JP3037756B2 - Vaccine against mumps containing jeryl-lynn virus strain - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Mumps is essentially a childhood disease that usually appears with only a few signs. However, in some cases, the clinical consequences of mumps infection are severe. For example, in the UK, mumps is the most common cause of meningoencephalitis in children under the age of 15, and is a cause of permanent childhood sensory paralysis. Although 30-40% of spontaneous mumps infections are asymptomatic, salivary gland failure is felt uncomfortable, and mumps causes miscarriage and male orchitis in the first three months and complications of the above nerves in the adult population. The potential cause has prompted the application of mass vaccine programs in many countries.

The mumps virus belonging to the family Paramyxoviridae is composed of single-stranded genomic RNA having a negative sense, and the order of the genes is 3'NPFMSH-HN-L.
5 '(N = nucleocapsid protein, P = phosphoprotein, M
= Matrix protein, F = fusion protein, SH = small hydrophobic protein that may be expressed, HN = hemagglutinin neuraminidase, L = large protein). Of the various mumps strains, Jeryl-Lin (Je
ryl-Lynn) (B-level) is a live attenuated variant characterized by sequence analysis of the F, P, HN, M genes.

Until recently, two mumps viruses had been approved for vaccination against mumps. These are Urabe Am9 and Jeryl-Lin. However, the Ulabe strain was disqualified in September 1992 due to the report of unacceptable levels of side effects [European Journal of Pediatrics (Euro)
pean Journal of Pediatrics) (1993, 152: 387
page]. The Jeryl-Lin strain has been marketed under the trade name "MumpsVax" by Merck Sharp and Dohme for many years. The Jeryl-Lin strain was obtained from a clinical sample of a mumps patient by amniotic inoculation into fertilized chicken eggs (Proc. Sosa. Expermental Biolo Medi (Proc. Soc. Exptl. Biol. .Me
d.) Volume 123 (3) (1966)).

Afzal et al. Recently reported that the Jeryl-Lin strain used in the mumps vaccine in the UK is in fact a mixture of two viruses, designated JL-2 and JL-5. (Journal of
General virology (J.Gen.Virology) 1993
74, 917).

Takeuchi et al., Virology
(1991) 181: 364-366 report that substantial nucleotide sequence changes at the SH gene level may exist in different mumps strains.

Afzal et al. Now manufacture the commercially available vaccine "Mumpsbucks" under carefully controlled conditions, including cell banking and passaging restrictions that are expected to maintain the ratio of the two strains between batches. Emphasize that. However, there is no guarantee that this balance between the two strains will be maintained if the Jeryl-Lin strain is further passaged. Moreover, it is difficult to evaluate the proportion of the two strains in any batch of vaccine.

We surprisingly found that Afzal et al.'S JL-2
And additional isolates different from both JL-5 were identified. The difference is that the SH gene and its surrounding region, more specifically, the 3 'side is linked to the SH coding sequence and the 5' side is
The untranslated intercistronic region leading to the HN gene was determined by nucleotide sequence analysis. In clinical trials, this isolate has a higher zero conversion (zero conversion).
sion) and has the highest mumps antibody average titer among commercially available mumps vaccines.

Accordingly, we provide an attenuated jeryl-phosphorus mumps strain comprising the nucleotide sequence shown in FIG. This sequence encodes the N terminus of the SH and HN genes. This strain is referred to herein as SBB JL-1.

In FIG. 1, the SH gene coding region and SH-
Figure 3 shows the cDNA sequence of the JL-1 mumps virus isolate over the HN intergenic region.

Further, the present invention provides a method for producing substantially uniform immunogenic geryl-
A mumps vaccine comprising a phosphorus isolate is provided.

Substantially homogenous means that the isolate has a different jeryl-phosphorus isolate than that defined by the sequence of the region shown above.
It is not contaminated by more than 10%, preferably another geryl- as defined by the sequence of the region shown above.
It means that the contamination of the phosphorus isolate is less than 5%, most preferably less than 1%. In a preferred embodiment of the invention, the vaccine comprises a pure and homogeneous jeryl-phosphorus isolate, i.e. free of other jeryl-phosphorus mumps isolates that are different in the region shown in FIG. .

