CN106994182A - Prevent and treat vaccine of Coxsackie virus associated diseases and preparation method and application - Google Patents
Prevent and treat vaccine of Coxsackie virus associated diseases and preparation method and application Download PDFInfo
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- CN106994182A CN106994182A CN201710182230.8A CN201710182230A CN106994182A CN 106994182 A CN106994182 A CN 106994182A CN 201710182230 A CN201710182230 A CN 201710182230A CN 106994182 A CN106994182 A CN 106994182A
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1009—Picornaviridae, e.g. hepatitis A virus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32311—Enterovirus
- C12N2770/32321—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32311—Enterovirus
- C12N2770/32334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Abstract
The invention discloses vaccine of prevention and treatment Coxsackie virus associated diseases and preparation method and application.The preparation method of prevention and treatment Coxsackie virus associated diseases vaccine disclosed by the invention, including:The type strain of Coxsackie virus A 6 of inactivation is prepared into the vaccine as active component;The type strain of Coxsackie virus A 6 be China Committee for Culture Collection of Microorganisms's common micro-organisms center deposit number be CGMCC No.13393 strain.The present invention has the effect for treating and preventing CVA6 associated diseases using the CVA6 strains WF057R vaccines prepared and CVA6 antiserums, and CVA6 strains WF057R female antibody that passes also has protective effect to Neonatal Mouse.
Description
Technical field
The present invention relates to vaccine and its preparation side in biological technical field, preventing and treating Coxsackie virus associated diseases
Method and application.
Background technology
Coxsackie virus (Coxsackievirus, CV) is single strand plus RNA virus, belongs to Picornaviridae, enteron aisle disease
Poison category.Coxsackie virus particle is icosahedron cubic symmetry, and spherical in shape, diameter about 23~33nm, nucleocapsid is exposed, no coating
And projection;Coxsackie virus genome is encoded before a longer polyprotein only comprising an open reading frame (ORF)
Body.The ORF is divided into the Structural protein VP1~VP4 in 3 areas of P1, P2 and P3, wherein P1 areas coding virus, the viral clothing of composition
Shell.P2 and P3 areas encode non-structural protein.
Coxsackie virus is the infant within the main pathogens for causing hand foot and mouth disease (HFMD), especially 3 years old,
It can result in its serious neurological complication.Causing HFMD enterovirus (EVs) mainly includes four types:EV-A、
EV-B, EV-C and EV-D.The type of Coxsackie virus A 6 (CVA6) belongs to enterovirus A groups, totally 16 hypotypes, wherein hypotype A groups 11
Individual and B groups 5.According to the difference of genotype, CVA6 is divided into five branches of A~E, and E genotype is divided into E1 again and raq gene is sub-
Type.From CVA6 the first explosions in 2008 since Finland, E2 hypotypes turn into the main genotypes of world-wide prevalence.Clinical number
According to showing, compared to classical HFMD cases, more serious respiratory system and nerve is frequently can lead to after E2 subtype Cs VA6 infection people
Systemic disease.Importantly, virus infection can cause serious herpangina, or even cause neonatal death.
Meanwhile, the subtype virus are easy to other type enterovirus mixed infections in epidemic season, treatment and diagnosis band to disease
Come many difficult.
Before 2010, HFMD is mainly caused by EV71 and CA16 two types enteroviruses, the report on CVA6
It is considerably less.In recent years, CVA6 is popular in worldwide, especially pan-Pacific area, including TaiWan, China, Singapore,
Japan and Thailand etc., many area CVA6, which have replaced EV71 and CA16 to turn into, causes HFMD Major Epidemic strain.From 2011 to
2015, the popular enterovirus in other countries and regions such as Europe and North America was also CVA6.At present, in many of China's Mainland
Explosion type prevalence is presented in city CVA6, such as the ground such as Shenzhen, Shanghai and Qingdao, its morbidity and mortality are raised year by year.So,
E2 types CVA6 has been increasingly becoming in worldwide popular and can cause the HFMD pathogen of severe complication.
Clinical manifestation caused by other type enteroviruses (non-EV71 and non-CA16) is slight, pathogenic weaker, and
Concern of the people to CVA6 is not caused.Nearest report shows that popular E2 types CVA6 infection in the recent period can cause serious maincenter
Nervous system (CNS) acute complicationses, such as aseptic meningitis, BBE and AFP Cases.At present on hand
The pathogenesis of sufficient stomatosis is not clear, and particularly CVA6 pathogenesis is not yet reported, screens anti-CVA6 medicines and vaccine lacks
The animal infection modal of weary stabilization, the exploitation of anti-CVA6 medicines and vaccine is also restricted.
The content of the invention
The technical problems to be solved by the invention are how to treat and/or prevent the type associated diseases of Coxsackie virus A 6.
In order to solve the above technical problems, being activated present invention firstly provides one kind prevention and/or treatment Coxsackie virus
The preparation method of thing disease vaccine.
The preparation method of animal disease vaccine caused by prevention and/or treatment Coxsackie virus provided by the present invention, including:
The type strain of Coxsackie virus A 6 is prepared into the vaccine as active component;
The type strain of Coxsackie virus A 6 can be in China Committee for Culture Collection of Microorganisms's common micro-organisms
The deposit number of the heart is CGMCC No.13393 strain, the entitled WF057R of the strain.
In the above method, the type strain of Coxsackie virus A 6 can be the type strain of Coxsackie virus A 6 of inactivation.
The above method specifically may include the type strain of Coxsackie virus A 6 and immunologic adjuvant being mixed to get the vaccine.
In the above method, the immunologic adjuvant can be complete Freund's adjuvant or incomplete Freund's adjuvant.
In the above method, the vaccine can be the vaccine of Animal diseases caused by preventing and/or treating the type of Coxsackie virus A 6.
In order to solve the above technical problems, present invention also offers the vaccine by described method preparation.
In order to solve the above technical problems, present invention also offers Animal diseases caused by preventing and/or treating Coxsackie virus
Product, the product for it is following 1) or 2):
1) product of the type strain of Coxsackie virus A 6 is contained;
2) antiserum prepared using the type strain of Coxsackie virus A 6.
The product can be only using above-mentioned A1) as active component, can also be by A1) there is the material of identical function with other
It is combined together as active component.
Above-mentioned 1) described product can be medicine or vaccine.
The antiserum can be that the antiserum that animal obtains is immunized using the type strain of Coxsackie virus A 6.The animal
It can be mammal.The mammal concretely mouse (such as ICR mouse).
In order to solve the above technical problems, preparing prevention and/or treatment COxsackie disease present invention also offers methods described
Application in Animal diseases product caused by malicious.
The product can be medicine or vaccine.
In order to solve the above technical problems, preventing and/or controlling present invention also offers the vaccine by described method preparation
Application in Animal diseases caused by treating Coxsackie virus.
In the present invention, the animal can be mammal.The mammal concretely mouse (such as ICR mouse).
In the present invention, Animal diseases caused by the Coxsackie virus can be Animal diseases caused by the type of Coxsackie virus A 6, but
It is not limited to Animal diseases caused by the type of Coxsackie virus A 6.
