CN100479858C - A recombined smallpox vaccine - SARS vaccine and preparation method thereof - Google Patents

A recombined smallpox vaccine - SARS vaccine and preparation method thereof Download PDF

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CN100479858C
CN100479858C CNB2006100030110A CN200610003011A CN100479858C CN 100479858 C CN100479858 C CN 100479858C CN B2006100030110 A CNB2006100030110 A CN B2006100030110A CN 200610003011 A CN200610003011 A CN 200610003011A CN 100479858 C CN100479858 C CN 100479858C
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mva
sars
vaccine
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秦川
魏强
高虹
涂新明
陈志伟
张林琦
何大一
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Institute of Laboratory Animal Science of CAMS
Aaron Diamond AIDS Research Center
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Aaron Diamond AIDS Research Center
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Abstract

The invention provides a reorganization pox seedling virus vaccine SARS-CoV (ADS-MVA) for preventing or treating SARS, and also the method for carrying out preventive vaccine anti-SARS-CoV infection in the body of Chinese rhesus monkey infection models. By constructing SARS vaccine using modified attenuation virus vaccine (MVA) as vector, fundamental immunity and toxic material elimination are realized on rhesus monkey. The invention can screen ideal vaccines for the prevention and treatment of SARS.

Description

A kind of recombined smallpox vaccine-SARS vaccine and preparation method thereof
Technical field
The present invention relates to a kind of recombined smallpox vaccine-SARS vaccine and preparation method thereof, especially relate to a kind of attenuation vaccine MVA after the modification and SARS vaccine ADS-MVA of sars coronavirus S gene and preparation method thereof of comprising.
Background technology
SARS from November, 2002 first Guangdong Province find and report since, very fast popular in the world, WHO be called severe acute respiratory syndrome (Severe Acute RespiratorySyndrome, SARS).This disease is mainly by the air spittle closely and contact transmission closely, has general crowd's susceptible, propagates characteristics such as fast, the anxious and certain case fatality rate of morbidity.At present, total confirmed SARS cases in the whole world and suspected case 5663 examples, by the end of dead 372 examples on May 15th, 2004, China has 5316 examples by the end of on May 26th, 2004, dead 315 examples, doubtful 1573 examples are cured 2742 examples.Human beings'health in the serious threat that is widely current of SARS, and studies show that both at home and abroad, and the SARS cause of disease is a kind of variation coronavirus, and length is the positive single strand RNA virus of 29742bp.
China has all obtained certain progress to the research of aspects such as SARS cause of disease, diagnosis, source, has set up the SARS infected animal model in our previous work.At the beginning of 2003 5 months; we have infected Rhesus Macacus; systematically carried out virusology; serology and pathological research; importantly in the monkey body that infects, detected high neutralizing antibody of tiring; this result has confirmed that SARS virus can bring out body and produce protection antibody; thereby development SARS virus vaccine is possible in theory; the monkey that infects is reliable as the animal pattern of vaccine evaluation; this work has solved limit drug screening in the quite a while; the bottleneck problem of research such as vaccine evaluation; especially established important foundation for the research of aspects such as vaccine evaluation; the control fully of this disease depends on the development of effective vaccine.
The SARS vaccine that comprises SARS associated coronavirus S gene and eukaryon expression plasmid is disclosed in China's application 200410044291.0, and the SARS vaccine that comprises adenovirus vector and SARS associated coronavirus S gene etc. disclosed in the China application 200410044285.5, but there is safety hidden danger in various degree in existing SARS vaccine, and their effectiveness waits further to observe and checking.
Summary of the invention
The present invention of existing SARS vaccine has overcome existing many technical problems, success develop recombined smallpox vaccine-SARS vaccine with good immunogenicity, safety and effectiveness, and, obtained satisfied effect in animal infection modal China Rhesus Macacus body with the experiment that this vaccine prevents pathogenic SARS-CoV to infect.Especially compare with the deactivation SARS vaccine in the clinical trial, this vaccine need not to contact SARS-CoV, and is safer reliable.
A first aspect of the present invention relates to a kind of SARS-CoV vaccine (ADS-MVA) that makes up on attenuated vaccinia virus MVA basis.
Above-mentioned SARS-CoV vaccine is inserted into MVA disappearance III district with SARS associated coronavirus S gene and obtains.