In one embodiment of the present invention, there is provided a vaccine comprising homogeneous SBB JL-1 free of JL-2.

Pure isolates provide a product without potential batch-to-batch variability and easy to meet consistent quality guidelines.

Commercially available mumps bucks were used for chicken embryo fibroblasts (CE
By passage in F) and then selecting a pure culture by limiting dilution and testing of the resulting isolate or by isolation of individual plaques, it is also possible to obtain a homogeneous geryl-phosphon according to the invention. Good. Other suitable cell lines include Vero cells and MRC5 cells. At this time, there is a need for a method that can be used to detect a small population of known variant viruses in the population. Such test methods include the Maprec assay proposed by Chumakov for attenuated poliovirus (WO 92/09758 and PNAS 1991, 88: 199-203), and the direct sequence of viral plaques. Determination and differential hybridization of viral plaques.

Advantageously, the vaccine according to the invention may comprise other components such as attenuated measles virus and / or attenuated rubella virus, their killed virus or subunits, in order to provide protection against measles and / or rubella infection. It contains. Trivalent mumps, measles and rubella vaccines are well known in the art, and the mumps isolates of the invention will be formulated into trivalent vaccines in a manner similar to those vaccines already used.
Additionally or alternatively, the vaccines of the present invention contain live varicella-zoster attenuated virus to provide protection against chickenpox or shingles. May be. Preferred specific examples of varicella-zoster virus are
Andre FE, Postgraduate MED J. (1985) No. 61
Volume (Supplement 4), pp. 113-120 or Veska Tea
ri T.) et al., Acta paedi scandi
Ediatr. Scand.) 80: 1051-1057 (1991). Preferably,
The vaccine of the present invention is tetravalent and provides protection against mumps, rubella, measles and varicella-zoster virus.

The invention also provides a method for producing a complete virus vaccine, for example, by lyophilizing the virus in the presence of a suitable stabilizing agent or by mixing the virus strain of the invention with a suitable carrier or adjuvant. . It may also be advantageous to formulate the virus strains of the present invention in liposomes or carrier particles. Alternatively or additionally, 3de-O-acyl monophosphoryl lipid A
(Manufactured by Ribi Immunochem) or saponin derivative QS21 (Cambridge Biotech (Ca)
An immunostimulant such as mbridge Biotech) may be included in the formulation.

In a further aspect, the present invention provides a method of treating mumps infection in a human comprising administering to a human subject in need thereof an immunologically effective amount of the vaccine of the present invention.

The method of administration of the vaccine of the present invention may be by any suitable route that delivers an immunoprotective amount of the strain and other immunogenic components of the vaccine to the subject. However, preferably, the vaccine is administered parenterally by the intramuscular or deep subcutaneous route. If desired, oral administration or other parenteral routes, ie, other modes of administration, such as intradermal, intranasal or intravenous, may be used.

Suitable immunoprotective and non-toxic amounts of such vaccines can be readily determined by those skilled in the art. That is, a suitable immunoprotective and non-toxic amount of the strain of the present invention contained in the vaccine of the present invention may be within the effective amount of the antigen in a conventional whole virus vaccine. However, particular dosage levels for particular patients may include patient age, general health, gender and diet; timing of administration; route of administration; synergy with any other drug administered; Depends on various factors, including the degree of Of course, if necessary, the administration can be repeated at appropriate intervals. Typically, in monovalent vaccine administration,
At least 3.7logTCID50, more usually 4.5logTCID50
Will be included in a single dose. In the trivalent mumps, measles and rubella vaccine, the mumps component will be present at about 4.8 log TCID50 and will offset the interference of the other two viral components.