The present invention has screened one plant of representative CVA6 strain WF057R, the vaccine and CVA6 prepared using the strain
Antiserum has the effect for treating and preventing CVA6 associated diseases, and CVA6 strains WF057R mother passes antibody to Neonatal Mouse
Also there is protective effect.
Biomaterial preservation explanation
The Classification And Nomenclature of biomaterial:The type of Coxsackie virus A 6 (Coxsackievirus)
The strain number of biomaterial:WF057R
Depositary institution's title of biomaterial:China Committee for Culture Collection of Microorganisms's common micro-organisms center
The depositary institution of biomaterial is referred to as:CGMCC
The depositary institution address of biomaterial:Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences microorganism
Research institute, postcode:100101
The preservation date of biomaterial:On December 26th, 2016
The collection of biomaterial is registered on the books numbering:CGMCC No.13393
Brief description of the drawings
Fig. 1 is clinical manifestation, changes of weight and the life that different infectious conditions combine lower inoculation CVA6 strain WF057R suckling mouses
Deposit rate change.A-C is respectively 5 age in days suckling mouse Different vaccine dosages and the combination of different vaccination approach;D-F is various dose through IM ways
Footpath is inoculated with 3,7,9 age in days suckling mouses.
Fig. 2 is the change that 5 age in days suckling mouses are inoculated with different tissues inner virus carrying capacity after CVA6.A-C dosage of inoculation is
105.5TCID50/ only, A is that 5 age in days suckling mouses each tissue virus load after intramuscular injection infection CVA6 viruses changes with infection number of days
Situation, B is that each tissue virus load is with infection number of days situation of change after abdominal cavity infectable infection CVA6 viruses for 5 age in days suckling mouses, and C is
5 age in days suckling mouses are each after infecting CVA6 viruses through intracerebral injection to organize virus load with infection number of days situation of change;D-F Inoculant
Amount is 107TCID50/, D is that virus load is respectively organized after 5 age in days suckling mouses infect CVA6 viruses through intramuscular injection with infection day
Number situation of change, E, which is that 5 age in days suckling mouses are each after abdominal cavity infectable infection CVA6 viruses, organizes virus load with infection number of days change feelings
Condition, F is that virus load is respectively organized after 5 age in days suckling mouses infect CVA6 viruses through intracerebral injection with infection number of days situation of change.Wherein,
The log truth of a matter is 10.
Fig. 3 is that 5 age in days suckling mouses infect 310LD50The expression of inflammatory cytokine becomes in 1~5dpi peripheral bloods after dosage CVA6
Change.CVA6, which is represented, connects malicious group, and NC is represented and do not connect malicious group, and the C of ordinate represents expression quantity.
Fig. 4 is HE dyeing and the IHC results that Neonatal Mouse infects cerebral tissue after CVA6 strains WF057R.A:5dpi HE
Dyeing;B:HE negative controls;C:5dpi IHC dyeing;D:IHC negative controls.A and B amplification 200X, C and D amplifications 400X.
Fig. 5 is HE dyeing and the IHC results that Neonatal Mouse infects heart tissue after CVA6 strains WF057R.A:5dpi HE
Dyeing;B:HE negative controls;C:5dpi IHC dyeing;D:IHC negative controls.A~D amplifies 200X.
Fig. 6 is HE dyeing and the IHC results that Neonatal Mouse infects lung tissue after CVA6 strains WF057R.A:5dpi HE
Dyeing;B:HE negative controls;C:5dpi IHC dyeing;D:IHC negative controls.A~D amplifies 200X.
Fig. 7 is HE dyeing and the IHC results that Neonatal Mouse infects skeletal muscle after CVA6 strains WF057R.A:5dpi HE dyes
Color;B:HE negative controls;C:5dpi IHC dyeing;D:IHC negative controls.A~D amplifies 200X.
Fig. 8 is HE dyeing and the IHC results that Neonatal Mouse infects CVA6 strain WF057R rear intestinals.A:5dpi HE dyes
Color;B:HE negative controls;C:5dpi IHC dyeing;D:IHC negative controls.A~D amplifies 200X.
Fig. 9 is the protective effect of CVA6 antiserums in vivo by passive immunity to suckling mouse.
Figure 10 is that protective effect of the female mice mother's biography antibody to suckling mouse is immunized in CVA6 inactivated whole virus.
Figure 11 is the therapeutic effect to different onset degree suckling mouse in CVA6 antiserums body.Wherein, CVA6 represents CVA6 pairs
According to a group suckling mouse.
Embodiment
The present invention is further described in detail with reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the scope being not intended to be limiting of the invention.Experimental method in following embodiments, unless otherwise specified, be
Conventional method.Material, reagent, instrument used etc., unless otherwise specified, are commercially obtained in following embodiments.
Quantitative test in following examples, is respectively provided with three repetition experiments, results averaged.
(Chen Guoqing, Wang Yao, Xu Xiaoqing, Shao Rong mark enterovirus EV 71s and CVA6 separation to RD cells in following embodiments
Analysis of Influential Factors [J] the Jiangsu preventive medicine of effect, 2015,26 (4):4-5.) it is people's rhabdomyoma RD cells
(Rhabdomyoma cell), is given by the anti-system of Shandong Province disease prevention and control center viral infectious, through Shandong Province
The public can obtain the biomaterial, the life at applicant after viral infectious anti-system in disease prevention and control center is agreed to
Thing material is only attached most importance to used in the related experiment of duplicate invention, can not be used as other purposes.
MEM maintaining liquids in following embodiments are Gibco products.
Suckling mouse, female mice and adult rats in following embodiments are Beijing Vital River Experimental Animals Technology Co., Ltd.
SPF grades of ICR mouse.Wherein, the body weight of 5 age in days suckling mouses is 4 ± 0.5g.
The different places clinical separation strain of Coxsackie virus in following embodiments
WH15066/Shandong/China/2015、DY003R/Shandong/China/2015、
DY005R/Shandong/China/2015 and LW03R/Shandong/China/2015 are Coxsackie virus A 6
Type (CVA6) strain, wherein WH15066/Shandong/China/2015 genome sequence are classified as No. GenBank in NCBI
The sequence that U is obtained is replaced with for the T in KY126092.1 sequence,
DY003R/Shandong/China/2015 genome sequence is classified as is by No. GenBank in NCBI
T in KY126089.1 sequence replaces with the sequence that U is obtained, DY005R/Shandong/China/2015 genome sequence
For be KY126090.1 by No. GenBank in NCBI sequence in T replace with the sequence that U is obtained,
LW03R/Shandong/China/2015 genome sequence is classified as is by No. GenBank in NCBI
T in KY126091.1 sequence replaces with the sequence that U is obtained, and the public can obtain these biomaterials at applicant, these
Biomaterial is only attached most importance to used in the related experiment of duplicate invention, can not be used as other purposes.
The separation and identification of embodiment 1, type (CVA6) strain of Coxsackie virus A 6 WF057R
Inventor was in the isolated type of a Coxsackie virus A 6 from the brothers mouthful infant excrement of Shandong Province one in 2015
(Coxsackievirus) strain, WF057R is named as by the strain.The type of Coxsackie virus A 6 (Coxsackievirus) strain
WF057R is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (referred to as on December 26th, 2016
CGMCC, address is:Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), deposit number is CGMCC No.13393.
