CN101092609A - Vaccine against mumps containing a JERYL-LYNN virus strain - Google Patents
Vaccine against mumps containing a JERYL-LYNN virus strain Download PDFInfo
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Abstract
A new mumps vaccine is presented, comprising a homogeneous pure isolate derived from the Jeryl-Lynn strain of mumps virus. In a preferred embodiment of the invention the vaccine produces higher seroconversion and antibody titres than known commercial vaccines.
Description
Related application
The application is to be on November 15th, 1994 applying date, and application number is dividing an application of 94194807.2 application.
Technical field
The present invention relates to a kind of Mumps Vaccine, it contains a kind of homogeneous purifies and separates strain of deriving and from the Jeryl-Lynn strain of mumps virus.
Background technology
Mumps is a kind of children diseases that take a disease basically, has only slight clinical symptom usually.But the clinical effectiveness that mumps infects in some case is serious.For example, Britain's mumps be the most commonly encountered diseases of suffering from meningoencephalitis of children below 15 years old because of, also be a cause of disease of the permanent sensorineural hearing loss of children.Although 30～40% natural parotitis infects symptom does not appear, but can involve sialisterium, and in the crowd of growing up, except nervosa complication above-mentioned, mumps also can cause first trimenon miscarriage and testitis, these all are the not ccontaining doubtful facts, and it makes many countries all implement the premunitive plan of crowd.
Mumps virus belongs to Paramyxoviridae, it is made of the minus strand strand geneome RNA of about 15.3kb, gene order is: (N is a nucleocapsid protein to 3 ' N-P-M-F-SH-HN-L5 ', P is a phosphorprotein, M is a stromatin, and F is a fusion rotein, and SH is the little hydrophobic proteins of potential expression (potentially expressed), HN is the hemagglutinin neuraminidase, and L is a large protein).In numerous mumps virus strains, (the B-level is a kind of attenuated live mutation B-level) to Jeryl-Lynn, through sequential analysis, it is characterized in that F, P, HN, M gene.
There are two kinds of mumps virus strains to get permission to be used for anti-popular parotitic preventive vaccination recently: Urabe Am 9 and Jeryl-Lynn.But Urabe Am 9 is cancelled in September, 1992 after having reported a routine unacceptable side effect level [European Journal of Pediatrics (1993) 152:387].
The Jeryl-Lynn virus strain is sold with the trade(brand)name of " MumpsVax " by Merck Sharp and Dohme for many years always.It is to obtain clinical samples on one's body from a routine patient who suffers from mumps, (Proc.Soc.Exptl.Biol.Med.123 (3) (1966)) that gets through the inoculation of chicken embryo amnion.
People such as Afzal report that recently (J.ofGen.Virology 1993
74917) in fact be the mixture of JL-2 and these two kinds of viruses of JL-5 as the Jeryl-Lynn virus strain of Mumps Vaccine, in Britain.
People such as Takeuchi, Virology (1991)
181P364-366 reports that also different mumps virus strains can produce the nucleotide sequence variation of substantive (substantial) on the SH gene level.
People such as Afzal also emphasize, present " MumpsVax " vaccine commodity are to prepare under the condition of strictness control, these conditions comprise a cell bank (cell bank), the limit that goes down to posterity, and the condition of tending to keep the ratio of two kinds of varients between the product that difference is criticized.Yet, can't guarantee to keep this balance between above-mentioned two kinds of varients along with further going down to posterity of Jeryl-Lynn strain.And the ratio of very difficult these two kinds of varients of estimation in any a collection of vaccine.
Summary of the invention
The invention provides a kind of vaccine, it comprises the immunogenicity Jeryl-Lynn strain isolated of homogeneous (substantially homogenous) basically, and described virus strain is the strain of attenuation Jeryl-Lynn mumps virus, and it comprises nucleotide sequence as shown in Figure 1.The N-terminal and the SH gene of this sequence encoding HN gene.This virus strain is called as SBB JL-1 in this article.Compare with existing Mumps Vaccine commodity, this strain isolated can induce zero higher conversion in clinical trial, and has the highest parotitis antibody geometric mean titer.
What is called homogeneous basically is meant that according to the qualification to sequence section shown in Figure 1, described strain isolated is no more than 10% by the degree that another kind of Jeryl-Lynn strain isolated pollutes, and preferably is less than 5%, more preferably less than 1%.In a preferred embodiment of the present invention, this vaccine comprises a pure lines Jeryl-Lynn strain isolated, and promptly this vaccine is not polluted by other discrepant Jeryl-Lynn mumps virus strain isolated in constant gene segment C shown in Figure 1.
One embodiment of the invention provide a kind of vaccine, and it comprises the purifying SBB JL-1 that no JL-2 pollutes.
This purifies and separates strain does not have the inconvenience that the potential difference of inferior strain between difference is criticized is brought, and it provides a kind of easier consistent quality standard (consistent quality guideline) of guaranteeing to meet.