Example 1) Primary sequence of SH gene Commercially available mumpsbacks virus has about 3.0 logTCID50.
DMEM containing 0.5% fetal calf serum using inoculum
Bio-rich medium (dMEM Biorich medium) (50/50 v
/ v) confluent monolayer Vero grown in 25 cm ² flasks
Passaged in cells. Seven days after culturing at 34 ° C., the infected cells were collected, and collected by Ferre and Garduno.
(Nucleic Acid R
esearch) 1989, 17: 2141).
Was extracted into 100 mcl of water and treated with diethyl pyrocarbonate at 100 ° C. for 5 minutes. 5 mcl of this extract was reverse transcribed by adding the following reagents: RNAsin40 unit (Boehringer Mannhei, Germany)
m), 4mcl of 5X concentrated reverse transcriptase buffer (Bethesda Research Laboratories)
s.) 4mM deoxynucleotide triphosphate mixture 2mcl, 10pmole NH2 oligonucleotide primer, 1mcl Moloney murine leukemia virus (MMLV) reverse transcriptase (Bethesda Laboratories, 200 units per mcl) and Water for a final volume of 20 mcl. Oligonucleotide NH2 has homology to the F gene of the mumps virus, Urabe strain. Mix at 37 ° C
Incubate for 45 minutes, then heat at 95 ° C. for 5 minutes. The oligonucleotides NH8 and NH14 were then used as primers, and an additional 1000
The cDNA was amplified by two consecutive rounds of the PCR reaction using the doubling dilution as the second round starting material. Each PCR
The round consisted of 25 cycles of 1 minute at 94 ° C, 1 minute at 53 ° C, and 1 minute at 72 ° C. NH8 or NH14 as fluorodideoxynucleotide terminator and primer
And then analysis of the product with an automated sequencer from Applied Systems (373A DNA sequencer) according to the manufacturer's protocol and recommended methods, followed by primers The PCR product corresponding to the SH gene was sequenced from both directions. Unknown bases were observed at many positions in the sequence and were confirmed on both strands. The resulting sequence is described in Takeuchi et al. (Virology 1991) for Jeryl-Lin.
181: 364-366) is 17 out of 361 bases.
Differed in the number of bases and included four unassigned bases. Further, the sequence can be found in Afzal et al. (J. Gen. Virol. 1993, Vol. 74: 917-920) for the JL-5 isolate.
9) out of the 319 bases from the part of the sequence
It contained four unassigned bases. Not pre-passaged in Vero cells but using random primers for viral RNA
Is reverse transcribed into cDNA, followed by oligonucleotide NH30
Unknown bases were also observed when the same region was directly sequenced from 4.0 to 5.0 log TCID50 mumps backs recovered by ultracentrifugation after PCR amplification using bis and NH31bis as primers.

2) Cloning of SH gene Vero cells were infected with Mumpsbacks virus, and total RNA was prepared as described above. RNA was reverse transcribed using random primers and PCR amplification was performed using oligonucleotides NH22 and NH23 as primers (to facilitate cloning of the amplified DNA fragment, NH22 is a Hind III restriction site in the primers). And NH23 has a BamHI restriction site in the primer). The amplified DNA was cleaved with Hind III and BamHI endonuclease and cloned into vector pUC9. Eleven clones containing the insert corresponding to the mumps SH gene region were recovered. All eleven had sequences corresponding to the sequence of Takeuchi et al. (Cited above). In addition, 5 clones had Dde I restriction sites not found in the other 6 clones. The insert corresponding to the JL-5 sequence was not recovered. This result and the unknown base from sequencing were determined by JF defined by Afzal et al.
-2 variant virus formed a substantial or detectable part of the mumpsbucks virus.

3) Direct passage from Mumpsbacks Mumpsbacks virus was further passaged directly in chicken embryo fibroblasts (CEF). At the third passage, virus was recovered from five different lots, including lot MJ05, used to prepare lyophilized sample for animal injection, MJ05A42. Infect Vero cells using the five virus preparations, collect RNA, and initiate the reverse transcription reaction using random primers.
Standards were prepared for DNA sequencing by amplification with NH18 and NH14. All five lots showed the same sequence as JL-2 (Takeuchi et al.), With no unknown bases.

To investigate this further, direct sequencing on viral plaques was used as described above. The five lots were used to infect Vero cell culture to obtain plaques,
NH30bis and NH31bis were processed for sequencing using as primers. Thirteen out of a total of 26 plaques for the five lots showed the same sequence as Takeuchi et al. For JL-2, and five showed two bases at positions 270 and 279 as shown in FIG. Very similar except for differences, eight plaques showed sequences with unknown bases indicating a mixture of viruses.

