected cell culture obtained by the previous step at a higher MOI and an incubation temperature of about 1°C to about 3°C lower than the previous step for five or more times Subsequent to CPE until complete CPE is obtained in each subculture, and (iv) Step (iii) is repeated in a stepwise manner with a system of incrementally lowering the incubation temperature until the selected target temperature for optimal replication of the virus is reached. In one embodiment, the target temperature is about 26°C to about 29°C, preferably about 28°C. In one embodiment, reducing the incubation temperature incrementally is a temperature reduction of about 1°C to about 2°C.

In one embodiment, the cell culture infected with the wild-type virus is subcultured once or twice at a temperature of about 36°C to about 38°C, preferably about 37°C, until a complete cytopathic effect (CPE) is obtained. Prepare the reference stock of the parental wild-type virus. Place an aliquot of the culture supernatant containing the generated virus in a vial or other suitable storage device. The supernatant of this culture serves as a reference stock for the parental wild-type virus. The reference parent wild-type virus line is used for the subsequent attenuation 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 a complete cytopathic effect (CPE), the clear culture supernatant containing the virus is obtained and the supernatant is transferred to each continuous fresh new culture of cells (such as Vero cells) The virus is subcultured in the bottle and in each step of the process. In another embodiment, the virus-containing culture supernatant is delivered at a higher infection rate (MOI) of 20 at the beginning of each successive change to a lower incubation temperature. After noticing that the virus can cause complete CPE in Vero cells within 3 days after inoculation, inoculate a new culture flask containing freshly confluent single-layer Vero cells with virus of the same MOI for at least three subcultures, and then reduce to 5 To the lower MOI of 10. Once it is noted that the virus can cause complete CPE within 3 days after inoculation with a lower MOI of 5 to 10, and after at least three subcultures at an MOI of 5 to 10, the attenuation process then proceeds to a continuous lower incubation temperature. stage. Days required for each succession The number depends on the speed at which the virus adapts to each stage of increasing the incubation temperature. Those who are familiar with this technique will easily 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 Picornaviridae 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 Coxsackie virus A16 (CA16). The method of the present invention is suitable for producing cold-suitable temperature-sensitive virus strains of any of these viruses, including but not limited to the cold-suitable 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 cell is a Vero cell (ATCC CCL-81). In one embodiment, the cells are maintained by routine subculture in a medium suitable for cell growth. In the example where the cells are Vero cells, the Vero cells are cultured in Dulbecco modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). In one embodiment, Vero cells maintained in DMEM supplemented with 1% FCS are used to produce parental wild-type virus, virus culture, attenuation, titration, and assessment of temperature-sensitive phenotypes. In another embodiment, DMEM is supplemented with 1% FCS to adapt the virus strain to replicate in successively lowering incubation temperatures.

The present invention also relates to cold temperature virus strains produced by the methods described herein. The cold temperature virus produced by the method described herein is suitable for vaccine production using techniques well known to those skilled in the art. These vaccines are suitable for administering vaccines to individuals to provide immunity against parental virus strains by using techniques well known to those skilled in the art.

The method of the present invention is suitable for the production of temperature-sensitive viruses suitable for cold of the Picornaviridae and Enteroviruses. This applicability has been demonstrated in this article by the production of temperature-sensitive strains EV71 (TLLα) and EV71 (TLLβ) suitable for cold. These temperature-sensitive strains are obtained after successive subcultures in cell cultures under increasing incubation temperature. . EV71 (TLLβ) virus strain It maintains phenotype and genetic stability under specific in vitro cell culture conditions and does not exhibit neurotoxicity in monkeys after intravenous inoculation. This applicability has also been demonstrated herein by generating a temperature-sensitive CA16 suitable for cold, which is obtained after successive subcultures in cell cultures at increasing incubation temperature.

The present invention also provides a kit for immunizing individuals with the temperature-sensitive enterovirus 71 strain suitable for cold described herein. The kit includes the cold-suitable enterovirus 71 temperature-sensitive strain described herein, a pharmaceutically acceptable carrier, an applicator, and instruction materials for its use. The present invention includes other embodiments of sets known to those skilled in the art. These guidelines can provide any information suitable for guiding the administration of the temperature-sensitive enterovirus 71 strain suitable for cold described herein.