Homogeneous of the present invention (homogenous) Jeryl-Lynn can followingly obtain: the MumpsVax commodity are gone down to posterity in chick embryo fibroblast (CEF) and screen pure growth, described screening can be to carry out limiting dilution and check the gained strain isolated, or carries out single plaque and separate.Other suitable clone comprises Vero cell and MRC5 cell.This will seek survival in the method that can effectively detect in the colony than the known viruse variant of small proportion.This class inspection method comprises that the Maprec at attenuated polio viruses that is proposed by people such as Chumakov detects (WO 92/07958 and PNAS 1991,88; 199-203), directly virus plaque is checked order in addition and virus plaque is carried out differential hybridization.
Preferred vaccine of the present invention also comprises other composition, such as attenuated measles virus, and/or the attenuation rubella virus, their deactivation form or subunit's form, thereby the provide protection that can provide anti-measles and/or rubella to infect.Tervalent parotitis, measles and Rubella Vaccine are widely known by the people in this area, and the parotitis strain isolated among the present invention also can be mixed with trivalent vaccine according to the mode similar to existing those vaccines.Further, perhaps alternatively, vaccine of the present invention can comprise the attenuated varicella zoster live virus, so that anti-varicella (varicella, chicken pox) or zoster (Zoster, provide protection shingle) to be provided.In a preferred embodiment, varicella zoster virus is Andre F E PostgraduateMED J. (1985) 61 (Suppl.4), 113-120 or Veskari T etc., Acta paediatr.Scand.80:1051-1057,1991 disclosed OKa strains.Preferred vaccine of the present invention is a quaternary, can provide material for anti parotitis virus, the provide protection of rubella Measles virus and varicella zoster virus.
The present invention also provides the method for preparing whole virus vaccine, for example under the condition that has suitable stabilizers to exist with viral freeze-drying, or virus strain of the present invention mixed with suitable carriers or adjuvant.Also preferably virus strain of the present invention is formulated in the liposome or with carrier granule and prepares.Further; perhaps alternatively; also can add immunologic stimulant in this preparaton; single phosphinylidyne lipoid A (3 de-O-acyl monophosphoryl Lipid A, Ribi Immunochem) or saponin (saponin) derivative QS21 (Cambridge Biotech) as 3-deoxidation acylations.
On the other hand, the present invention also provides a kind of method that human mumps infects for the treatment of, comprise to have in requisition for patient use the vaccine of the present invention of immune effective dose.
The method of application of vaccine of the present invention can be that any can transport other immunogenic components in the virus strain of immunoprotection amount and the vaccine suitable pathways to the experimenter.But preferably use described vaccine through the non-enteron aisle mode of muscle or deep subcutaneous route.Can also adopt other method of application as required, as oral or, promptly in intracutaneous, the nose or vein through other parenteral approach.
This kind vaccine can provide immanoprotection action nontoxic again suitable dosage; can measure out at an easy rate by those skilled in the art; meaning promptly; contained in the vaccine of the present invention, not only caused immunoprotection but also avirulent virus quantity of the present invention, drop within the scope of the antigen significant quantity in traditional whole virus vaccine.But the concrete dosage level that should be understood that any concrete patient will depend on multiple factor, comprise the age, general health level, sex and diet; The time of medication, the approach of medication; Synergistic effect with any other medicines of using; And want the protective reaction degree that reaches.Certainly, if necessary, can be with the proper spacing repetitive administration.In univalent vaccine, be typically every dose of 3.7log TCID at least
50Virus, and every dose of 4.5log TCID more usually
50In parotitis, measles, rubella trivalent vaccine, the mumps virus composition will reach 4.8log TCID
50About, to compensate the interference of other two kinds of plain compositions of disease.
Description of drawings
Fig. 1 has shown the SH gene coding region of JL-1 mumps virus strain isolated and the cDNA sequence of SH-HN intergenic region section.
Embodiment
1) the SH gene is tentatively checked order
At 25cm
2On the individual layer Vero cell that covers with in the culturing bottle, add 0.5% foetal calf serum, inoculate about 3.0log TCID with dMEM Biorich substratum (50/50V/V)
50MumpsVax virus commodity, go down to posterity.34 ℃ of incubations 7 days are gathered in the crops infected cell then, with Ferr é and Garduno (Nucleic Acids Research 1989,17; 2141) method is extracted RNA, places 100mcl water, handles 5 minutes in 100 ℃ with diethyl coke hydrochlorate.Get the so following reagent of extract adding of 5mcl and carry out reverse transcription: the RNAsin of 40 units (Boehringer Mannheim, Germany), the spissated reversed transcriptive enzyme damping fluid of 4mcl 5X (Bethesda Besearch Labs), the mixture concentration of four kinds of deoxynucleoside triphosphates of 2mcl is 10mM, 10pmole NH2 Oligonucleolide primers, 1mclMoloney murine leukemia virus (MMLV) reversed transcriptive enzyme (Bethesda Research Labs, every mcl200 unit), adding water, to make final volume be 20mcl.The F gene of oligonucleotide NH2 and mumps virus Urabe strain has homology.With mixture 37 ℃ of incubations 45 minutes, then in 95 ℃ of heating 5 minutes.Make primer with oligonucleotide NH8 and NH14 cDNA increased through continuous two-wheeled PCR reaction, with the first round reactant of 1000 times of dilutions of 1mcl as second initiator of taking turns reaction.Every PCR of wheel is made up of 25 circulations, and each circulation all is 94 ℃ of heating 1 minutes, and 53 ℃ were heated 1 minute, and 72 ℃ were heated 1 minute.There is being the acid of fluorine di-deoxynucleoside to stop under the situation of thing, make primer with NH8 or NH14 and further carry out pcr amplification, schedule of operation and explanation that its product is furnished with by instrument with Applied Biosystems automatic sequencer (373ADNA Sequencer) are analyzed, thereby from both direction the PCR product corresponding to the SH gene are checked order.Found that in described sequence, have a plurality of positions to exist difference to read result (ambiguity), be not always the case on verified two chains.(Viroloy 1991, and 181:364-366) Bao Dao Jeryl-Lynn sequence is compared, and 17 differences are arranged in 361 bases, do not determine (unassign) base comprising 4 for people such as institute's calling sequence and Takeuchi.People (J.Gen.Virol.1993,74 such as gained gene order and Afzal; 917-920) sequence of Bao Dao JL-5 strain isolated is compared, and 9 differences are arranged in 319 bases, wherein has 4 not determine base.Another kind of situation is not go down to posterity on the Vero cell, but gather in the crops 4.0～5.0log TCID with ultracentrifugation
50MumpsVax virus, with random primer these viral RNA reverse transcriptions are become cDNA again, carry out pcr amplification with oligonucleotide NH30bis and NH3 1bis as primer then, directly same sector is checked order at last, the result can find that still difference reads the result.