Embodiment 2, utilize COxsackie A6 virus stains WF057R prepare CVA6 infected animal models
First, WF057R culture
RD cells are inoculated in into culture medium 1, and (culture medium 1 is that hyclone, penicillin and strepto- are added into MEM maintaining liquids
The mass percentage concentration of the liquid that element is obtained, wherein hyclone is 10%, and the mass percentage concentration of penicillin is 1%, strepto-
The mass percentage concentration of element in 1%), in temperature be 37 DEG C, 5% concentration C O2Cell culture incubator in cultivate, obtain RD cells
Nutrient solution.The COxsackie A6 virus stains WF057R of embodiment 1 is inoculated on the RD cells in RD cell culture fluids, to cell
When there is induced cytopathic effect (Cytopathic effect, CPE) area more than 80%, cell culture fluid is collected, is obtained
CVA6 strain WF057R virus liquids, carry out Viral Quantification by limiting dilution assay in 96 orifice plates, determine virus TCID50After be sub-packed in
In centrifuge tube, -80 DEG C of refrigerators are stored in.
2nd, the preparation of CVA6 infected animal models
Pass through intramuscular injection (Intramuscular injection, IM), intraperitoneal injection (Intraperitoneal
Injection, IP) and cranial cavity injection (Intracerebral injection, IC) three kinds of different routes of infection respectively with
104、105.5、107TCID50/ three kinds of different infective doses are combined, and exploration sets up 3 age in days suckling mouse CVA6 infection models most
Good route of inoculation and infective dose.It is determined that after optimal route of inoculation, then through this approach respectively with 105.5With 107TCID50/ only two
Plant infective dose combination and explore pathogenic situations of the CVA6 to big age in days (5,7,9 age in days) suckling mouse, it is final to determine to set up CVA6 models
Optimal age in days, route of inoculation and infective dose, infectious condition combination be shown in Table 1.
Using document (Mao Q, Wang Y, Gao R, Shao J, Yao X, Lang S, Wang C, Mao P, Liang Z,
Wang J.A neonatal mouse model of coxsackievirus A16for vaccine evaluation.J
Virol.2012;86(22):Method in 11967-76.), the half for calculating 5 age in days suckling mouses according to Reed&Muench formula is caused
Dead amount LD50(Reed LJ,Muench H.A simple method of estimating 50percent end-
points.Am.J.Hyg.1938,27:493-497.).1 day (1day post after suckling mouse infection CVA6 strains WF057R
Infection, 1dpi)~12dpi Continuous Observations infection the clinical manifestation of suckling mouse, changes of weight and survival rate change.Every group equal
Negative control group (NS) is set, i.e., isometric physiological saline is injected with identical route of inoculation.
Note:Intramuscular inoculation, IM;Intraperitoneal inoculation, IP;Cranial cavity is inoculated with, IC;Physiological saline, NS.
Clinical score standard:To evaluate the order of severity that Strain is caused a disease to suckling mouse, different clinical manifestations is assigned not
Same score value, this research is using conventional clinical score standard (table 2).
Table 2, clinical criteria scoring
As a result show, body weight, survival rate and the Clinical scores of each mouse of the identical age in days of control group are without significant difference.5 days
Age suckling mouse is 10 through IM, IP and IC infective dose4TCID50CVA6 strains WF057R experimental group average weight is with respect to negative control
Group does not show Body weight loss (A in Fig. 1), and mean clinical scores show transient symptom (B, three kinds of vaccination ways in Fig. 1
It is that 0), final survival rate is respectively 30%, 50% and 100% (C in Fig. 1), and this shows not in 10-12dpi Clinical scores
It is adapted to use 104TCID50Dosage infects 5 age in days suckling mouses to set up CVA6 animal models.5 age in days suckling mouses are through IM, IP infective dose
105.5TCID50Experimental group average weight show to be remarkably decreased with respect to negative control group, 9dpi 105.5TCID50- IM organize and
105.5TCID50- IP group body weight have dropped 51.47% and 22.42% (A in Fig. 1) respectively relative to negative control group body weight;
105.5TCID50- IM is organized and 105.5TCID50- IP groups about 4dpi shows rear myasthenia, as infection number of days increase occurs successively
Single hind limb paralysis, double hind leg myoparalysis, dying and small part suckling mouse such as gradually recover one's health at the symptom, and 105.5TCID50- IC groups
Mean clinical scores only show transient rear myasthenia symptom (B in Fig. 1);105.5TCID50- IM groups, 105.5TCID50- IP groups
With 105.5TCID50It is respectively 0,10% and 40% (C in Fig. 1) that-IC, which organizes final survival rate, and this shows to infect 10 through IM5.5TCID50
Dosage CVA6 strains WF057R is more adapted to set up 5 age in days CVA6 infection models.5 age in days suckling mouses are through IM, IP and IC infective dose
For 107TCID50Experimental group average weight there is body weight with respect to negative control group and be remarkably decreased, it is complete in 6dpi~7dpi after infection
Portion is dead, and the time-to-live is shorter, is unfavorable for the foundation (C in Fig. 1) of infection model.105.5TCID50(equivalent to 310LD50) through IM
After approach inoculation different days (3,7,9) suckling mouse, symptom is obvious after the inoculation of 3 age in days suckling mouses, mean clinical scores 5 (E in Fig. 1),
Time-to-live is short, all death (F in Fig. 1) in 7 days after infection;7 and 9 age in days suckling mouses are due to the big reason of age in days, compared to control group
Changes of weight is small (D in Fig. 1), and the death rate is low (F in Fig. 1), is not suitable for the foundation of model.Therefore, selection is inoculated with 5 through IM approach
Age in days suckling mouse, dosage are 105.5TCID50A/(310LD50) it is used as the condition for setting up suckling mouse infection model.Repeated experiment is proved
Clinical symptoms typical case after 5 age in days suckling mouses is inoculated with the conditions of the dosage, the approach, mean disease score is 5,9 after suckling mouse infection
It is all dead, and individual disease time and the death rate are stable, there is very high repeatability.
3rd, nucleic acid extraction, virus load detection and virus variation detection
10 are inoculated with respectively for 5 age in days suckling mouses5.5TCID50With 107TCID501dpi after the suckling mouse of dosage, infection, 3dpi,
Dead 3 of 5dpi each components other places, take brain, heart, lungs, spleen, muscle of posterior limb, enteron aisle and blood, are taken after being fully ground on 0.01g
Each tissue abrasive material is stated to be respectively placed in 1.5mL sterile centrifugation tubes, plus 4 DEG C of centrifugations of 0.01M PBS to 1mL, 9000rpm
10min, takes centrifuged supernatant TRIzol methods to extract viral nucleic acid, detects that virus load changes with time in each tissue,
It is preliminary to judge that CVA6 strains WF057R tissue tropisms in vivo and carrying capacity change.