Unless otherwise specified, the practice of the present invention adopts the conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture, and transgenic biology. Within the technical skills. 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 Press, Cold Spring Harbor, New York); Nucleic Acid Hybridization (BDHames & SJHiggins, eds. 1984); Transcription And Translation (BDHames & SJHiggins, eds. 1984); Culture Of Animal Cells (RIFreshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); Methods In Enzymology (Academic Press, Inc., NY); Methods In Enzymology , Volumes 154 and 155 (Wu et al. eds); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, Edited, Academic Press, London, 1987); Handbook Of Experimental Immunology , Volume I-IV (eds by DMWeir and CC Blackwell, 1986); Riott, Essential Immunology , 6th 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 Interf erence: Technology and Application , CRC, 2004.

Instance

The present invention is described with reference to the following examples, which are provided in an illustrative manner and are not intended to limit the present invention in any way. Use standard techniques well known in the art or techniques specifically described below.

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

Cells, viruses and cold adaptation process : Vero cells (ATCC CCL-81) will be maintained by conventional subculture in DMEM supplemented with 10% Fetal Calf Serum (FCS). It is used for virus culture, attenuation, titration and evaluation of temperature-sensitive phenotype. According to the previously described technique, the three EV71 isolates belonging to genotypes C1, B3 and B4 were isolated from the individual oral secretions, stool and brainstem specimens of patients with hand, foot and mouth disease (HFMD) in Vero cells. The spots were purified once (Dougherty, 1964). After plaque purification, virus stocks indicated as respective parent wild-type virus strains were prepared after two subcultures in Vero cells at 37°C. Store all virus stocks and the virus strains of their respective generations in a freezer at minus 80°C.

The freshly confluent young monolayer Vero cell line in DMEM containing 1% FCS was used to adapt the virus strain to replicate at a continuous lower incubation temperature starting from the initial low incubation temperature of 34°C. Once the virus has achieved a complete cytopathic effect (CPE), the clear culture supernatant containing the virus is delivered to each successively fresh new culture flask of Vero cells. At the beginning of each successive change to a lower incubation temperature, the virus-containing culture supernatant was delivered with a higher infection rate (MOI) of 20. After noticing that the virus can cause complete CPE in Vero cells within 3 days after inoculation, inoculate a new culture flask containing freshly confluent single-layer Vero cells with virus of the same MOI for at least three subcultures, and then reduce to 5 To the lower MOI of 10. Once it is noted that the virus can cause complete CPE within 3 days after inoculation with a lower MOI of 5 to 10, after at least three subcultures, the attenuation process then proceeds to the next continuous lower incubation temperature. In this example, the lower incubation temperatures used to develop the temperature-sensitive enterovirus 71 strain suitable for cold are 34°C, 32°C, 30°C, 29°C, and 28°C.

Virus titration : According to the method described in the Polio Laboratory Manual 2004 of the World Health Organization, the virus titration concentration was determined by microtitration analysis in Vero cells and the virus titration concentration was calculated as per the Reed and Muench method (1938). 50% cell culture infection dose (CCID ₅₀ ) per milliliter. In short, after treatment with an equal amount of chloroform to disperse virus aggregates, a 10-fold serial dilution of the clear virus supernatant was prepared in DMEM containing 1% FCS. Vero cell monolayers of 96 well flat bottom tissue culture plates ⁽¹⁰⁴ cells per well) were serially diluted by lines 100μl of each virus stock material and inoculated at 5% CO ₂ environment in each of the respective incubation temperature Incubate for 5 days, and then observe the presence of CPE.

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 (replication kinetics) the virus strain takes to cause complete CPE in infected cells and the second method assesses the titer concentration of the virus strain in cells grown at each specific test temperature. In short, 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 with 1% FCS). Subsequently, the culture medium in each flask was equilibrated to the specific temperature to be tested by placing it in each incubator for 1 hour, and then inoculated with the virus strain at a dose of 10 infection rate (MOI). If no CPE was noticed at the end of the 10-day culture, the supernatant was transferred to a flask of a new monolayer of Vero cells and similarly incubated for another 10 days. If CPE is not noticed after the second generation, it is considered as virus-free. In the second method, a Vero cell suspension with a density of 10 ⁴ cells per 100 μl was inoculated into each well of three 96-well cell culture plates and incubated at 37°C under 5% CO ₂ . After incubating for 10 hours, each cell culture plate was equilibrated to the specific temperature to be tested by placing it in each incubator for 1 hour. Subsequently, the cells in each well were inoculated with 100 μl of 10-fold serial dilution of the virus strain, and then transferred to an incubator at each temperature and incubated for 5 days, and then the presence of CPE was observed.