2) the SH gene is cloned
With MumpsVax virus infection Vero cell, prepare total RNA more as stated above.This RNA carries out reverse transcription with random primer, and carries out pcr amplification with oligonucleotide NH22 and NH23 as primer.(NH22 comprises Hind III restriction site, and NH23 comprises the BamHI restriction site, thereby is convenient to the dna fragmentation of amplification is cloned).Behind the DNA usefulness HindIII and these two kinds of restriction endonuclease cuttings of BamHI with amplification, be cloned on the carrier pUC9.Obtain 11 clones at last, they respectively have insertion corresponding to mumps virus SH constant gene segment C.All 11 clones have the corresponding sequence of sequence with (in above-mentioned quoted passage) report of Takeuchi etc.In addition, have 5 clones to have the DdeI restriction enzyme site, and other 6 clones do not have.Do not find to insert son accordingly with the JL-5 sequence.This result, and the difference that obtains of order-checking reads the result, all hints: the JL-2 varient that is identified by people such as Afzal is the part of the important or easy detection of in the MumpsVax virus.
3) directly go down to posterity from MumpsVax
MumpsVax also directly goes down to posterity on chick embryo fibroblast (CEF).From cultivating, the third generation of the cell that comprises 5 different batches of lot MJ05 reclaims virus, so that the freeze-drying sample of preparation MJ05A42 is used for injecting animal.With these 5 batches viral prepared product vero cells infections, reclaim RNA, increase as primer with NH8 and NH14 as stated above, in order to dna sequencing, unique different be to start reverse transcriptase reaction with random primer.All 5 batches of viruses all demonstrate the identical sequence with JL-2 (people such as Takeuchi), and do not have difference to read the result.
In order further to investigate this point, as stated above virus plaque is directly checked order.With above-mentioned 5 batches of virus infection Vero cell cultures, the gained plaque is handled in order to order-checking, wherein use NH30bis and NH31bis makes primer.Surveyed 26 plaques in 5 batches altogether, wherein 13 plaques provide the identical sequence of JL-2 sequence with report such as Takeuchi; 5 plaques provide the sequence closely similar with JL-5, have only two bases of the 270th and 279 that difference is arranged; The sequence of 8 plaques has difference to read the result, hints that it is a virus mixture.