The invention still further relates to a kind of preparation method of SARS-CoV vaccine, it comprises:
(1) make up shuttle plasmid, this plasmid comprises the SARS-CoV antigen gene, and attenuated vaccinia virus MVA disappearance III district's gene code region sequence or with the coding region outside the flanking sequence of both sides sequence homology;
(2) in the permission cell of vaccinia virus, make shuttle plasmid and attenuated vaccinia virus MVA strain homologous recombination;
(3) by selection markers, recombinant virus is carried out single speckle screening, obtain pure highly attenuated vaccine ADS-MVA virus to separate.
The invention still further relates to the SARS-CoV vaccine (ADS-MVA) that the preparation method that adopts above-mentioned SARS-CoV vaccine makes.
In addition, the present invention relates to the application of aforementioned attenuation recombiant vaccine SARS-CoV vaccine ADS-MVA in treatment or prevention SARS-CoV infection and disease.
The present invention relates to the application of method in making up the recombinant MVA expression alien gene of aforementioned structure attenuation recombinant vaccinia SARS vaccine ADS-MVA.
The invention further relates to the application of method in making up recombinant MVA expression foreign pathogens antigen preparation pathogen detection and diagnostic reagent that makes up aforementioned attenuation recombinant vaccinia SARS vaccine ADS-MVA.
In addition, the present invention relates to the method that aforementioned attenuation recombinant vaccinia SARS-CoV vaccine ADS-MVA prevents pathogenic SARS-CoV to infect in Chinese Rhesus Macacus body, it is characterized in that, in the monkey challenge test, Rhesus Macacus all is 5 * 10 at the 0th and 28 day inoculation ADS-MVA twice, twice dosage of inoculation respectively 8TCID50, the vaccine of half be by intramuscular inoculation, in addition half through nasal cavity give with, every the animal in 4 week backs is with 10 5TCID50 is by intranasal inoculation virus SARS-CoV PUMC01.
The invention still further relates to a kind of pseudovirus and make the method for neutralization test, it is characterized in that, with pcDNASopt9 and two plasmid co-transfection 293T of pNL4-3Luc+Env-Vpr-cell of S gene that carries SARS-CoV respectively and the main core gene of HIV-1, with the preparation pseudovirus; The neutralizing antibody activity detects with pseudovirus invasion test, and the pseudovirus of Xi Shi blood serum sample and isodose is cultivated down at 37 ℃ by a certain percentage, and serum/virus mixture adds respectively in the 293-ACE2 cell, and the infection cell cracking detects the luciferase activity.
Consider that the S gene that changes over to may change the cytotaxis of ADS-MVA, this may influence the safety range of vaccine.We have detected the growth characteristics of ADS-MVA in mammal, we infect several mammal cell lines with ADS-MVA, and 293, Hela and Vero cell.Compare (Fig. 2 A) with observed typical autgmentability growth pattern in the CEF cell, the mammalian cell that ADS-MVA infects only limits in the discrete cell.Therefore, although S albumen is still expressed, the same with the MVA of wild type, ADS-MVA does not duplicate diffusion in mammalian cell.In addition, ADS-MVA goes down to posterity in the CEF cell 9 times, but these go down to posterity and do not cause the disappearance of S protein expression, and prompting ADS-MVA hereditary stability is fine
In vaccine development, recombined smallpox vaccine-SARS vaccine has many advantages, might become ideal a kind of vaccine, and the present invention utilizes the vaccine (MVA) after the modification to be vector construction SARS vaccine.Can cause hospital infection and death unlike traditional vaccine.MVA is not because contain host gene in the viral genome, and can not and duplicate in most mammalian cells in human body, and MVA DNA gene expression do not have influencedly, can synthesize in human body cell in early days and late gene.The more important thing is that MVA has done more than one hundred million times at variola and has been applied to large-scale vaccine test and clinical trial, does not in use find side effect, even the monkey of high risk patient or experiment immunization defective.In the present invention, total length SARS-CoV peplos S glycoprotein gene has changed MVA disappearance III district gene over to, we select S albumen to be because it has mediated viral invasion as the main target protein of cell and humoral immunization, the new reorganization ADS-MVA that produces can be through causing high-caliber neutralizing antibody in mice, rabbit and monkey after twice immunity, the neutralizing antibody that causes in Rhesus Macacus can prevent the invasion of SARS virus.Measure through vaccine immunogenicity, vaccine safety is measured, the vaccine effectiveness is measured, and TPPA, detection of dynamic in the virus load body, indexs such as SARS pathology degree variation are determined, recombinant vaccinia SARS vaccine has good immunogenicity, safety and effectiveness, can be used as a potential safe useful preventative vaccine at the infection of human and animal's SARS virus, is safe for control SARS screens ideal vaccine, should carry out clinical trial as early as possible.