Genetic stability and temperature sensitivity analysis : In order to evaluate the genetic stability of virus strains cultured in a specific culture environment and cell type, the virus strains were subcultured 20 times in cell culture under 5 MOI of virus inoculum. The incubation was carried out at an incubation temperature of 28°C. At the end of the 20 subcultures, the temperature-sensitive phenotypic characteristics of the virus strains were evaluated by culturing at the incubation temperature of 28°C, 37°C, and 39.5°C as described above. Then sequence and analyze the complete nucleotide sequence of each genome relative to the complete genome of each parent wild-type virus.

Perform the inversion of temperature sensitivity analysis on a stable temperature-sensitive virus strain suitable for cold. The selected virus strains were cultured in a single layer of Vero cells at 37°C under 5% CO ₂ for 5 times. In each passage, a virus inoculum of 10 MOI was used. The growth characteristics of the virus strain obtained in each subculture in Vero cells were evaluated at 28°C, 37°C, and 39.5°C incubation temperature by a similar method as described above for temperature sensitivity analysis. Sequence and analyze the entire genome of the virus strains of each successive generation at a culture temperature of 37°C.

RNA extraction, RT-PCR and sequencing : Viral RNA Extraction Kit (Qiagen, Germany) was used to extract viral genome RNA from the culture medium of infected cells under complete CPE. First-strand synthesis was performed using Superscript II RNA polymerase (Invitrogen, USA) with EV71-specific primers, and the subsequent PCR was performed using 18 degenerate primer pairs using GoTaq Green PCR mix (Promega, USA). The generated fragments were sequenced using BigDye Terminator sequencing kit (Applied Biosystems, USA). Carry out 5'RACE to use standard T4 DNA ligase (Fermentas, USA) to 5'-cordycepin (cordycepin) capped adaptor DT88 (5'-GAA GAG AAG GTG GAA ATG GCG TTT TTG G- Cordyceps militaris-3'; SEQ ID NO: 1) was joined to the 5'end of EV71 cDNA to determine the 5'-UTR virus sequence, and then a primer complementary to DT88 (5'-CCA AAA CGC CAT TTC CAC CTT was used) CTC TTC3'; SEQ ID NO: 2) and EV71 specific primers (5'-ATT CAG GGG CCG GAG GAC TAC-3'; SEQ ID NO: 3) were subjected to standard PCR. 3'-RACE was also performed to determine the 3'-UTR virus sequence using oligo-dT primers (Li et al., 2005).

Molecular cloning and plastid purification : The fragments with fuzzy sequences were cloned into pZero-2 plastids (Invitrogen, USA) and transformed into TOP10 E. coli cells (Invitrogen, USA). A commercially available Plasmid Miniprep Kit (Qiagen, Germany) was used to extract plastids containing the cloned EV71 fragment from at least 10 colonies from each transformant, and then the sequence of the cloned fragment was determined.

Use European Molecular Biology Open Software Suite (EMBOSS; http://mobyle.pasteur.fr/cgi-bin/portal.py?#forms::merger) (Rice et al. 2000) to merge the obtained fragment sequences. Use the program BioEdit Sequence Alignment Editor version 7.0.9.0 (Hall, 1997) to align the combined sequence with the reference sequence of the EV71 virus strain 3799-SIN-98 (GenBank accession number DQ341354.1).

Monkey research : The monkey research on the safety and immunogenicity of the selected EV71 temperature-sensitive strain (EV71: TLLβP20) suitable for cold was contracted to a researcher based in Animal Facility of DUKE-NUS, Singapore. 7 Macaca fascicularis (3 females (2202F, 2207F, 2891F) and 4 males (0791M, 2247M, 2889M, 2890M) without Mycobacterium tuberculosis and monkey immunodeficiency virus ( Macaca fascicularis ), with The average body weight of 3.23Kg (range 2.44 to 4.11, SD=0.7) is used to study the safety and immunogenicity of stable temperature-sensitive EV71: TLLβP20 suitable for cold. All 7 monkeys were pre-screened for the absence of binding to EV71 (indirect immunofluorescence method) 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. Virus inoculation and observation, animal care and autopsy were carried out in accordance with the guidelines of the committee.