4) directly virus plaque is checked order
Three kinds of diluents of MumpsVax, estimate that every 0.50ml equal portions have 100,50 and 10 virions, infect the individual layer Vero cell that has been paved with and has discarded nutrient solution in the 5cm Petri culture dish with them, wash ware with dMEM Biorich (50: the 50 V/V) substratum (Biorich) that does not contain serum.Make virus absorption 30 minutes at 34 ℃.Cover cell with the agar that remains on 42 ℃, agar layer comprises 2.5 mldMEM Biorich substratum, 0.5% foetal calf serum, and the low gelatinization temperature agarose of 2.5ml 3% (W/V).After the curing, this agar layer is covered with 3ml dMEM Biorich substratum (containing 0.5% foetal calf serum), and at 34 ℃ of incubations.Behind the incubation 7 days, discard the liquid nutrient medium on surface, add the diffusion of 0.03% (W/V) neutral red solution after 1 hour, just can see plaque.Remove liquid and agar then, a dried nylon membrane is pressed in the bottom of culture dish with finger.This film addend is dripped 2x SSC soak, mention again.Film was soaked 5 minutes in 2x SSC, in the 2x SSC that contains 0.2% (W/V) SDS, soaked 30 minutes again, under UV light, expose 3 to 5 minutes then, make viropexis on film.Cut 20 independently plaques from nylon membrane, these diaphragms are immersed in the 100mcl water, add 1mcl RNAsin (Boehringer Mannheim, 40 units), 65 ℃ were heated 30 minutes.100mcl liquid is transferred in the new test tube, added 10mcl 3M sodium-acetate, add 250mcl ethanol again, make the nucleic acid precipitation.Mixture is spent the night or placed 1 hour in-70 ℃ in-20 ℃, centrifugal then.After the drying precipitate, add following solution and carry out reverse transcription: the spissated reversed transcriptive enzyme damping fluid of 4mcl 5x (Bethesda ResearchLabs), 2mcl 0.1 M dithiothreitol (DTT), 1mcl deoxynucleoside triphosphate mixture (PerkinElmer-Cetus, 10mM concentration), 1mcl N6 random primer oligonucleotide (New England Biolabs, concentration 100mcg/ml), 11mcl water.Add 1mcl MMLV reversed transcriptive enzyme then, 37 ℃ of incubations 1 hour, 95 ℃ of incubations are 5 minutes then.Get this mixture 10mcl, add each 500ng of Oligonucleolide primers NH30bis and NH31bis, and add PCR damping fluid and 1mcl Stoffel archaeal dna polymerase (PerkinElmer-Cetus, 10 μ g/100 mcl final volume.With 30 circulations of this mixture heating up, every circulation be 95 ℃ 1 minute, 60 ℃ 1 minute, 72 ℃ 1 minute.The gained fragment is carried out purifying with Magicprep test kit (PromegaBiotech A7170) according to its explanation.Make primer with NH30bis or NH31bis, adopt the acid of fluorine di-deoxynucleoside to stop thing and carry out the asymmetric PCR amplification, the method and the reactant that provide with producer on Applied Biosystems (373A) automatic sequencer check order by the on-radiation method.Found that in these 20 plaques from MumpsVax, 19 JL-2 sequences with people's (above) such as Takeuchi report have 11 bases different in 275 bases, have 2 bases different with the JL-5 sequence of people's (above) report such as Afzal.There is 1 plaque to provide the different results that reads.This result hint: MumpsVax may comprise one or more discrepant varient of finding with Afzal etc. of JL-5 dominant strain in this section.Two bases that JL-5 is different with the plaque that is checked order are in the 279th and 290 position, in the intergenic region section between SH and HN coding region.
5) plaque hybridization
For attempt more directly measuring MumpsVax and the culture of deriving in the ratio of JL-5 and JL-2 form variation body, adopted a kind of plaque hybridizing method.With MumpsVax virus and the MJ05 virus infection Vero cell monolayer that goes down to posterity, obtain plaque, print on the nylon membrane, press the preceding method fixed nucleic acid.Nylon membrane earlier in the following solution of 200ml in 65 ℃ of prehybridizations 3 hours: 5x SSC (SSC is 0.15M sodium-chlor 0.01M Trisodium Citrate pH 7.2), (it is 0.2%W/VFicoll 400 to the spissated Denhardts solution of 10x, 0.2% calf serum, 0.2% polyvinyl chloride, 0.1% (W/V) SDS, the 50mcg/ml salmon sperm DNA.Then in 50ml solution in 65 ℃ of slight vibrations 2.5 hours so that hybridize, solutions employed have the composition identical with aforementioned solution, be preheated to 65 ℃, also added radioactive probe and cold competition probe solution.Oligonucleotide as the varient specific probe is BC 252 (can hybridize with the JL-5 varient) and BC 253 (can hybridize with the JL-2 varient).They all will activate (kination) and be labeled as γ in comprising the solution of following ingredients
32The oligonucleotide that P-ATP:100ng is to be marked, the spissated kinase buffer liquid of 3mcl 10x (comprising: 0.5M Tris-HCl pH7.6,0.1M MgCl
2, 50mM dithiothreitol (DTT), 1mM spermidine and 1mM EDTA pH8.0), 3mcl
32(Amersham International, 3000Ci/nmole is 10mCi/mcl) with 2mcl T for P-ATP
4Polynucleotide kinase (Boehringer Mannheim) is supplied 30mcl with sterilized water.With this mixing solutions in 37 ℃ of incubations after 30 minutes, in 5 minutes termination reactions of 95 ℃ of heating, add weight ratio then and be 100: 1 cold competition oligonucleotide probe, promptly every 100ng label probe will add the cold competition oligonucleotide of 10mcg, then mixture is added in the hybridization solution.After the hybridization, with film in the 100ml solution identical with the hybridization solution composition in 65 ℃ 30 minutes the washing once, again in the following solution of 100ml 65 ℃ wash 2 30 minutes: SSC 5x, 0.1%SDS.Make the film drying then, and film is exposed on x-ray film with intensifying screen.As MumpsVax during with this technology for detection, with the plaque of JL-5 varient specific b C 252 oligonucleotide hybridizations greatly more than with the plaque of BC 253 hybridization.When checking MJ05, though originally equate with the plaque base of two kinds of probe hybridizations, the plaque of hybridizing with BC253 is relative more more than the plaque of hybridizing with BC252.