TRIzol methods are extracted through reverse transcription acquisition cDNA after viral RNA, and reverse transcription is in ABI SimpliAmpTM Thermal
Carried out in Cycler PCR instruments, use TAKARA PrimeScriptTMRT reagent Kit (Perfect Real Time),
Cumulative volume is 10 μ L, wherein:5 × PrimeScript Buffer (for Real Time) 2 μ L, PrimeScript RT
The μ L of 0.5 μ L, Random 6mers of the Enzyme Mix I 1 and μ L of sample to be tested RNA 6.5.Reverse transcription reaction condition:42 DEG C,
45min(×1);85 DEG C, 5s (× 1);4 DEG C, ∞.Quantifying for virus load is carried out by quantitative fluorescent PCR, in ABI
Carried out on 7500FAST quantitative real time PCR Instruments, reaction system cumulative volume is 25 μ L, wherein:GoldStarTaqMan Mixture
(CW2625M, Beijing CoWin Bioscience Co., Ltd.) 12.5 μ L;CVA6 fluorescent quantitation specific forward primers CVA6-F
(25μM)0.625μL;The μ L of specific Down Stream primer CVA6-R (25 μM) 0.625;The μ L of CVA6 specific probes (25 μM) 0.625;
RNase-Free water 8.625μL;The μ L of cDNA templates amount 2 to be measured.Quantitative fluorescent PCR reaction condition:95 DEG C, 10min (×
1);95 DEG C, 15s (× 40);60℃, 1min (× 40), in each circulation 60℃, 1min detects fluorescence after terminating.Its middle and upper reaches
Primer CVA6-F sequences:CCTGAATGCGGCTAATCC;Anti-sense primer CVA6-R sequences:TTGTCACCATWAGCAGYCA;Probe
Sequence:5’FAM-CCGACTACTTTGGGWGTCCGTGT-3’BHQ1.Will be using CVA6-F and CVA6-R to CVA6 strains
WF057R nucleic acid enters PCR primer (dsDNA) and the carrier T structure plasmid that performing PCR amplification is obtained, and detection plasmid concentration is used as standard
It is standby after product, 10 times of doubling dilutions.The each dilution factor of carrier criteria product is all provided with three repetitions when carrying out quantitative fluorescent PCR every time
Hole, takes the average value of three repeating hole Ct values as the final Ct values of correspondence dilution factor.
For different infective stages and the carrying capacity distribution in suckling mouse body in Different Organs after infection of research virus, use
310LD50Dosage (105.5TCID50/ only), through IM approach infect 5 age in days suckling mouses, 1,3 and 5dpi detection virus load, find sense
Contaminate restrovirus carrying capacity amplitude of variation in each tissue larger.Each tissue viral level in 1~3dpi periods is generally presented fast
Speed is raised and 5dpi restrovirus content rate of climb downward trends.1dpi virus in blood nucleic acid contents are very low, but 3dpi
Quickly reach peak value 6log (copy number/mL blood), the time of occurrence of this viremia virusemia, with the clinical manifestation of suckling mouse (including
Behavior, Body weight loss etc.) occur time be corresponding.Viral level is up to 7~8log (copies in 5dpi hearts and lungs
Number/mg is organized), viral high content causes lungs serious viral pneumonia occur.Viral level in skeletal muscle (muscle of posterior limb)
Up to 10log (copy number/mg tissues), is 10 of viral level in other histoorgans of each time point2~104Times, show
Skeletal muscle is virus replication the most active position in suckling mouse body, also indicates that virus enters hematological system through intramuscular routes, passes through
Blood flow spreads all over rapidly whole body.Viral level highest in different vaccination approach muscle of posterior limb, illustrates that WF057R plants have strong flesh
Meat tissue preferendum, it is the place (A~C in Fig. 2) that virus is mainly replicated also to illustrate muscle.10 are being infected through IM approach4LD50Agent
Amount (107TCID50/ only) after, 1~5dpi viruses changes of contents trend and infective dose 310LD in each tissue50Dosage
(105.5TCID50/ only) variation tendency is similar, but virus load is notable in high dose virus inoculation hindbrain, blood, lungs, spleen
Raise (P<0.05), and IP and IC routes of infection virus load and low dosage inoculation change not significantly (Fig. 2).In Fig. 2, log's
The truth of a matter is 10;When sample is blood, log (copy number/mL) represents log (copy number/mL blood);When sample is other groups
When knitting, log (copy number/mL) represents log (copy number/mg tissues).
In vivo whether can producer mutation, PCR amplifications CVA6 at it after 5 age in days suckling mouses in addition, being infected for detection virus
Strain WF057R infects the complete VP1 fragments of CVA6 viruses in suckling mouse 1,3 and each tissues of 5dpi, and sequencing after product is reclaimed passes through
The softwares of Mega 5.05 carry out sequence alignment, and whether detection nucleotides and amino acid sequence undergo mutation.Expand VP1 upstream region of gene
Primer VP1-F is:5’-GGAGATAGGGTGGCAGATGT-3’;Anti-sense primer VP1-R is:5’-
AAGGGTGGTGATCGATGTGC-3’.As a result show, viral sequence does not change in each tissue.
4th, infection model plasma levels of cytokines expression situation of change
5 age in days suckling mouses infect 310LD through IM50(105.5TCID50/ only) 1~5dpi of collection peripheries after CVA6 strains WF057R
Blood plasma, different infection ranks are detected by cell factor ELISA detection kit (Hangzhou Lian Ke Biotechnology Ltd.)
IFN-γ, TNF-α, the change of IL-1 β, IL-4, IL-6, IL-10, IL-13 and IL-18 expression quantity in section suckling mouse model blood plasma,
To understand the effect that above-mentioned inflammatory cytokine plays immunopathogenesis damage in severe CVA6 infective stages.The inspection of each cell factor
Surveying lower limit is respectively:IFN-γ(1.74pg/ml)、TNF-α(1.63pg/ml)、IL-1β(1.03pg/ml)、IL-4(0.22pg/
ml)、IL-6(1.17pg/ml),IL-10(1.17pg/ml),IL-13(1.17pg/ml),IL-18(0.21pg/ml)。
Research shows, CVA6 strain WF057R initial infection Evaluation of Cytokines in Peripheral Blood IFN-γ, TNF-α, IL-6, IL-
10th, IL-13 and IL-18 (2dpi and 3dpi except) expression quantity has different degrees of high expression (figure relative to negative control group
3).Wherein, cell factor IFN-γ and IL-6 show the expression trend similar to the mankind (Han J, Wang Y, Gan X,
Song J,Sun P,Dong XP.Serum cytokine profiles of children with human
enterovirus 71-associated hand,foot,and mouth disease.J Med Virol.2014.;Lin
TY,Chang LY,Huang YC,et al.Different proinflammatory reactions in fatal and
non-fatal enterovirus 71infections:implications for early recognition and
therapy.Acta Paediatr.2002;91:632-5.), burst is presented to increase, peak value is in more than 2000pg/ml, and
The phenomenon reduced afterwards is now first raised with the increase of infection number of days.IL-10 expression quantity is to rise to highest in initial infection (2dpi)
40.13pg/ml, is then presented the trend steadily reduced;Equally, IL-13 expression quantity is in initial infection high level expression, but with
Infection number of days increase expression quantity is quickly reduced.The later stage (5dpi) is infected, TNF-α expression quantity gradually increases, and reaches 102pg/ml;
IL-18 is in whole infective stage expression quantity relative to negative control without significant difference (p>0.05);IL-1 β and IL-4 are from beginning to end
Reached without detectable content table.Data above illustrates, induction of cell factor after CVA6 strains WF057R infection suckling mouses ---
IFN-γ, IL-6, TNF-α, IL-10 and IL-13 expression, induce the immune response and inflammatory reaction of suckling mouse, IFN-γ and IL-
6 be probably CVA6 suckling mouse experimental infections Earlier period of inflammation reaction main predisposing factors, and cytokine TNF-α, IL-10 and
IL-13 also may play a significant role in the immunopathogenesis damage that CVA6 strains WF057R infects.