1 ml of 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 under 10 ⁷ CCID ₅₀ , and the other 3 monkeys (2890M, 2202F and 2247M) were given 10 ⁸ CCID ₅₀ and the seventh A monkey served as a negative control. Observe the monkey's clinical diseases, especially neurological manifestations, twice a day, and record its body temperature with 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. On the 4th day after inoculation (PI), two monkeys (one from each inoculum with a different virus dose) were killed under deep anesthesia. During autopsy, various parts of central nervous system (CNS), non-neural tissue and blood are 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 during autopsy. After collecting blood samples for the evaluation of anti-EV71 antibodies, the remaining two monkeys were given respective equivalent booster doses of EV71: TLLβP20 on the 14th day after vaccination. On the 16th day after receiving the booster dose, they were killed without pain and the CNS tissue was collected for histopathological study during autopsy.

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

Antigen detection and virus isolation from monkeys : Collect blood samples in non-additive tubes 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, an aliquot of the PBMC suspension was inoculated onto the wells of a Teflon-coated slide to use a commercial detection monoclonal antibody (Cat. No. 3360, Light Diagnostics, USA) detects EV71 antigen by indirect immunofluorescence analysis. Virus isolation was performed by seeding the PBMC suspension into the wells of a 24-well cell culture plate containing a single layer of Vero cells. Virus isolation was performed on each serum sample by inoculating 50 μl and 100 μl of serum into each well of a 24-well culture plate containing a single layer of Vero cells. The monkey tissues were washed slightly with sterile PBS exchanged twice and homogenized by grinding with a mortar and pestle in a Class II biological safety cabinet. The tissue homogenate (10%, w/v) prepared in DMEM was clarified by centrifugation at 1000 g for 10 minutes. The clarified supernatant was filtered through a 0.22 micron syringe filter and the virus was separated by inoculating 100 μl and 200 μl of the filtrate. A 10% stool suspension was prepared in PBS and clarified by centrifugation at 1000 g for 10 minutes. After filtering through a 0.22 micron syringe filter, virus isolation was performed by inoculating 100 μl and 200 μl stool filtrate. All virus isolation work was performed on monkey samples in duplicate, with one group of inoculated cell cultures grown at 28°C and another group at 37°C.

Molecular detection and whole genome sequencing : Commercial viral RNA extraction and purification kit (Qiagen, Germany) is used to extract viral genome RNA from serum, PBMC and clarified tissue homogenate. A commercial one-step RT-PCR kit (Qiagen, Germany) and common oligonucleotide primer pair (sense: 5'-CACCCTTGTGATACCATGGATCAG-3' (SEQ) to amplify the proximal third of the VP1 gene of all EV71 genotypes 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-organization. 18 pairs of sequence-specific primers based on the whole genome sequence EV71:TLLβ were used to amplify and sequence the EV71 whole genome present in the sera of two monkeys obtained on the 4th and 8th day after immunization. Any PCR amplified fragments that failed to give good sequence reads by direct sequencing were cloned into pZero-2 (Invitrogen, USA) and transformed into TOP10 E. coli. At least ten colonies were selected from each transformant and sequenced on purified plastids carrying inserts.

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

It was slightly modified according to the method described in the Polio Laboratory Manual 2004 of the World Health Organization. Vero cells were used to determine the monkey neutralizing antibody titer by microneutralization analysis. The concentration of each genotype of EV71 used for neutralization is 100 CCID ₅₀ per 100 μl. Use 96-well flat-bottomed culture plates for virus neutralization analysis. Serial 2-fold dilutions of each serum sample were prepared in duplicate in an amount of 100 μl DMEM (1% FCS) starting at a dilution of 1:10. An equal amount (100 μl) of 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 incubation, 100 μl of the 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 a 5% CO ₂ environment. The disc is read daily for the presence of CPE affecting the Vero cells in each well for up to 8 days. The titrated concentration of neutralizing antibody in each serum sample was determined by the hole that did not show the highest dilution of CPE.

Example 2 The results of the phenotype and genotype characteristics of the temperature-sensitive strain of Enterovirus 71 suitable for cold

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

The three original parental EV71 viruses used to obtain virus strains suitable for cold caused complete CPE in Vero cells within 3 days after inoculation with a virus inoculum of 10 MOI and an incubation temperature of 37.5°C. In 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 derived from the first two passages in Vero cells cultured at 37.5°C did not cause in Vero cells at a virus inoculum of 10 MOI and an incubation temperature of 28°C. CPE. Nor was CPE noticed after blind subculture at the end of 10 days incubation at 28°C. The presence of negatively stained suspension cells in the culture supernatant at the end of the 10-day culture by indirect immunofluorescence using a commercial detection monoclonal antibody against EV71 further supports the absence of virus replication in the inoculated Vero cells.