Description of drawings
Fig. 1 represents an example of the construction method of SARS vaccine ADS-MVA.
Fig. 2 represents S albumen and GFP (green fluorescent protein) coexpression in the CEF cell that ADS-MVA infects, and wherein A shows S albumen, and B shows GFP.
Fig. 3 represents to contain the structure and the expression of dna vaccination of the S genetic fragment of optimization.
Fig. 4 represents the testing result of the anti-SARS-CoV specificity neutralizing antibody reaction after the ADS-MVA immunity BALB/c mouse.
Fig. 5 represents the testing result of the anti-SARS-CoV specificity neutralizing antibody reaction after the ADS-MVA immunity New Zealand white rabbit.
Fig. 6 represents the testing result of the anti-SARS-CoV specificity neutralizing antibody reaction behind the ADS-MVA immunity Rhesus Macacus.
Fig. 7 represents the testing result of the anti-SARS-CoV specificity neutralizing antibody reaction after the dna vaccination immunity New Zealand white rabbit.
Fig. 8 show merge human Fc is arranged ACE2 receptors bind zone (RBR-Fc) to New Zealand white rabbit (A), BALB/c mouse (B), the absorption and the removing of the anti-SARS-CoV specificity neutralizing antibody of Rhesus Macacus (C).
The specific embodiment
Embodiment 1
Make up pZC3d and insert carrier: (Bernie Moss and Lynda doctor Wyatt of NIH provide importing the pLW7 carrier by the synthetic pH5 promoter of DNA, with reference to Development of areplication-deficient recombinant vaccinia virus vaccine effectiveag ainstparainfluenza virus 3 infection in an animal model.Linda S.Wyatt.et.al.Vaccine.Vol.14, No.15,1451-1456.1996), make up the pZC3d carrier that a new generation contains double-promoter.In this carrier, the S gene is under strong synthetic promoter pSYN, reporter gene GFP gene is under weak promoter pH5 (Fig. 1), two genes change in the identical insertion sequence, (Bernie Moss and Lynda doctor Wyatt of NIH provide GFP as acting on behalf of the recombinant MVA carrier that labelling is used to carry the S gene, with reference to Vaccine Protocols, Second Edition, August 2003, pps.51-68, ISBN:1-59259-399-2Series:Methods in Molecular Medicine; Volume #:87; ByCaroline Staib and Gerd Sutter).These two promoteres of pSYN and pH5 all are the specific promoteres of vaccine, and new carrier allows polygenes to be inserted into single recombinant MVA virus.As pLW7, the insertion site of pZC3d is the disappearance III district (referring to Fig. 1) of MVA gene.
Embodiment 2
Make up and purification ADS-MVA: the method that adopts flush end to be connected, GFP gene and S is gene constructed in pZC3d.The S gene is from the cDNA of SARS-CoV HKU39849 Strain, and this virus is separated from Hong Kong (the S gene is preserved in GenBank, preserving number AY278491).The ADS-MVA of reorganization produces in chick embryo fibroblast (CEF) by the method for homologous recombination: at first, infect CEF cell (1MOI) with parental generation MVA.After 90 minutes, use Effectene (Qiagen Cat:301427), use the pZC3d transfection with a group cell; After 48 hours, positive cell group is picked out by the fluorescence that GFP sends under fluorescence microscope; The ADS-MVA of reorganization is able to purification by 8 subinfection CEF passages.Thus, use pZC3d, we recombinate the SARS total length S gene of wild type and GFP in the MVA III district, obtain ADS-MVA (referring to Fig. 1).Use the same method in order to compare, to adopt, we will import same loci through modifying HCV E1E2 gene, make up another recombinant virus ADC-MVA.