5th, pathological examination (HE) and immunohistochemical detection (IHC)
After CVA6 infection model optimum combination conditions are set up in final determination, with this condition, (IM approach is inoculated with 5 ages in days breast
Mouse, dosage are 105.5TCID50A/(310LD50)) set up and put to death 3 in suckling mouse CVA6 infection models, 1~5dpi daily
WF057R infects suckling mouse, takes its brain, heart, lungs, spleen, muscle of posterior limb, enteron aisle and blood;Blood normal temperature 10,000rpm is centrifuged
After 10min, upper plasma is taken to be stored in -20 DEG C;Remaining neutral formalin of internal organs 10% makes after fixing 48h through FFPE
Paraffin section is to carry out HE dyeing and immunohistochemistry (immunohistochemistry, IHC) detection.For immune group
Change detection, paraffin section is by 60 DEG C of roasting piece 1h, the dewaxing of alcohol gradient, EDTA antigen retrievals, dehydration, primary antibody CVA6 Anti-TNF-αs
Body (1:200 dilutions) 4 DEG C be incubated overnight, plus secondary antibody (1:1000 dilutions, Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge) room temperature
It is incubated after 30min and DAB colour developings is added dropwise, haematoxylin is anti-blue.Negative control group section primary antibody uses negative serum.Detected under microscope
As a result.Wherein, primary antibody CVA6 polyclonal antibodies be using CVA6 VP1 albumen twice be immunized 8 week old ICR adult rats obtain it is many
Clonal antibody.
Brain, the heart, lungs, the hind leg of the 5 age in days suckling mouses to infecting CVA6 strains WF057R through intramuscular injection are dyed by HE
Flesh and enteron aisle carry out pathological examination, find later period of infection, occur in that the damage and inflammatory reaction of organs.
1) brain:Pathological examination shows that obvious pathological change locally occurs for brain, and brain starts local meninx occur in 3dpi
And brain parenchym oedema (in Fig. 1 shown in A arrows).Brain SABC finds that a large amount of viral antigens occurs in Cerebral cortex since the 3dpi,
Cerebral cortex (C in Fig. 4) is distributed in as the growth viral antigen of infection number of days diffuses, negative control group brain tissue slice is not sent out
Now obvious pathological change (B in Fig. 4) and antigen dyeing (D in Fig. 4).
2) heart:HE dyeing finds that blood vessel dilatation hyperemia, regional nodes locally just occur in suckling mouse infection CVA6 5dpi hearts
Locally there is interstitial edema, cardiac muscle fibre dissolving, local companion steatosis and capillary vessel leak, and visible red in infiltration, heart
Cell hypoxia is showed (in Fig. 5 shown in A arrows).And SABC inspection finds that CVA6 antigens are distributed (Fig. 5 in heart in diffusivity
Middle C), negative control group heart sections do not find obvious pathological change (B in Fig. 5) and specific antigen dyeing (in Fig. 5
D)。
3) lungs:HE dyeing find CVA6 infection can cause suckling mouse occur viral pneumonia, show as diffusivity pulmonary parenchyma,
Alveolar and interstitial fibrosis and a large amount of lymphocytic infiltrations and alveolar collapse, collapse (in Fig. 6 shown in A arrows).SABC is sent out
Existing CVA6 antigens diffusivity distribution and whole lungs (C in Fig. 6), negative control group lungs section do not find that obvious pathology becomes
Change (B in Fig. 6) and specific antigen dyeing (D in Fig. 6).
4) skeletal muscle:HE dyeing shows that suckling mouse infection CVA6 3dpi skeletal muscle occurs between a small amount of lymphocytic infiltration, part
Matter oedema and a small amount of proliferation of fibrous tissue, as a large amount of lymphocytic infiltrations, interstitial edema occurs in infection number of days increase muscle of posterior limb
And proliferation of fibrous tissue scope expands, and start to occur local necrosis and muscle bundle fracture (in Fig. 7 shown in A arrows).Immune group
Change display relative to negative control group (D in Fig. 7) CVA6 infection after 3dpi muscle of posterior limb viruses start to increase and using spot distribution as
It is main, (C in Fig. 7) is distributed in muscle of posterior limb journey diffusivity with the increase CVA6 of infection number of days, negative control group muscle of posterior limb is cut into slices not
It was found that obvious pathological change (B in Fig. 7).
5) enteron aisle:HE dyeing finds that suckling mouse starts interstitial edema occur through intramuscular injection infection CVA6 3dpi enteron aisles, with
There is congested and lymphocytic infiltration (in Fig. 8 shown in A arrows) in the increase for infecting number of days, but does not find enteron aisle necrosis.Immune group
Change and check discovery, phase diffusivity is distributed in intestinal mucosa and intestinal villus (C in Fig. 8) after infection through intramuscular injection CVA6, negative right
Obvious pathological change (B in Fig. 8) and specific antigen dyeing (D in Fig. 8) are not found according to group intestine colibacillosis.
6th, other strains is pathogenic
According to the method for step one, by COxsackie A6 virus stains WF057R and original strain KM114057 viruses,
KP144344 viruses and KJ541157 viruses are inoculated with RD cells respectively, and inoculum concentration is 500TCID50, prepare the different strains of CVA6
Virus liquid.
Wherein, KM114057 viruses are the type of Coxsackie virus A 6 containing RNA shown in sequence A, and sequence A is by NCBI
T in KM114057.1 (No. Genebank) replaces with the sequence that U is obtained;KP144344 viruses are to contain RNA shown in sequence B
The type of Coxsackie virus A 6, sequence B is that the T in KP144344.1 in NCBI (No. Genebank) is replaced with into the sequence that U is obtained;
KJ541157 viruses are the type of Coxsackie virus A 6 containing RNA shown in sequence C, and sequence C is by KJ541157.1 in NCBI
T in (No. Genebank) replaces with the sequence that U is obtained.
As a result find, cell infects this KM114057, KP144344 and KJ541157 virus (500TCID respectively50) produce afterwards
Raw CPE time will be longer than strain WF057R, and virus titer is respectively 8.2 × 10 in 48 hour cell supernatants after infection7、2.7
×108With 3.6 × 108TCID50/ ml, less than same time strain WF057R virus titer 1.8 × 109TCID50/ ml, thus this three
Strain is less than strain WF057R to the adaptability of cell.