6) pure Jeryl-Lynn strain isolated is carried out purifying
For obtaining pure JL-5 and JL-2 varient strain isolated, portion is indicated that titre is 4-6logTCID
50Commercialization MumpsVax (the lot number 92A06 of infectious unit, from Merck Sharp andDohme, leave Public Heath Laboratory Services in, Porton Down, Wiltshire, UK, registration number is Jeryl-Lynn Mumps strain:V93110585, on November 5th, 1993) sample carries out limiting dilution, then on 96 hole droplet plates with the inoculum size infected chicken embryo fibroblast of every approximately hole 0.1 infectious unit.With these plates 34 ℃ of incubations 11 days so that virus multiplication.There are 17 holes cytopathic effect to occur in whole 192 inoculation holes, viral growth has been described.These holes are inoculated in another batch CEF cell culture.Make the about 4.9 log TCID of titre
50Second pass filter 0.8 μ m filter membrane for thing, and with 42, centrifugal 1 hour of 000rpm is with virus precipitation 100mcl H
2O suspends again, with this determine the characteristic (identity) of isolating virus.(65 ℃ of incubations 30 minutes add the 3M sodium-acetate (pH4.5) of 1/10 volume again, add 2.5 times of volume of ethanol then for BoehringerMannheim, 40 units/mcl) to add 1mcl RNase inhibitor.-20 ℃ of precipitations are spent the night or-70 ℃ of precipitations 1 hour, then in Eppendorf desk-top (bench-up) whizzer 4 ℃ centrifugal 30 minutes, and will precipitate drying.
The gained viral RNA is carried out reverse transcription, method is to add the following mixture of 20mcl in described precipitation: 4mcl 5x core RT damping fluid (Bethesda Research Labs), 2mcl 10nM deoxynucleoside triphosphate mixture (Perkin Elmer, Cetus), 1mcl random primer N6 (Biolabs, 100mcg/ml), 11mcl H
2O, 1mcl MMLV reversed transcriptive enzyme (Bethsda Research Labs).37 ℃ of incubations are after 1 hour, and 95 ℃ are experienced 5 minutes to suppress reversed transcriptive enzyme.Get the mixture 10mcl after the heating, in final volume 100mcl,,, carry out 30 round-robin pcr amplifications by following program with primer NH30bis and NH31bis: 95 ℃ 1 minute, 60 ℃ 1 minute, 72 ℃ 1 minute.The gained fragment is carried out purifying with Magic Prep (Promega) by the experimental arrangement that producer provides.
6 only are inoculated in the Vero cell with the strain isolated of JL-5 probe reaction, obtain plaque.Be transferred on the nylon membrane again, make these films and BC 252 and BC253 oligonucleotide hybridization, described two kinds of oligonucleotide are all as described above by on the activation band
32The P mark.Hybridization is in 5x SSC, and oligonucleotide and the cold competition oligonucleotide of 10mcg with about 100ng tape label in final volume 50ml, carried out 2.5 hours in 65 ℃.Every kind of strain isolated has been tested about 200 plaques, and none and JL-2 probe (oligonucleotide BC253) react.All plaques are all reacted with BC 252.
One strain virus strain isolated is arranged, is derived from the 9H2A hole on the droplet plate, after further be accredited as SBB strain JL-1, it was passed for two generations again on the CEF cell.Last goes down to posterity after (count from original M umpsVax material and passed for four generations altogether), with this virus infection Vero cell, obtains plaque, be transferred on the nylon membrane, by be with through activation
32The oligonucleotide BC 252 of P mark and BC253 are hybridized and are tested.Detected more than 2000 plaque with JL-2 specific probe BC253, none and this probe reaction.With the plaque that oligonucleotide BC 252 has tested lesser amt, all are positive.To the 4th generation on the CEF cell the go down to posterity viral aggregation of the JL-1 strain that obtains in the thing directly check order, method is that virus is centrifugal, uses ethanol sedimentation, carries out reverse transcription with random primer by preceding method then.CDNA increases by PCR reaction, adopts oligonucleotide NH14 and BC265 to make primer, press follow procedure 30 circulations of increasing: 94 ℃ 1 minute, 60 ℃ 1 minute, 72 ℃ 1 minute.The gained dna fragmentation is gone up at Magic Prep post (Promega Biotech) and is carried out purifying by the explanation of producer, on Applied Biosystems 373A automatic sequencer, illustrate and use NH14 according to producer, BC265, NH30bis and NH31bis check order as primer.So obtain sequence as shown in Figure 1.Surprisingly, the intergenic region section of sequence between SH and HN gene coding region of the JL-5 strain isolated that obtains of this sequence and Afzal etc. has 6 sites different.
Second is accredited as 10H5F and only also checks order with the virus isolated strain of JL-5 probe reaction, method is with s-generation virus infection Vero cell, plaque is gone on the nylon membrane, carry out pcr amplification as primer, then order-checking with NH14 and BC265 oligonucleotide.So obtain a sequence, it is identical with the sequence of above-mentioned 9H2A, but has 6 bases different in SH-HN intergenic region section with disclosed JL-5 strain isolated sequence.