5 MOI之病毒接種體及28℃之培育溫度下之接種的3日內於維羅細胞中引起完全CPE。病毒株進一步在28℃之培育溫度下繼代直至第100次繼代。在第100次繼代，將來源於自患者之口液、腦幹組織及大便標本分離之原始EV71的病毒株分別指示為EV71：TLL、EV71：TLLα及EV71：TLLβ。在溫度敏感性分析中，所有三種適於冷之病毒株使用10 MOI之病毒接種體在28℃之培育溫度下在2日內於維羅細胞中引起完全CPE，但在37℃之培育溫度下需要4日以達到完全CPE。在39.5℃之培育溫度下，所有三種病毒株在培養10日之後均未注意到CPE。然而，EV71：TLL在盲目繼代至培育於39.5℃下之維羅細胞之新燒瓶中之後於培養之第10日給出2+ CPE。即使在另外兩次盲目繼代至培育於39.5℃下之維羅細胞之新燒瓶中之後，EV71：TLLα及EV71：TLLβ仍未注意到CPE。在各10日培養結束時自藉由EV71：TLLα或EV71：TLLβ接種之培養物上澄液收集之懸浮細胞(包括來源於盲目繼代之彼等細胞)藉由EV71偵測單株抗體未給出陽性免疫螢光染色。自藉由EV71：TLL接種之培養物上澄液收集之約1%懸浮細胞給出陽性染色，然而，在10日培養之後未注意到CPE。 After more than 90 subcultures under successively reduced cultivation temperatures, all three subcultured EV71 virus strains were able to

5 The virus inoculum of MOI and the inoculation at the incubation temperature of 28 ℃ caused complete CPE in the Vero cells within 3 days. The virus strain was further subcultured at a cultivation temperature of 28°C until the 100th subculture. At the 100th generation, the virus strains derived from the original EV71 isolated from the patient’s oral fluid, brainstem tissue and stool specimens were designated as EV71: TLL, EV71: TLLα, and EV71: TLLβ, respectively. In the temperature sensitivity analysis, all three virus strains suitable for cold use 10 MOI virus inoculum to cause complete CPE in Vero cells within 2 days at an incubation temperature of 28°C, but it is required at an incubation temperature of 37°C 4 days to achieve full CPE. At an incubation temperature of 39.5°C, all three virus strains did not notice CPE after 10 days of cultivation. However, EV71:TLL gave 2+ CPE on the 10th day of culture after blindly subcultured to a new flask of Vero cells grown at 39.5°C. Even after two other blind passages to new flasks of Vero cells grown at 39.5°C, EV71:TLLα and EV71:TLLβ still did not notice CPE. At the end of each 10-day culture, the suspension cells (including those cells derived from blind succession) collected from the culture supernatants inoculated with EV71:TLLα or EV71:TLLβ were detected by EV71. Positive immunofluorescence staining. Approximately 1% of the suspended cells collected from the culture supernatant inoculated with EV71:TLL gave positive staining, however, no CPE was noticed after 10 days of culture.

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

By subcultured EV71:TLLα and EV71:TLLβ in Vero cells at an incubation temperature of 28°C and a virus inoculum of 10 MOI for another 20 generations, the stability of the two virus strains to adapt to cold was evaluated. After another 20 generations, EV71:TLLα caused complete CPE in cells grown at 28°C within 2 days after seeding, but failed to cause CPE in cells grown at 37°C, even after 2 blind passages. Its ability to maintain a virus titer of 1×10 ⁸ CCID ₅₀ /ml at an incubation temperature of 28°C. EV71: TLLβ maintains the same cold-suitable phenotype in terms of growth kinetics and virus titer concentration at 28°C and 37°C incubation temperatures after another 20 generations. The stability of the cold adaptation of EV71:TLLβ was further evaluated by subcultured under the same culture conditions for another 20 generations (another 40 generations from the 100th generation) and it was found that it maintained a temperature-sensitive phenotype similar to that suitable for cold.

The evaluation of the reversal of the temperature-sensitive phenotype suitable for cold was performed by 6 consecutive passages of the virus in the Vero cells cultivated at 37°C under each complete CPE on EV71:TLLβP20. The growth characteristics of the virus derived from each individual culture cultivated at 37°C in terms of growth kinetics and virus titer concentration at incubation temperatures of 28°C, 37°C, and 39.5°C are shown in Table 1. The virus cannot produce viable infectious particles in Vero cells at an incubation temperature of 39.5°C (lack of positive immunofluorescence staining cells) after 3 consecutive subcultures in cells incubated at 37°C and cannot be used in the sixth time. Repeated subcultures caused complete CPE in cell culture at an incubation temperature of 39.5°C.