According to the preparation method of CVA6 infected animal models in step 2, COxsackie A6 virus stains WF057R is replaced respectively
Original strain KM114057 viruses, KP144344 viruses and KJ541157 viruses are changed to, 10 are injected by IM approach5.5TCID50Agent
The strain of amount prepares CVA6 infected animal models.
As a result find, by IM approach respectively by 105.5TCID50Three strain virus of dosage infect 5 age in days suckling mouses, 9 after infection
The death rate of its suckling mouse is respectively 80%, 80% and 100%.Integrated comparative virus it is pathogenic, strain WF057R in vivo and
It is better than KM114057, KP144344 and KJ541157 virus in experiment in vitro.
Embodiment 3, the antiserum produced by inactivating WF057R as the CVA6 vaccines of activity can protect mouse and treatment
CVA6 associated diseases
1st, the preparation of CVA6 vaccines is inactivated
By the CVA6 strain WF057R virus liquids (10 after the dilution of embodiment 25TCID50/ ml) press 1:4000 (V/V) ratio
Example obtains viral dilution liquid after being diluted using formalin, and viral dilution liquid is incubated into inactivation of viruses 72 hours in 37 DEG C, obtained
Inactivation of viruses liquid;Inactivation of viruses liquid is mixed in equal volume with complete Freund's adjuvant, complete emulsion, as CVA6 vaccines is made,
The content of inactivation of viruses is 5 × 10 in CVA6 vaccines4TCID50/ml.CVA6 vaccine infection titres are detected using microtitrimetry
To observe its inactivating efficacy.
Virus microtitre method:1 × 10 is pressed per hole4The μ l nutrient solutions of cell/100 (cell in the nutrient solution is RD cells,
Culture medium is the culture medium 1 of embodiment 2) RD cells are inoculated in the flat Tissue Culture Plate in 96 holes, cell length to the 90% of every hole
More than, above-mentioned CVA6 vaccines are used into the MEM culture mediums containing 2% hyclone by (extension rate point after 10 times of volume gradient dilutions
Not Wei 10 times, 102Again, 103Again with 104Times), RD cells are inoculated in 100 μ l/ holes, each dilution factor is inoculated with 8 holes, observation daily
Record cytopathy.
As a result find, cytopathy does not occur for the RD cells for being inoculated with different dilution factor CVA6 vaccines, shows, above-mentioned CVA6
WF057R in vaccine has been inactivated.
2nd, CVA6 vaccines bacterium is examined
The CVA6 vaccines of step 1 are aseptically inoculated with nutrient broth and nutrient agar panel, 37 DEG C of incubators respectively
Culture is observed after 2 days, it is found that bacterium inspection is feminine gender without bacterial growth.
3rd, CVA6 vaccine safeties are tested
3.1 adult rats safety testings
Experiment in triplicate, repeats comprising the following steps that for experiment every time:
8 week old healthy adult mouse (male and female are random) 20 are taken, experimental group and control group, every group 10 is randomly divided into.Experiment
The CVA6 vaccines of group suckling mouse subcutaneous injection step 1, co-injection three times is 1 week per double injection interval time, three inoculations
Amount is 200 μ l/;The mixed liquor that control group injection complete Freund's adjuvant is mixed to get in equal volume with PBS, co-injection three times,
It it is 1 week per double injection interval time, three inoculum concentrations are 200 μ l/.Continuous Observation 14 days, observation adult rats whether there is
Morbidity, dead and changes of weight.
Visible through 8 week old adult rats safety testing results, 3 experimental group adult rats are time different through subcutaneous 1,2 and 3 respectively
Immune time injects CVA6 vaccines, and control group adult rats body weight indifference, without morbidity, dead, without visually can after execution
See lesion.
3.2 suckling mouse safety testings
Experiment in triplicate, repeats comprising the following steps that for experiment every time:
5 age in days ages healthy suckling mouse (male and female are random) 20 is taken, experimental group and control group, every group 10 is randomly divided into.Experiment
The CVA6 vaccines of group suckling mouse subcutaneous injection step 1, co-injection three times is 1 week per double injection interval time, three inoculations
Amount is 50 μ l/;Control group, which injects complete Freund's adjuvant and the mixed liquor that PBS is mixed to get in equal volume, (please examine control group
The liquid of injection), co-injection three times is 1 week per double injection interval time, and three inoculum concentrations are 50 μ l/.It is continuous to see
Examine 14 days, observation suckling mouse whether there is morbidity, dead and changes of weight.
It is visible through 5 age in days suckling mouse safety testing results, 3 group experimental group Neonatal Mouses respectively through subcutaneous 1,2 and 3 times not
CVA6 vaccines are injected with immune time, and control group suckling mouse body weight indifference, without morbidity, dead, without visually can after execution
See lesion.
4th, the preparation and titration of antiviral serum
4.1 CVA6 are sero-fast to be prepared
8 week old healthy adult mouse (male and female are random), the CVA6 vaccines of subcutaneous injection step 1 are taken, injection volume is 200 μ l/
Only;CVA6 vaccine injections gather blood after 35 days, supernatant, as CVA6 antiserums, sterile bar are taken after 10,000rpm/min centrifugations
Dispense, freeze standby in -80 DEG C under part.Using the sero-fast antibody titer of microneutralization measuring.
4.2 few cells neutralization tests
The CVA6 antiserums of step 4.1 are utilized into the MEM culture mediums containing 2% hyclone from 1:100 proceed by 2 times
Gradient volume dilution, totally 10 dilution factors, dilution ratio is respectively 1:102、1:2×102、1:22×102、1:23×102、1:24
×102、1:25×102、1:26×102、1:27×102、1:28×102With 1:29×102, obtain the anti-blood of CVA6 of different dilution factors
Clear dilution.
By the CVA6 strain WF057R virus liquids (500TCID of embodiment 250/ ml) 96 orifice plates, 50 μ l/ holes are added to, then distinguish
Add the CVA6 antiserum dilutions of above-mentioned different dilution factors, 50 μ l/ holes, per the dilution of one dilution factor in hole, every kind of dilution
Three multiple holes of dilution of degree, 37 DEG C of incubation 1h;RD cell culture fluids are inoculated with into every hole, and (concentration is 2 × 105Individual/ml), 100
μ l/ holes, 37 DEG C of culture 7d, observation cytopathy situation (CPE) calculates neutralize antibody titers using Reed-Muench methods.
According to above-mentioned few cells neutralization test method to continuously detection 6 times of the sero-fast neutralization titers of CVA6, calculate flat
Variance yields AVE and coefficient of variation CV is extracted, the repeatability of this method is verified.
2 times of serial dilutions are carried out to CVA6 antiserums on the RD cells cultivated in vitro, CVA6 strain WF057R are added, led to
The lesion calculating for crossing RD cells obtains the sero-fast antibody titers of CVA6 for 1,024 (AVE>0.5, CV≤8%).