Embodiment 3
Immunofluorescence test: in order to detect the S glycoprotein of cell surface expression, we have done immunofluorescence test.Easy steps is the CEF cell to be taped against in the 1st day that (there are 2 x 10 in each hole on 6 orifice plates 6Individual cell), dull and stereotyped through Con A (100 μ g/ml) pretreatment 30 minutes, PBS cleans twice.The 2nd day, with ADS-MVA (1:10) the infection CEF cell of the serial dilution after the reorganization, after two hours, cell culture medium was replaced with hyclone (FCS) DMEM that contains 2%, in 37 ℃ of 5% CO 2Hatched 48 hours, metainfective cell is with heat-inactivated SARS patients serum (WH, 1:500) hatch 1 hour, use goat anti-human igg (H+L) (the Molecular Probes of AlexaFluor594 labelling then, U.S.A.) hatch 30 minutes, cell is given a baby a bath on the third day after its birth time with PBS, and positive group differentiates that with the immunofluorescence fibrescope result is referring to Fig. 2 A.Also dyeing in contrast in each test of CEF cell infection control group A DC-MVA.
Embodiment 4
The preparation of ADS-MVA Strain: this Strain uses the CEF cell through previously described method purification.The ADS-MVA of purification prepares with the amplification of 2000ml rolling bottle culture technique in the CEF cell.
Embodiment 5
Western blot analyzes: in order to determine the zone of the contained neutralizing antibody decision of total length S glycoprotein epi-position among the ADS-MVA, we have prepared a collection of dna vaccination, and they contain the S gene of different length.For S albumen is better expressed in mammalian cell, we optimize the codon sequence of S gene with engineered method.The targeting sequencing of S gene gene (the tissue plasminogen activator of tissue plasminogen activator, Tpa) targeting sequencing is replaced, and this targeting sequencing can promote protein expression by promoting protein to transfer to from endoplasmic reticulum on the Golgi apparatus.These dna vaccinations prove and can express in mammalian cell in the transient expression test.Main method is, with dna vaccination mammals expression vector pcDNA-S200, pcDNA-S400, pcDNA-S600, pcDNA-S800, pcDNA-Sopt9, or pcDNA3.1 transfection 293T cell.After 48 hours, (1 x 10 of the cell after the transfection 6) wash in the back placement dry ice with 200 μ l cell pyrolysis liquid (50mM Tris-HCl (pH8.0), 137mMNaCl, 2mM EDTA with PBS, 0.5% NP-40,10% glycerol, and 1 μ g/ml of each of pepstatin, leupeptin and pefabloc) cracking 30 minutes.Cell debris is in 4 ℃, 14, centrifugal 10 minutes of 000rpm, remaining lysis supernatant electrophoresis on 10% sds page.The albumen that separation obtains is transferred on the nitrocellulose filter, do the test of the antibody marking, the result is referring to Fig. 3, S albumen is also in conjunction with 400 amino acid whose rabbit antibody of N-terminal, S albumen molecular weight be 160 and the 250Kd indicia band in occur, this is bigger than actual prediction value, and this change may be because due to the protein translation post-treatment.Male blood serum sample is from the pcDNA-S400 serum that electrotransfection technology immune rabbit obtains in body.400 aminoacid of pcDNA-S400 coding S gene N-terminal, negative serum is from the animal of the health of using placebo injection.For S albumen is better expressed in mammalian cell, we optimize the codon sequence of S gene with engineered method.The targeting sequencing of S gene gene (the tissue plasminogen activator of tissue plasminogen activator, Tpa) targeting sequencing is replaced, this targeting sequencing can promote protein expression by promoting protein to transfer to from endoplasmic reticulum on the Golgi apparatus, we can express in people 293T cell by these protein of Western blot test demonstration, in order to ensure producing antibody response, we change the DNA plasmid in the rabbit over to by electrotransfection technology in the body.
Embodiment 6
Virus inoculation method sars coronavirus (SARS-CoV) separates from Chinese patient SARS, the patient is the SARS ward patient of BJ Union Hospital, isolating virus is cultivated on the Vero cell through Concord Hospital and is gone down to posterity, be defined as sars coronavirus through RT-PCR and electron microscopic morphology observation, viral TCID50 is 10 6/ ml.This virus is by our called after PUMC-01 strain, and there is certain difference in its sequence and Beijing 4 strains (BJ01-4), and with Hong Kong SU10 strain, Singapore and Canadian SARS-CoV strain have nearer evolutionary relationship (Genbank Accession No., AY350750).Sars coronavirus via intranasal application collunarium inoculation, all zooperies are all used by relevant laboratory animal institute laboratory animal in the P3 Animal Lab. and are tested rules and carry out.