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

P1: Succession 1

IFA: Indirect immunofluorescence analysis

The whole genomes of EV71: TLLα and EV71: TLLβ, its respective virus strains after another 20 generations (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. EV71: TLLα, EV71: The variation of the nucleotide number of each fragment of the gene between TLLαP20 and the original parent wild-type is shown in Table 2. The changes in the number of nucleotides between the original parent wild-type, EV71: TLLβ, EV71: TLLβP20 and EV71: TLLβP40 (another 40 generations) are shown in Table 3. The whole genome of the virus strain derived from the temperature-sensitive reversal study of 6 consecutive generations at the incubation temperature of 37°C was also sequenced and the nucleotide number changes relative to EV71:TLLβP20 are shown in Table 4.

Example 3 The result of monkey study of temperature-sensitive strain of human enterovirus 71 (EV71: TLLβP20) suitable for cold

Clinical findings : The general observation of the clinical state of the monkeys in the study and the measurement of body temperature were carried out twice a day in the morning and evening. Throughout the process, it was noted that all monkeys were active and eating normally. None of the monkeys had any minor local nerve defects, such as limb weakness, tremor, or abnormal movement. Except for one monkey (2247M) given an intravenous dose of 1×10 ⁸ CCID ₅₀ /ml, none of the monkeys had a low-grade fever peak (39.3°C) on the 3rd day after vaccination. None of the monkeys had weight loss when they were rescaled when they were killed under deep anesthesia.

Necropsy and histological findings : All monkeys on autopsy did not notice overall postmortem lesions. No abnormal histological findings were seen in all monkey tissues collected for histopathological examination.

Virology research : Virus isolation of monkey serum, PBMC, stool samples and all autopsy tissues was performed using Vero cells at culture temperatures of 28°C and 37°C. Despite blind subculture after 10 days of culture, no virus was isolated from any of the monkey samples. Another blind subculture in Vero cells was performed on serum, PBMC samples and their tissue homogenates that tested positive for EV71 by RT-PCR. Although the virus could not be isolated, it was collected from two monkeys 2891F (EV71 received 1×10 ⁷ CCID ₅₀ : TLLβP20) by indirect immunofluorescence analysis using commercial monoclonal antibodies (Figure 1a) on the 4th day after vaccination. And 2890M (EV71 receiving 1×10 ⁸ CCID ₅₀ : TLLβP20) of several PBMCs of heparinized blood detected EV71 antigen. No viral antigen was detected in the PBMC collected from the heparinized blood of the monkey collected on the 8th day after vaccination.

The EV71-specific genome sequence was detected in serum samples of all monkeys collected on the 4th and 8th day after vaccination by RT-PCR. Among non-neuronal tissues, the EV71 genome sequence was only detected in the spleen homogenates of 2 monkeys (2202F and 2891F) (Figure 1b). The EV71 genome sequence was not detected in any of the neuronal tissue homogenates. For two monkeys (2889M and 2890M) serum samples (collected on the 4th and 8th day after vaccination) EV71: The genome of TLLβP20 is extracted and fully sequenced. Compared with the genome of EV71: TLLβP20, the number of nucleotides (NT) and corresponding amino acid (AA) mutations or reversals in each of the genome fragments of the virus strain present in the serum is shown in the table 5 in.

Monkey humoral immune response : Using internally prepared infected Vero cells as antigens, the presence of binding antibodies (IgM and IgG) in the serum of monkeys given intravenous EV71:TLLβP20 was analyzed by indirect immunofluorescence analysis. Anti-EV71 IgM present in the blood of two remaining monkeys (2889M and 2890M) collected on the 14th day after vaccination (the equivalent intravenous booster dose was previously given) and the 30th day after vaccination (the 16th day after booster) The titer of IgG and IgG are shown in Table 6.

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

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

Materials and methods : Vero cells (ATCC CCL-81) maintained by conventional subculture in Dulbecco modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) were used for virus culture , Attenuation, titration and evaluation of the temperature sensitivity of Coxsackie virus A16. The parental wild-type Coxsackie virus A16 (CA16) used in the attenuation process is derived from oral secretions of patients with hand, foot and mouth disease (HFMD). The virus was plaque purified once according to the previously described technique. After plaque purification, virus stocks indicated as respective parent wild-type virus strains were prepared after two subcultures in Vero cells at 37°C. Store all virus stocks and the virus strains of their respective generations in a freezer at minus 80°C.