7) immunogenicity
Immunogenicity with the 6 gained JL-1 strains of monkey test implementation example.With the JL-1 of a kind of MJ11A42 by name virus freeze-dried products (the 4th generation of MumpsVax go down to posterity thing, obtaining after 6 days in 34 ℃ of growths on the CEF cell), with 4.2log TCID
50Dosage by one group of 4 cercopithecus aethiops of subcutaneous injection mode immunity.Every group of 4 monkey of three groups injected following material in addition: (a) concentration is 4.3log TCID
50
MumpsVax; (b) freeze-dried products MJ21A42, concentration is every dose of 4.3log TCID
50, gather in the crops after 9 days through 32 ℃ of growths, derived from the directly biography three generations of MumpsVax in the CEF cell, (c) freeze-dried products MJ05A42, concentration is every dose 4.3 TCID
50, be in the CEF cell in the viruses of 34 ℃ of growths results after 7 days, directly go down to posterity derived from three times of MumpsVax, this three generations is different from the thing that goes down to posterity of MJ21A42 goods.
Before the injection (the 0th day), and after the preventive vaccination the 28th and 42 day extracted blood specimen respectively, commercialization Enzygnost Anti-Parotitis Virus test kit (Behringwerke AG with Behring, Marburg, Germany), test the existence of the IgG antibody of material for anti parotitis virus in the blood specimen according to producer's suggestion.As shown in table 1, in described animal body, induce the material for anti parotitis antiviral antibody titre higher from pure JL-1 virus strain deutero-goods than other goods that comprise MumpsVax.In addition, these serum are carried out the twice serial dilution, make Test Virus, reduce in (reduction) test in plaque and test with MumpsVax.Those serum of having injected the animal of MJ11A42 reduce than the plaque number that other serum produces.
8) clinical study
Further in clinical trial,, test the JL-1 virus strain with about 15 months big seronegativity children.Use pure JL-1 strain as the parotitis composition, or the virus of using MumpsVax to obtain as on the CEF cell, directly going down to posterity as described in the embodiment 6, preparation measles, parotitis and rubella trivalent vaccine, and lyophilize.Also used M-M-R in the test
II vaccine commodity, it is produced by Merck and CoInc, can be from Merck Frossr Inc Kirkland, Quebec, Canada obtains; It contains Jeryl-Lynn (B-level) strain as the parotitis composition.
The titre of mumps virus is after measured in these three kinds of vaccine products: lot number is that the vaccine of MJR111D42 (containing pure JL-1 strain) is every dose of 4.5log TCID, lot number is that the vaccine of MJR121C42 (containing the MumpsVax virus that goes down to posterity) is every dose of 4.7logTCID, and lot number is the commercialization M-M-R of 80391OU
The II vaccine is every dose of 4.5logTCID.
Before the inoculation and inoculation back was extracted the blood specimen that is tried children on the 42nd day.
The existence of the IgG antibody of material for anti parotitis virus is tested with embodiment 6 described commercial kits.As shown in table 3, in three kinds of goods, pure JL-1 strain MMR is vaccine-induced to go out the highest frequence of seroconversion and the highest geometric mean titer.
Table 1
| In July, 93 injection | Parotitis ELISA test | |||||
| Explanation | Monkey | The 0th day | The 28th day | AMT (arithmetical mean titre) | The 42nd day | AMT (arithmetical mean titre) |
| Pure JL-1 (MJ11 A42) | KU542 544 547 551 | ＜230 ＜230 ＜230 ＜230 | 3300 4300 3300 2600 | 3375 | 2800 4100 3900 2200 | 3250 |
| (MJ21 A4) | 545 550 552 553 | ＜230 ＜230 ＜230 ＜230 | 770 2300 3300 3500 | 2468 | 510 2100 2500 2900 | 2003 |
| MSD92A06 MumpVax | 548 549 554 555 | ＜230 ＜230 ＜230 ＜230 | 3100 890 600 1700 | 1573 | 690 2300 490 1300 | 1195 |
| (MJ05A42) | 556 557 558 559 | ＜230 ＜230 ＜230 ＜230 | 1000 3100 ＜230 750 | ＜ 1270 | 1400 2600 ＜230 440 | ＜ 1168 |
Table 2
| Used oligonucleotide | |
| Code | Sequence (by 5 '-3 ' direction) |
| NH2 NH8 NH14 NH22 NH23 NH30 NH31 NH30bis NH31bis BC265 BC252 BC253 | GTA GCA CTG GAT GGA TCT GTG TTG TAT TGT GAT CC GTC GAT GAT CTC ATC AGG TAC CGG TAG AAG CTT GTC GAT GAT CTC ATC AGG TAC CGC TGA GGA TCC TCT GTG TTG TAT TGT GAT CC ATC TCC TAG GGT CGT AAC TTT GGA TGC AGC TTG TTC AAT CTC CTA GGG TCG TAA CGT CTC GTG A TTT GAA TGC AGC TTG TTC TAG CGT CCG ACA TTA TGA ATA GTT TCG AGG GCT CC ATA TCG CAC CGC CGT CTT ATA GTT AAT AGT C ATA CCG AAC CGC CGT ATT ATG GTT AAT GGT C |
Table 3
The experimenter of serum antibody response feminine gender is to serum conversion and the geometric mean titer (GMT) of mumps virus
| Vaccine | Time | The example number | Serum is changed routine number | Geometric mean titer |
| MUR111D42 MJR121C42 803910U | The inoculation before the inoculation after the 42nd day the inoculation before the inoculation after the 42nd day the inoculation before the inoculation after the 42nd day | 15 15 13 13 17 17 | 0 15 0 11 0 16 | - 1434 - 971 - 1247 |
The explanation of biomaterial preservation:
Following biomaterial is preserved in European cell culture preservation center.