Embodiment 7
Immune animal: with ADS-MVA the 0th, 3 week respectively through the female BALB/c mouse at 6~8 monthly ages of intramuscular injection (i.m.) immunity, be divided into following several groups according to the difference of immunizing dose: ADS-MVA 5 x 10 6TCID50 or 5 x 10 7TCID50, ADC-MVA, normal saline matched group, two week back do injections for the second time, blood sampling sample analysis after the mice euthanasia.At the 0th and 28 day, 2 rabbits were with 1 x 10 8The TCID50ADS-MVA immunity, other two usefulness, 1 x 10 8TCID50 ADC-MVA immunity.8 Rhesus Macacus were immunity twice in the 0th and 28 day, and wherein 4 inoculation ADS-MVA inoculate ADC-MVA for other 4.Initial immunizing dose is 1 x 10 8TCID50, secondary immunity dosage are 3 x 10 8TCID50.In each immunity two weeks of back, take analysis of blood.Dna vaccination inoculation, every group has 2 rabbits to use electroporation technology in the body at the 0th and 28 day, inoculate 400 simple μ g DNA plasmids (pcDNA-S200 ,-S400 ,-S600and-S800).And behind the last dna immunization 2 months, these rabbits were with 1 x 10 8Per 4 weeks of TCID50 ADS-MVA are strengthened once.The matched group rabbit is with the similar DNA plasmid inoculation of expression of HCV E1E2, and matched group is also strengthened in the same way with ADC-MVA, in every group, all takes blood serum sample and at immunity 2 all post analysis.
In the monkey challenge test, totally 16 3 years old Rhesus Macacus are used for experiment, divide two groups of vaccine immunity and contrasts, and every group each 8,8 monkeys of vaccine immunity group are numbered 0411,0412,0413,0414,0415,0416,0417 and 0418; 8 of matched groups are numbered 0419,0420,0421,0422,0423,0424,0425 and 0426.Rhesus Macacus is provided by China Medical Sciences Academy Medical Biology Institute, detects with monkey national standard microorganism SPF level by experiment before the inoculation, checks to show that SARS virus antibody is negative.16 Rhesus Macacus are respectively inoculation twice in the 0th and 28 day, and wherein 8 inoculation ADS-MVA inoculate ADC-MVA for other 8.Both dosage of inoculation all is 5 x 10 8TCID50.The vaccine of half gives by the mode of muscle (i.m) inoculation, and half gives through nasal cavity in addition, and every the animal in 4 week backs is with 10 5TCID50 SARS-CoV PUMC01 passes through intranasal inoculation.The S gene order (AY350750) that it should be noted that vaccine SARS-CoV HKU39849 Strain and SARS-CoV PUMC01 is in full accord, and this experiment is finished in the P3 of Institute of Experimental Animals, Chinese Academy of Medical Sciences laboratory animal facility.
Embodiment 8
We have done the activity of neutralization test with the humoral immunization of detection vaccine generation, the advantage of this test is to eliminate the harmful effect of using the SARS-CoV that lives to produce in traditional neutralization test, we have detected the neutralization activity that produces in the serum behind the ADS-MVA immune animal.Hot deactivation animal serum neutralizing antibody activity detects with pseudovirus invasion test, this experiment is to carry out (with reference to Chen.Z. according to the method for work of describing in the document, P.Zhou, et.al.Genetically divergent strains of simianimmunodeficiency virus use CCR5 as a coreceptor for entry.J.Virol.71:2705-14,1997.).Pseudovirus is with two plasmid pcDNASopt9 that carry optimized S gene and HIV-1 major gene respectively and pNL4-3Luc +Env -Vpr -Cotransfection 293T cell obtains, and the pseudovirus of Xi Shi blood serum sample and isodose was cultivated 1 hour down at 37 ℃ by a certain percentage, then serum/virus mixture is added respectively in the 293-ACE2 cell, and after 56 hours, the infection cell cracking detects the luciferase activity.