The young, freshly confluent monolayer Vero cell line in DMEM containing 1% FCS was used to adapt the original wild-type CA16 to replicate at a continuous lower incubation temperature starting from the initial lower incubation temperature of 34°C. Under the complete cytopathic effect (CPE), the virus was subcultured to each successive new culture flask of Vero cells. The virus was subcultured at a higher rate of infection (MOI) of 20 when it was changed to a continuous lower incubation temperature and after noticing that it could cause complete CPE within 3 days after inoculation, it was reduced to 5 to 10 after at least three subcultures. The lower MOI. After noting that the virus can cause complete CPE within 3 days after inoculation with an MOI of 5 to 10, proceed to the next continuous lower incubation temperature after at least three more subcultures. The culture supernatant of each successive subculture is stored in a refrigerator at -80°C for future analysis.

Virus titration : 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 as per the Reed & Muench method (1938). 50% cell culture infection dose (CCID ₅₀ ) per milliliter. In short, after treatment with an equal amount of chloroform to disperse virus aggregates, a 10-fold serial dilution of the clear virus supernatant was prepared in DMEM containing 1% FCS. Vero cell monolayers of 96 well flat bottom tissue culture plates ⁽¹⁰⁴ cells per well) were serially diluted by lines 100μl of each virus stock material and inoculated at 5% CO ₂ environment in each of the respective incubation temperature Incubate for 5 days, and then observe the presence of CPE.

Temperature sensitivity analysis : Two methods were used to evaluate the growth characteristics of cold-suitable 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 (replication kinetics) the virus strain takes to cause complete CPE in infected cells and the second method assesses the titer concentration of the virus strain in cells grown at each specific test temperature. In short, 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 with 1% FCS). Subsequently, the culture medium in each flask was equilibrated to the specific temperature to be tested by placing it in each incubator for 1 hour, and then inoculated with the virus strain at a dose of 10 infection rate (MOI). If CPE is not noticed at the end of the 10-day culture, the supernatant is transferred to a flask of a new monolayer of Vero cells and incubated similarly for another 10 days. If CPE is not noticed after the second generation, it is considered as virus-free. In the second method, a Vero cell suspension with a density of 10 ⁴ cells per 100 μl was inoculated into each well of three 96-well cell culture plates and incubated at 37°C under 5% CO ₂ . After incubating for 10 hours, each cell culture plate was equilibrated to the specific temperature to be tested by placing it in each incubator for 1 hour. Subsequently, the cells in each well were inoculated with 100 μl of 10-fold serial dilution of the virus strain, and then transferred to an incubator at each temperature and incubated for 5 days, and then the presence of CPE was observed.

Example 5 The result of virus characteristics in cells of cold human Coxsackie virus A16 temperature-sensitive strain

5 MOI之病毒接種體及28℃之培育溫度下之接種的3日內於維羅細胞中引起完全CPE，但在37℃之培育溫度下達到完全CPE需要4日。在39.5℃之培育溫度下，未注意到CPE。 Under the 100th successive generation of the continuously lowered incubation temperature, the cold-suitable CA16 virus strain can be

5 The virus inoculum of MOI and the inoculation at the incubation temperature of 28°C cause complete CPE in the Vero cells within 3 days, but it takes 4 days to reach the complete CPE at the incubation temperature of 37°C. At an incubation temperature of 39.5°C, no CPE was noticed.

The incubation temperature of 28°C and 37°C is used to analyze the virus replication titration concentration of the temperature sensitive strain of CA16 suitable for cold. When the titration plate is incubated at 28°C and 37°C, respectively, the titration concentration of the temperature-sensitive CA16 strain suitable for cold is 1×10 ⁸ CCID ₅₀ /ml and 1×10 ⁷ CCID ₅₀ /ml.

Unless otherwise stated herein or obviously contradictory in the context, the terms "a/an" and "the" are used in the context of describing the present invention (especially in the context of the scope of the following patent applications) And similar indicators should be interpreted as covering the singular and plural. Unless otherwise indicated, the terms "include", "have", "include" and "contain" are all interpreted It is an open-ended term (meaning "including (but not limited to)"). Unless otherwise indicated herein, the description of the value range herein is only intended to serve as a shorthand method for individually referring to each independent value belonging to the range, and each independent value is incorporated into this specification as if individually described herein. For example, if the range 10-15 is revealed, 11, 12, 13, and 14 are also revealed. Unless otherwise indicated herein or clearly contradicted by context, all methods described herein can be performed in any suitable order. Unless otherwise claimed, the use of any and all examples or illustrative language (eg, "such as") provided herein is only intended to better illustrate the invention and not to limit the scope of the invention. The language in this specification should not be regarded as indicating that any unclaimed element is essential for practicing the present invention.