| The classification name | Preservation date | Deposit number |
| The Jeryl-Lynn mumps virus | 1993.11.05 | ECACC：V93110585 |
Sequence table
(1) physical data:
(i) applicant: Smithkline Beecham Biologicals sa
(ii) invention exercise question: vaccine
(iii) sequence number: 13
(iv) address:
(A) addressee: Smithkline Beecham
Corporate Intellectual Property
(B) street: SB House L/5 Great West Road
(C) city: Brentford
(D) state: Middlesex
(E) country: United Kingdom
(F) postcode: TW8 9BD
(the v) readable form of computer:
(A) media type: Floppy disk
(B) computer: IBM PC compatible type
(C) operating system: PC-DOS/MS-DOS
(D) software: PatentIn Release#1.0, Version#1.25
(vi) application materials:
(A) application number:
(B) applying date:
(C) classification number:
(viii) agency/proxy's data:
(A) name: Dalton, Marcus JW
(ix) telecommunication data:
(A) phone: 44 181 975 4093
(B) fax: 44 181 975 3688
(2) data of SEQ ID NO:1:
(i) sequence signature:
(A) length: 393bp
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: DNA (genome)
(iii) false plan the: not
(iv) antisense: not
(vi) originate:
(A) organism: mumps virus
(B) strain system: Jeryl lynn-1
(xi) sequence explanation: SEQ ID NO:1
TGAATCTCCT AGGGTCGTAA CGTCTCGTGA CCCTGCCGTC GCACTATGCC GGCAATCCAA 60
CCTCCCTTAT ACCTAACATT TCTAGTGCTA ATCCTTCTCT ATCTCATCAT AACCCTGTAT 120
GTCTGGACTA TATTGACTAT TAACTATAAG ACGGCGGTGC GATATGCAGC ACTGTACCAG 180
CGATCCTTCT CTCGCTGGGG TTTTGATCAC TCACTCTAGA AAGATCCCCA ATTAGGACAA 240
GTCCCGATCC GTCACGCTAG AACAAGCTGC ATTCAAATGA AGCTGTGCTA CCATGAGACA 300
TAAAGAAAAA AGCAAGCCAG AACAAACCTA GGATCATAAC ACAATACAGA ATATTAGCTG 360
CTATCACAAC TGTGTTCCGG CCACTAAGAA AAT 393
(2) data of SEQ ID NO:2
(i) sequence signature:
(A) length: 15bp
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: DNA (genome)
(iii) false plan the: not
(iv) antisense: not
(vi) originate:
(A) organism: nh2
(xi) sequence explanation: SEQ ID NO:2
GTAGCACTGG ATGGA 15
(2) data of SEQ ID NO:3
(i) sequence signature:
(A) length: 20bp
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: DNA (genome)
(vi) originate:
(A) organism: NH8
(xi) sequence explanation SEQ ID NO:3
TCTGTGTTGT ATTGTGATCC 20
(2) data of SEQ ID NO:4:
(i) sequence signature:
(A) length: 21bp
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: DNA (genomic)
(vi) originate:
(A) organism: NH14
(xi) sequence explanation: SEQ ID NO:4
GTCGATGATC TCATCAGGTAC 21
(2) data of SEQ ID NO:5
(i) sequence signature:
(A) length: 33bp
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: DNA (genomic)
(vi) originate:
(A) organism: NH22
(xi) sequence explanation: SEQ ID NO:5
CGGTAGAAGC TTGTCGATGA TCTCATCAGG TAC 33
(2) data of SEQ ID NO:6
(i) sequence signature:
(A) length: 32bp
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: DNA (genome)
(vi) originate:
(A) organism: NH23
(xi) sequence explanation SEQ ID NO:6
CGCTGAGGAT CCTCTGTGTT GTATTGTGAT CC 32
(2) data of SEQ ID NO:7
(i) sequence signature:
(A) length: 18bp
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: DNA (genome)
(vi) originate:
(A) organism: NH30
(xi) sequence explanation: SEQ ID NO:7
ATCTCCTAGG GTCGTAAC 18
(2) data of SEQ ID NO:8:
(i) sequence signature:
(A) length: 18bp:
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: DNA (genome)
(vi) originate:
(A) organism: NH31
(xi) sequence explanation SEQ ID NO:8
TTTGGATGCA GCTTGTTC 18
(2) data of SEQ ID NO:9:
(i) sequence signature:
(A) length: 28bp
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: DNA (genome)
(vi) originate:
(A) organism: NH 30 BIS
(xi) sequence description: SEQ ID NO:9
AATCTCCTAG GGTCGTAACG TCTCGTGA 28
(2) data of SEQID NO:10:
(i) sequence signature:
(A) length: 24bp
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: DNA (genome)
(vi) originate:
(A) organism: NH 31 BIS
(xi) sequence explanation: SEQ ID NO:10
TTTGAATGCA GCTTGTTCTA GCGT 24
(2) data of SEQ ID NO:11
(i) sequence signature:
(A) length: 29bp
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: DNA (genome)
(vi) originate:
(A) organism: BC265
(xi) sequence explanation SEQ ID NO:11
CCGACATTAT GAATAGTTTC GAGGGCTCC 29
(2) data of SEQ ID NO:12:
(i) sequence signature:
(A) length: 31bp
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: DNA (genome)
(vi) originate:
(A) organism: bc252
(xi) sequence explanation: SEQ ID NO:12
ATATCGCACC GCCGTCTTAT AGTTAATAGT C 31
(2) data of SEQ ID NO:13
(i) sequence signature:
(A) length: 31bp
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: DNA (genome)
(vi) originate:
(A) organism: BC253
(xi) sequence explanation SEQ ID NO:13
ATACCGAACC GCCGTATTAT GGTTAATGGT C 32
| Applicant or attorney docket PDXN5236D2 | International application no PCT/EP94/03801 |
Explanation about microbial preservation
(detailed rules and regulations 13 two)
| A. to specification sheets 8 Page or leaf, the 8 The explanation of the described little principal goods of row, | |
| Other are deposited in in the supplementary page B preservation item | |
| The title PUBLIC HEALTH LABORATORY SERVICES of depositary institution | |
| Depositary institution address (comprising postcode and name of the country) Porton Down, Wiltshire, United Kingdom (Britain) | |
| Preservation date on November 5th, 1993 | Deposit number Jeryl-Lynn mumps virus strain: V93110585 |
| C. have supplementary page in supplementary notes (in case of necessity) this column | |
| With regard to the designated state of Europe patent application, when authorizing the bulletin of Europe patent, up to this application out of court or when recalling till, can obtain the sample of the microorganism of this preservation. this sample is only provided the specified expert of people to the request sample. | |
| D. this explanation is done (if explanation is not done for all designated states) for following designated state | |
| E. remark additionally (in case of necessity) | |
| Following explanation will be subsequently provides (write out the classification of explanation, for example: the numbering of preservation ") to international office | |
PCT/RO/134 shows (in July, 1992)
Claims (6)
1, a kind of attenuation Jeryl-Lynn mumps virus strain is characterized in that the N-end of SH gene and HN gene comprises nucleotide sequence shown in Figure 1.
2, the purposes of attenuation Jeryl-Lynn mumps virus strain in the preparation vaccine, the strain of wherein said attenuation Jeryl-Lynn mumps virus is characterised in that the N-end of SH gene and HN gene comprises nucleotide sequence shown in Figure 1.
3, the purposes of claim 2, the strain of wherein said attenuation Jeryl-Lynn mumps virus are the immunogenicity Jeryl-Lynn strain isolateds of homogeneous basically.
4. claim 2 or 3 purposes, wherein said vaccine is a Mumps Vaccine.
5, claim 2 or 3 purposes, wherein said vaccine is a combined vaccine, this combined vaccine also contains the measles or the rubella virus of one or more attenuated measles virus or attenuation rubella virus or deactivation, or these viral subunits.
6,, also contain of the protection of a kind of medicament in addition to provide anti-varicella or banded acne rash to infect as the described vaccine of one of claim 2-5.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB939323820A GB9323820D0 (en) | 1993-11-19 | 1993-11-19 | Vaccine |
| GB9323820.2 | 1993-11-19 | ||
| GB9406480.5 | 1994-03-31 |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN94194807A Division CN1150819A (en) | 1993-11-19 | 1994-11-15 | Vaccine against mumps containing jeryl-Lynn virus strain |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101092609A true CN101092609A (en) | 2007-12-26 |
Family
ID=10745389
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200710103266 Pending CN101092609A (en) | 1993-11-19 | 1994-11-15 | Vaccine against mumps containing a JERYL-LYNN virus strain |
| CN 200410003915 Expired - Lifetime CN1763181B (en) | 1993-11-19 | 1994-11-15 | Vaccine against mumps containing a JERYL-LYNN virus strain |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200410003915 Expired - Lifetime CN1763181B (en) | 1993-11-19 | 1994-11-15 | Vaccine against mumps containing a JERYL-LYNN virus strain |
Country Status (3)
| Country | Link |
|---|---|
| CN (2) | CN101092609A (en) |
| GB (1) | GB9323820D0 (en) |
| ZA (1) | ZA949128B (en) |
-
1993
- 1993-11-19 GB GB939323820A patent/GB9323820D0/en active Pending
-
1994
- 1994-11-15 CN CN 200710103266 patent/CN101092609A/en active Pending
- 1994-11-15 CN CN 200410003915 patent/CN1763181B/en not_active Expired - Lifetime
- 1994-11-17 ZA ZA949128A patent/ZA949128B/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| CN1763181B (en) | 2010-12-01 |
| ZA949128B (en) | 1995-11-10 |
| CN1763181A (en) | 2006-04-26 |
| GB9323820D0 (en) | 1994-01-05 |
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| PB01 | Publication | ||
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| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
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Open date: 20071226 |