At first, 8 mices are used the ADS-MVA immunity.Mice M260 and M262 are with 5 x 10 6The vaccine immunity of TCID50, other 6, M263, M264, MM1, MM2, MM3 and MM4 are with 5 x 10 7The TCID50 immunity, all animals inoculate with intramuscular injection method, and twice immunity be 3 weeks at interval, take blood sample to do the neutralizing antibody test in 2 weeks the 2nd immunity back.At matched group, the proteic ADC-MVA of 4 mices (M265, M266, M267 and M268) inoculation expression of HCV E1E2, the vaccine dose that is used for matched group is every mice 5 x 10 7TCID50, we have also inoculated two mices (N1 and N2) in contrast with normal saline, have produced high-caliber neutralizing antibody in 8 mices with the ADS-MVA immunity.In high dose group, the serum of dilution more than 1000 times still can suppress or in and 50% virus (IC50).Mice in 2 matched groups does not produce neutralizing antibody.In addition, two ADS-MVA dosage groups have tangible dosage correlation (referring to Fig. 4), and therefore, ADS-MVA induces in mice and produced the specific neutralizing antibody of SARS-CoV.
In order to inquire in other strain animal, whether can produce similar neutralizing antibody, we with the ADS-MVA immunity two rabbits.Immune programme for children the 2nd time different when immune, be 4 weeks immunity at interval specifically, the selection of dosage is based on the result who detects MVA HIV-1 vaccine test before us in toy.In two rabbits (R524 and R525) of inoculation ADS-MVA, produce high-caliber SARS-CoV specificity neutralizing antibody.After diluting 10000 times, serum still can detect IC50.The rabbit (R520 and R521) of two matched groups of inoculation ADC-MVA does not produce SARS-CoV neutralizing antibody (referring to Fig. 5), and these data show do not have varietY specificity by the high-level neutralizing antibody that ADS-MVA produces.The animal of all tests can both well tolerate this vaccine, does not observe tangible disease in the experiment or loses weight.
In order to inquire into the probability that ADS-MVA uses in the mankind, we have further detected ADS-MVA in Rhesus Macacus.16 Rhesus Macacus have been used in this experiment altogether, use the ADS-MVA immunity for 8, use the ADC-MVA immunity for other 8, take blood serum sample to detect the appearance of neutralizing antibody continuously.DC-MVA compares with control group A, and the Rhesus Macacus of ADS-MVA immunity has produced high-caliber SARS-CoV neutralizing antibody.Therefore, except toy, ADS-MVA also can produce neutralizing antibody in Rhesus Macacus, consider that SARS-CoV mainly infects respiratory system, we have also detected the titre of the neutralizing antibody in lungs and the bronchus, lung and bronchial tissue's sample and serum are taked simultaneously, but all do not detect neutralizing antibody in all 8 Rhesus Macacus, even at minimum dilution factor (<1: 10) (referring to Fig. 6).
In addition, adopt electroporation technology in the body, use respectively dna vaccination S200 (R416, R417), S400 (R418, R419), S600 (R683, R684) and the dna vaccination (R518 of expression of HCV E1E2, R519) rabbit has been carried out immunity, method is the same, is 2 immunity, at interval 4 weeks, the result is referring to Fig. 7.
Embodiment 9
Clinical observation and blood testing: body temperature is (37.8~38.1 ℃) before 16 zooperies, behind vaccination and the contrast liquid, does not see fervescence.16 Rhesus Macacus have the fervescence phenomenon when the 1st~5 day of virus inoculation, be 38.6~39.2 ℃.The part control animal has dyspnea, and the minimizing of ingesting loses weight.Do not see clinical manifestations such as cough, watery nasal discharge, vomiting, diarrhoea.Leukocyte is at preceding 8 the vaccine monkey average out to of infection (7.6 ± 3.1) * 10 3, do not see obvious change behind vaccine immunity and the virus inoculation; 8 contrast monkeys average out to (6.9 ± 2.9) * 10 before infection 3, 2,5 behind the virus inoculation obviously reduced in the time of 7 and 14 days, average range (3.9 ± 1.8~5.4 ± 1.9) * 10 3AST each time point behind contrast monkey virus inoculation had remarkable rising during blood biochemical was measured, but had significance to rise other indexs such as ALT in the time of 7,14 days in vaccine monkey disease poison inoculation back, ALP, UREA, CRE, LDH, CK, TP and ALB do not see obvious change in whole experiment.