It should be understood that the methods and compositions of the present invention can be combined in the form of various embodiments, and only a few of these embodiments are disclosed herein. The embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the present invention. After reading the previous description, the changes in their embodiments may become obvious to those who are generally familiar with the art. The present inventor expects those familiar with the art to adopt these changes as appropriate, and the present inventor intends to practice the present invention in a manner different from that specifically described herein. Therefore, if permitted by applicable laws, the present invention includes all modifications and equivalents of the subject matter stated in the scope of the patent application attached to this document. In addition, unless otherwise indicated herein or otherwise clearly conflicting with the context, the present invention encompasses any combination of the above-mentioned elements in all possible variations thereof.

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【Biological Material Deposit】 Domestic deposit information [please note in order of deposit institution, date and number]

1. Food Industry Development Research Institute; May 08, 2013; BCRC970063

2. Food Industry Development Research Institute; May 08, 2013; BCRC970064

Foreign hosting information [please note in the order of hosting country, institution, date and number]

1. United States; American Type Culture Collection (ATCC); October 25, 2012; PTA-13284

2. United States; American Type Culture Collection (ATCC); October 25, 2012; PTA-13285

<110> Singapore Business Temasek Institute of Life Sciences Corporation

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Claims (19)

A temperature-sensitive strain of Enterovirus 71 suitable for cold, which comprises the nucleotide sequence shown in SEQ ID NO: 6 or 7. The temperature-sensitive strain of Enterovirus 71 suitable for cold as in Claim 1, which is used to elicit a protective immune response against Enterovirus 71 in individuals. For example, the temperature sensitive strain of Enterovirus 71 suitable for cold in Claim 1, which is used to prevent individuals from suffering from diseases related to Enterovirus 71. For example, the temperature-sensitive strain of Enterovirus 71 suitable for cold in Claim 1, which is used to delay the onset of the disease related to Enterovirus 71 or slow down the rate of the disease in individuals infected with Enterovirus 71. For example, the cold-suitable enterovirus 71 temperature-sensitive strain of claim 1, which is used for vaccine development. A composition comprising the enterovirus 71 temperature-sensitive strain suitable for cold as claimed in claim 1. The composition of claim 6, which further comprises a pharmaceutically acceptable carrier. The composition of claim 6, which further comprises an adjuvant. The composition according to any one of claims 6 to 8, which is a vaccine. The composition according to any one of claims 6 to 8, which is used to elicit a protective immune response against enterovirus 71 in an individual. The composition according to any one of claims 6 to 8, which is used to prevent an individual from suffering from diseases related to Enterovirus 71. The composition according to any one of claims 6 to 8, which is used to delay the onset of a disease related to the enterovirus 71 or to slow down the rate of the disease in an individual infected with the enterovirus 71. A use of a temperature-sensitive strain of Enterovirus 71 suitable for cold as claimed in claim 1 or a composition as claimed in any one of claims 6 to 9 for the production of an individual against enterovirus 71 of the protective immune response agent. A use of the temperature-sensitive strain of Enterovirus 71 suitable for cold as claimed in claim 1 or the composition as claimed in any one of claims 6 to 9 for the manufacture of an agent for preventing an individual from suffering from diseases related to enterovirus 71. A use of a temperature-sensitive strain of Enterovirus 71 suitable for cold as claimed in claim 1 or a composition as claimed in any one of claims 6 to 9 for the production of delayed and enterovirus 71 in individuals infected with enterovirus 71 The onset of related diseases or agents that slow down the rate of the disease. Such as the use of any one of claims 13 to 15, wherein the individual is a human individual. A kit for individual immunization by a temperature sensitive strain of Enterovirus 71 suitable for cold, comprising the temperature sensitive strain of Enterovirus 71 suitable for cold as claimed in claim 1 or a combination of any one of claims 6 to 9 Materials, pharmaceutically acceptable carriers and guidance materials for their use. Such as the set of claim 17, which further comprises an applicator. A nucleic acid composed of the nucleotide sequence shown in SEQ ID NO: 6 or SEQ ID NO: 7, which is used for vaccine development.

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