5th, protection of the CVA6 antiserums by passive immunity to suckling mouse
Passive immune protection for research CVA6 antiserums in suckling mouse is acted on, and the CVA6 antiserums of step 4.1 are utilized into life
Manage salt solution and carry out 10 times of serial dilutions (1:10,1:100,1:1,000 and 1:10,000) it is, that obtained different CVA6 antiserums are dilute
Release liquid and 4 age in days suckling mouses of experimental group 5 (every group 10) are inoculated into by way of intravenous injection in vivo, each experimental group injection one
The CVA6 antiserum dilutions of dilution gradient are planted, 1 negative control group suckling mouse (CVA6,10) injection is isometric to be isolated from not
The negative control sera of CVA6 healthy adult mouse is inoculated with, volume injected is by lethal dose 310LD after 50 μ l, 1h50
(105.5TCID50) CVA6 virus stains WF057R by intramuscular routes infect each group suckling mouse, 1 day (1days post after infection
Infection, 1dpi) to clinical manifestation, changes of weight and the survival rate situation of change of 12dpi Continuous Observations infection suckling mouse, adopt
Scored with the clinical score standard of table 2, regard 5 age in days suckling mouses of normal health as control (NC).According to experimental group suckling mouse
Survival rate, calculate 50% effective dilution factor ED of the CVA6 antiserums in suckling mouse body50I。
4 experimental groups are first inoculated with the WF057R for infecting lethal dose after the antibody of different dilution factors again, find 1:10 dilutions
CVA6 antiserums complete protective capability can be provided, there is mild clinical symptom in part suckling mouse, and score (Fig. 9, four houses five for 1
Enter), get well quickly;Injection 1:The sero-fast experimental group suckling mouse clinical scores of CVA6 of 100 dilutions are up to 3, most throughout one's life
It is 60% to deposit rate;Injection 1:The sero-fast experimental group suckling mouses of CVA6 of 1000 dilutions start clinic occur for 3 days after virus inoculation
Symptom, scoring in 10 days is all dead for the 5, the 11st day after infection;Compared with negative control group (CVA6 curves in Fig. 9), 1:10,
The CVA6 antiserums of 000 dilution almost without protective effect, after suckling mouse virus inoculation the 10th day it is all dead (Fig. 9).1:10 dilutions
CVA6 antiserums the protection of suckling mouse 100% can be provided, according to Reed&Muench formula (Reed and Muench, 1938)
The CVA6 antiserums that calculating obtains 163 times of dilutions can provide 50% protective rate, so CVA6 antiserums prevent in suckling mouse body
The ED of effect50I is 1.63.
6th, protective effect of the maternal antibody to suckling mouse
For the female immanoprotection action for passing antibody to Neonatal Mouse of research, the 8 subcutaneous multiple spots of week old female mice, multiple injection are walked
Rapid 1 μ l of CVA6 vaccines 200 carry out it is immune for the first time, the inactivation of viruses liquid of interval mode injecting step 1 same after 2 weeks with not
The μ l of complete emulsion 200 that complete Freund's adjuvant is mixed to get in equal volume are with booster immunization.After immune for the first time by female suckling mouse and
Male suckling mouse mating, young mouse birth in second immune latter 5~10 days.Choose 5 age in days suckling mouses point, 5 experimental groups (10/group).
It is alternative to take 8 week old female mices, according to the method described above, CVA6 vaccines and complete emulsion are replaced with into physiological saline,
To female mice injecting normal saline, the 5 age in days suckling mouses bred point, 5 control groups (10/group).
Each group suckling mouse is injected to the 310LD of lethal dose through IM approach respectively50(105.5TCID50) CVA6 virus stains
WF057R and different places clinic CVA6 separation strains WH15066/Shandong/China/2015 (300LD50)、DY003R/
Shandong/China/2015(300LD50)、DY005R/Shandong/China/2015(300LD50) and LW03R/
Shandong/China/2015(300LD50), the injection dosage of each strain is 310LD50, Continuous Observation 12d simultaneously records it
Changes of weight, clinical score (being scored using the clinical score standard of table 2), survival rate, antibody pair is passed to evaluate CVA6 mothers
Neonatal Mouse infects CVA6 protective effect.Using Mantel-Cox log-rank method comparative experiments groups and control group suckling mouse
Survival rate.
The suckling mouse that immune dams group breeds body weight after virus inoculation does not almost increase, and clinical symptoms are obvious, the 6th day
Start dead, all death in the 9th~10 day.The suckling mouse inoculation that the experimental group dams of immune WF057R inactivated vaccines breed is different
Clinical separation strain CVA6 (WF057R, WH15066, DY003R, DY005R and LW03R) occurs (in Figure 10 without clinical symptoms afterwards
Left figure), its changes of weight is not with meeting poison group suckling mouse indifference (P>0.05), final survival rate is 100% (right figure in Figure 10).
As shown by data, it is different to lethal dose that the present invention can provide suckling mouse using female biography antibody of the CVA6 vaccines of WF057R strains preparation
Local isolated strain CVA6 complete protective capability.
7th, clinical therapeutic efficacy of the antiserum to the suckling mouse that falls ill
To study clinical therapeutic efficacy of the CVA6 antiserums to different onset degree suckling mouse, first using IM approach to 5 days
Age suckling mouse inoculation lethal dose 310LD50(105.5TCID50) CVA6 virus stain WF057R, isolated rearing, concrete operations are same
The preparation of CVA6 infected animal models in embodiment 2.Started to screen infection suckling mouse in 4 days after infection, select clinical obtain
Respectively 1~2 and >=3 suckling mouse is respectively as early and late 2 experimental groups of falling ill (n=10/group).Using intravenous injection
Mode will using physiological saline according to 1:1、1:10 and 1:The CVA6 antiserums of the step 4.1 of 50 volume ratio dilution are noted respectively
In the experimental group suckling mouse body for being mapped to 3 different onset degree, volume injected is 50 μ l, and each experimental group injects different dilutions
The CVA6 antiserums of degree, a kind of CVA6 antiserums of dilution factor inject 10 suckling mouses in every group, and 1 CVA6 control groups suckling mouse is (only
CVA6 is inoculated with, antiserum, 10 are not injected) the isometric negative control sera of injection (is isolated from not being inoculated with CVA6 healthy adults
Mouse) as control, Continuous Observation 12d simultaneously records its changes of weight, clinical symptoms and survival rate, to evaluate CVA6 antiserums to hair
The clinical therapeutic efficacy of sick suckling mouse.5 age in days suckling mouses of normal health are injected to isometric physiological saline (NS) as control.According to
According to the survival rate of experimental group suckling mouse, the 50% effectively dilution that the CVA6 antiserums of calculation procedure 4.1 are acted in suckling mouse interior therapeutic
Spend ED50II.Using Mantel-Cox log-rank method comparative experiments groups and the survival rate of control group suckling mouse.