Embodiment 10
The antibody adsorption test: the solubility reorganization ACE2 receptors bind zone (S310-510) of having merged people Fc (RBR-Fc) is used to adsorb the antibody of specificity bind receptor calmodulin binding domain CaM.Then remove the RBR-Fc/ antibody complex with specific anti-human IgG Fc-agarose (Sigma).The serum of dilution and the RBR-Fc of solubility were at room temperature hatched 45 minutes, added the anti-human IgG Fc agarose of 10 μ l prewashing then in each hole.At room temperature microplate shakes and prevented from gel precipitation to adsorb in about 2 hours.Microplate at room temperature 3000 left the heart 10 minutes, remained unconjugated serum and used pseudovirus to cook neutralization test.Specificity in order to ensure test is provided with the parallel control group in microplate, comprise two groups of anti-human IgG gel and RBR-Fc.In other one group of experiment, owing to contain the monkey serum and the anti-people Fc antibody cross reaction that is coated on the carrier gel pearl of antibody, we have to take diverse ways, and we use a kind of chemical method of introducing in the product service manual to make Sepharose 4 Fast Flow beads (AmershamBiosciences) directly in conjunction with RBR-Fc.The RBR-Fc of solubility of no use absorption antibody, and be to use RBR-Fc in conjunction with pearl absorption with remove monkey antibody in conjunction with the RBR part, remaining experimental technique is identical with the front.
The serum of rabbit and mice has diluted 1:300~1:600 doubly according to the result of neutralizing antibody test, so that eliminate of the influence of too much neutralizing antibody to experimental result, solubility RBR-Fc can adsorb and remove the most of neutralizing antibodies in the rabbit immune serum, the sepharose 4B of RBR-Fc (-) group in and not effect of antibody.Ironically, the neutralizing antibody activity in the animal of dna vaccination immunity (R681.D and the R682.D) serum almost completely is eliminated.Correspondingly be, the animal of ADS-MVA vaccine immunity, in the serum reserve part after its absorption and active, prompting ADS-MVA can induce the broad-spectrum neutralizing antibody.ADS-MVA produces more broad-spectrum neutralizing antibody activity than S600DNA vaccine, and promptly may there be a plurality of neutralizing antibody active sites (referring to Fig. 8 A) in total length S albumen.
The ADS-MVA immune mouse is found same result.Most mice neutralizing antibodies is adsorbed by the RBR-Fc recombiant protein or removes, and therefore, our data show contains a main neutralizing antibody decision site (referring to Fig. 8 B) at the ACE2 calmodulin binding domain CaM.
In detecting the monkey serum test, go absorption and remove in the monkey serum and the bonded neutralizing antibody of RBR part with the bonded agarose 4 fast stream pearls (Sepharose4 Fast Flow beads) of RBR-Fc.Shown in Fig. 8 C, the pearl (-) that does not have a RBR-Fc in and the not effect of activity of antibody; And RBR-Fc pearl (+) can be removed most neutralizing antibody, and this result is similar to rabbit and mice serum result of the test.Comprehensive our experimental result, we reach a conclusion, and ADS-MVA causes neutralizing antibody, at first at the calmodulin binding domain CaM of SARS-CoV receptor ACE2.This conclusion all has embodiment in 3 species, point out the neutralizing antibody that is produced by ADS-MVA not have species specificity (referring to Fig. 8 C) simultaneously.
Embodiment 11
The SARS-CoV separation test: behind the SARS-CoV virus inoculation Rhesus Macacus, collected the Nasopharyngeal swabs specimen at the 2nd, 5,7 day, fully be immersed in the 1ml DMEM culture fluid, behind 0.22 μ m membrane filtration, inoculation Vero cell adsorbed 1 hour, and virus is separated.Euthanasia when 7 days and 30 days behind counteracting toxic substances, the lung tissue of every animal are done to be used for virus after the homogenate and are separated.Separate carrying out virus behind the lung tissue homogenate inoculation Vero cell.Adsorb after 1 hour, renew bright culture medium behind the eluting, culture observation cytopathy (CPE) 10 days if do not find CPE, with second batch of Vero cell of the blind inoculation of culture fluid, was observed 10 days again.If find CPE, with the SARS-CoV specific antigen in immunofluorescence test (method is the same) the detection Vero cell, to avoid occurring false positive.Separation detection result is referring to table 1
Embodiment 12
Reverse transcription polymerase chain reaction (RT-PCR): in the specimen samples of SARS-CoV virus inoculation Rhesus Macacus, detect viral RNA, total RNA uses MagNA Pure LC total nucleic acid separating kit (Roche Diagnostics) to separate from various specimen, the RNA that is separated to re-uses a kind of commercial SARS-CoV RT-PCR kit (Roche Diagnostics) and detects, and the result is referring to table 1.