As a result find, clinical symptoms fade away after suckling mouse (morbidity early stage) emergency injection antibody of Clinical scores 1~2,
Scoring is reduced to 0 (Figure 11), increased weight, 1:1、1:10 and 1:The final survival rate of experimental group of 50 dilution factors be respectively 100%,
100% and 40% (Figure 11).But Clinical scores are more than 3 (morbidity late period single hind leg occurs or double hind limb paralysis are even dying)
Each dilution factor of suckling mouse CVA6 antiserums to it all without any protective effect, and injection negative serum group (CVA6 after morbidity
Group) not notable (P of difference>0.05), the death rate is 100% (Figure 11).According to Reed&Muench formula calculate obtain 43 times it is dilute
The CVA6 antiserums released can provide 50% cure rate, so the effective dose 50 that CVA6 antiserums are acted in suckling mouse interior therapeutic
ED50II is 23.81.As shown by data, the CVA6 for each dilution factor of suckling mouse of nervous symptoms i.e. hind leg (single, double hind leg) paralysis occur resists
Serum is to it without any protective effect.With reference to the suckling mouse infection model being established above, whole body is presented in the suckling mouse for being scored above 3
The major injury of multi viscera and inflammatory reaction, nervous system have been destroyed, a large amount of neuronal necrosis, are swallowed by phagocyte;
Viral content is up to 10log in muscle of posterior limb, starts skeletal muscle fibre necrosis and muscle bundle fracture occur.So, now CVA6 resists
CVA6 antibody in serum is difficult the severe development for reversing suckling mouse, and for the suckling mouse of early stage, compared to injection negative serum
Therapeutic effect highly significant (the P of group antibody<0.005), gradually recovered one's health after treatment.
8th, the effect of other CVA6 viral vaccines
The preparation method of CVA6 vaccines is inactivated according to step 1, COxsackie A6 virus stains WF057R is replaced with into reality respectively
The KM114057 viruses, KP144344 viruses and KJ541157 viruses of example 1 are applied, other steps are constant, prepare CVA6 vaccines, i.e.,
KM114057 viral vaccines, KP144344 viral vaccines and KJ541157 viral vaccines.
By KM114057 viral vaccines, three kinds of vaccines of KP144344 viral vaccines and KJ541157 viral vaccines respectively according to
The method of step 4 35 days collection serum after the week old adult rats of subcutaneous vaccination 8, its antibody titer is detected by microneutralization experiment
Respectively 256,512 and 512 (continuous to detect 6 times), less than strain WF057R antibody titers (1024).
By the method for pricking even step 6, KM114057 viral vaccines, KP144344 viral vaccines and KJ541157 are utilized respectively
The week old female mice of three kinds of vaccine immunities of viral vaccine 8, the 5 age in days suckling mouses produced inject the 310LD of lethal dose through IM approach50
(105.5TCID50) CVA6 virus stain WF057R, as a result find three kinds of vaccine-induced female antibody that pass to suckling mouse lethal dose
(310LD50) WF057R protective rate is 70~80% (death rate is 20%~30%), it is impossible to complete protection is provided.
Based on previous work basis, inventor has done comprehensive pathology in this experiment to the suckling mouse of vaccine immunity group
Check, be especially noticed that whether there is the immunopathogenesis damage that vaccine immunity animal occurs after virus attack.Observe result table
Bright, vaccine immunity adult rats and suckling mouse do not find main organs Histopathology at viral metainfective different time points
Obvious change, only see extremely individual other inflammatory cell infiltration point in muscle of posterior limb.These minimal amount of inflammatory cell infiltration points
Pathological phenomena is after multiple analysis, it is believed that it is non-specific pathological phenomenon.Meanwhile, in nervous system and Main Tissues
In be showed no the presence that viral nucleic acid is also nearly no detectable in the pathological change of characteristic, each tissue.It has been confirmed that in epidemic disease
The immunoprotective effec formed after dams is immunized in seedling, is that can completely inhibit the propagation of virus in animal body, and finally may be used
To protect body to exempt to be infected by the virus caused pathological lesion completely.
Claims (10)
1. the preparation method of animal disease vaccine caused by prevention and/or treatment Coxsackie virus, including:By the type of Coxsackie virus A 6
Strain prepares the vaccine as active component;
The type strain of Coxsackie virus A 6 is the guarantor in China Committee for Culture Collection of Microorganisms's common micro-organisms center
Hide the strain that numbering is CGMCC No.13393.
2. according to the method described in claim 1, it is characterised in that:The type strain of Coxsackie virus A 6 is the COxsackie of inactivation
Viral A6 types strain.
3. method according to claim 1 or 2, it is characterised in that:The animal is mammal.
4. method according to claim 3, it is characterised in that:The mammal is mouse.
5. according to any described method in claim 1-4, it is characterised in that:Methods described is included in claim 1-3
Any type strain of the Coxsackie virus A 6 is mixed to get the vaccine with immunologic adjuvant.
6. method according to claim 5, it is characterised in that:The immunologic adjuvant is for complete Freund's adjuvant or not exclusively not
Family name's adjuvant.
7. according to any described method in claim 1-6, it is characterised in that:The vaccine is prevention and/or treatment Ke's Sa
The vaccine of strange virus Animal diseases caused by A6 types.
8. the vaccine that in claim 1-7 prepared by any described method.
9. the product of Animal diseases caused by prevention and/or treatment Coxsackie virus, be it is following 1) or 2):
1) product containing any type strain of Coxsackie virus A 6 in claim 1-3;
2) antiserum that in claim 1-3 prepared by any type strain of Coxsackie virus A 6 is utilized.
10. following X1) or X2):
X1) any described method Animal diseases caused by prevention and/or treatment Coxsackie virus is prepared are produced in claim 1-7
Application in product;
X2) application of the vaccine described in claim 8 in Animal diseases caused by preventing and/or treating Coxsackie virus.
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CN107739731A (en) * | 2017-10-12 | 2018-02-27 | 泰山医学院 | Virus composition and its application of strain are tamed comprising Coxsackie virus A 10 |
CN112522211A (en) * | 2020-11-12 | 2021-03-19 | 武汉生物制品研究所有限责任公司 | Coxsackie group A6 virus mutant strain and application thereof |
CN117230026A (en) * | 2023-08-24 | 2023-12-15 | 中国医学科学院医学生物学研究所 | Coxsackie virus A6 type CVA6-KM-J33 and application thereof |
-
2017
- 2017-03-20 CN CN201710182230.8A patent/CN106994182A/en active Pending
Non-Patent Citations (1)
Title |
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ZHENJIE ZHANG等: "A Neonatal Murine Model of Coxsackievirus A6 Infection for Evaluation of Antiviral and Vaccine Efficacy", 《JOURNAL OF VIROLOGY》 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107739731A (en) * | 2017-10-12 | 2018-02-27 | 泰山医学院 | Virus composition and its application of strain are tamed comprising Coxsackie virus A 10 |
CN107739731B (en) * | 2017-10-12 | 2020-07-21 | 山东第一医科大学(山东省医学科学院) | Virus composition containing Coxsackie virus A10 domesticated strain and application thereof |
CN112522211A (en) * | 2020-11-12 | 2021-03-19 | 武汉生物制品研究所有限责任公司 | Coxsackie group A6 virus mutant strain and application thereof |
CN117230026A (en) * | 2023-08-24 | 2023-12-15 | 中国医学科学院医学生物学研究所 | Coxsackie virus A6 type CVA6-KM-J33 and application thereof |
CN117230026B (en) * | 2023-08-24 | 2024-02-02 | 中国医学科学院医学生物学研究所 | Coxsackie virus A6 type CVA6-KM-J33 and application thereof |
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