The testing result of virus behind the Chinese Rhesus Macacus inoculation of table 1. SARS-CoV.
*Detect neutralizing antibody before the virus inoculation
*Isolated viral from lung tissue homogenate
* *The Nasopharyngeal swabs specimen is carried out RT-PCR
Embodiment 13
Histopathologic slide's preparation: in the animal that virus inoculation is put to death after the 7th day and month, get tissues such as lung, 10% formaldehyde fixed, paraffin embedding, after the routine pathology tissue slice is handled, H-E dyeing, microscopy.The result shows, the vaccine immunity group show counteracting toxic substances after 7 days 2 animals the interstitial lung inflammation appears, counteracting toxic substances after 35 days 1 animal the interstitial lung inflammation appears, and the visceral pleura edema occurs and ooze out, 3 animals are focal interstitial lung inflammation in addition; Matched group shows that all there was the interstitial lung inflammation in counteracting toxic substances in 4 animals after 7 days, and lesion degree is heavier, and each animal pathological changes range size is slightly variant.All there was the interstitial lung inflammation in counteracting toxic substances in 4 animals after 35 days, lesion degree than counteracting toxic substances after 7 days 4 animals light, each animal pathological changes range size is slightly variant.
From above pathology comparative result, to compare with model group, the pulmonary lesion of vaccine group animal is lighter, and the Preliminary experiment results prompting 1. relevant vaccine of this inspected SARS can alleviate the attack of SARS virus to the animal subject Rhesus Macacus; 2. this vaccine is not seen damaging pathological change to inspected other internal organs.

Claims (8)

1. SARS-CoV vaccine ADS-MVA who on attenuated vaccinia virus MVA basis, makes up, described SARS-CoV vaccine ADS-MVA is the MVA vaccine that the S gene of SARS-CoV is inserted into attenuated vaccinia virus MVA disappearance III district, it is characterized in that, described SARS-CoV vaccine ADS-MVA comprises the promoter pSYN of the S gene of SARS-CoV, and the S gene of SARS-CoV is positioned at the downstream of this promoter pSYN.
2. SARS-CoV vaccine ADS-MVA according to claim 1, wherein, described MVA disappearance III has also inserted selection markers in the district.
3. SARS-CoV vaccine ADS-MVA according to claim 2, wherein, described selection markers is the GFP gene, the promoter of described GFP gene is pH5, and the GFP gene is positioned at the downstream of this promoter pH5.
4. the preparation method of the described SARS-CoV vaccine of claim 1 ADS-MVA, it comprises:
A. make up shuttle plasmid, this plasmid comprises the S gene of SARS-CoV, and attenuated vaccinia virus MVA disappearance III district's gene code region sequence or with the coding region outside the flanking sequence of both sides sequence homology, the insertion site of described shuttle plasmid is the disappearance III district of attenuated vaccinia virus MVA;
B. in the permission cell of vaccinia virus, make shuttle plasmid and attenuated vaccinia virus MVA strain homologous recombination;
C. by selection markers, recombinant virus is carried out single speckle screening, obtain pure highly attenuated vaccine ADS-MVA virus to separate, wherein, described shuttle plasmid is the shuttle plasmid that the S gene of SARS-CoV is positioned at promoter pSYN downstream.
5. the preparation method of SARS-CoV vaccine ADS-MVA according to claim 4, wherein, described selection markers is GFP, and the S gene of SARS-CoV changes in the identical insertion sequence of shuttle plasmid with the GFP gene, and described selection markers is positioned at promoter pH5 downstream.
6. according to the made SARS-CoV vaccine ADS-MVA of the preparation method of claim 4 or 5 described SARS-CoV vaccine ADS-MVA.
7. the application of the preparation method of claim 4 or 5 described SARS-CoV vaccine ADS-MVA in making up the recombinant MVA expression alien gene.
8. application according to claim 7, wherein, the described application of preparation method in making up antigen preparation pathogen test of recombinant MVA expression foreign pathogens and diagnostic reagent that is applied as claim 4 or 5 described SARS-CoV vaccine ADS-MVA.
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