4) Direct sequencing of virus plaques After removing the medium and washing with serum-free dMEM BioRich 50: 50 / medium (Biorich), putatively 0.5%
Three confluent monolayers of Vero cells were infected in 5 cm Petri dishes using three mumps Bax dilutions containing 100, 50 and 10 virus particles per ml. 34 ℃
For 30 minutes to adsorb the virus. Next, contains 0.5% FCS
Cells were covered with 5 ml overlay agar containing 2.5 ml dMEM bio-rich medium and 2.5 ml 3% (w / v) low gelling point agarose and kept at 42 ° C. After solidification, contains 0.5% fetal calf serum d
The agar layer was covered with 3 ml of MEM bio-rich medium and incubated at 34 ° C. After 7 days of incubation, the surface liquid medium was removed, virus plaques were visualized by adding 0.03% (w / v) neutral red solution and allowing it to diffuse for 1 hour. The liquid and agar were then removed and the dried nylon filter pressed against the bottom of the dish with a finger. Then remove the filter with a few drops of 2X
Moistened with SSC and lifted. 5 on filter paper impregnated with 2XSSC
The virus was fixed to the filter by placing the filter on a filter paper impregnated with 2XSSC, 0.2% (w / v) sodium dodecyl sulfate for 30 minutes and then for 30 minutes.
Exposure to UV light for 3-4 minutes. Cut out 20 individual plaques from the nylon filter and filter out 10 pieces of membrane.
It was immersed in 0 mcl of water, added with 1 mcl of RNAsin (Boehringer Mannheim, 40 units), and then heated at 65 ° C for 30 minutes. 100 mcl of the liquid was transferred to a new tube, and the nucleic acid was precipitated by adding 10 mcl of 3M sodium acetate followed by 250 mcl of ethanol. The mixture was kept at -20 ° C overnight or at -70 ° C for 1 hour and then centrifuged. The pellet was then dried. Then, reverse transcription was performed by adding the following solution to the pellet: 5X
Concentrated reverse transcriptase buffer 4 mcl (Bethesda Research Laboratories), 0.1 M dithiothreitol 2 mcl, deoxynucleotide triphosphate mixture 1 mcl (Perkin Elmer-Cetus, 10 mM concentration) N
6 random primer oligonucleotide 1mcl (New England Biolabs)
s), 100 mcg per ml) and 11 mcl of water. Then, 1 mcl of MMLV reverse transcriptase was added and the mixture was incubated at 37 ° C. for 1 hour, then at 95 ° C. for 5 minutes. Next, 10 mcl of the above mixture was taken, and 500 ng of each of the oligonucleotide primers HN30bis and NH31bis, PCR buffer and 1 mcl of Stoffel DNA polymerase (Stoffel DNA polymerase) were used.
DNA polymerase) (Perkin-Elmer-Cetus,
10 μg in a final volume of 100 mcl), and add
The cDNA was PCR amplified by heating the mixture at 30 ° C. for 1 minute followed by 72 ° C. for 1 minute. MagicPrep kit according to supplier's instructions
(A71 manufactured by Promega Biotech)
The resulting fragment was purified using (70). NH30bi
s or NH31bis as a primer, using a fluorodideoxynucleotide terminator, using the supplier's method and reactants.
Sequencing was performed after further asymmetric PCR amplification by non-radiolabeling on a Biosystems (373A) automated sequencer. Of the 20 plaques from Mumpsbacks, 19 differ from the Takeuchi et al. (Cited above) JL-2 sequence by 11 out of 275 bases and the Afzal et al. (Cited above) JL-5 sequence. And two bases were different. One plaque showed an unknown base sequence. This result suggested that mumpsbucks contained a different variant in this region than the predominant JL-5 strain found by Afzal et al. The two bases that differ between JL-5 and the sequenced plaque are positions 270 and 279, which are located in the intergenic region between the SH coding sequence and the HN coding sequence.

5) Plaque Hybridization In order to more directly determine the proportion of JL-5 and JL-2 type variants in Mumpsbucks and the resulting cultures, a plaque hybridization method was used. Vero cell monolayers were infected with mumps bucks virus and passaged lot MJ105 virus to obtain plaques, which were then loaded on nylon membranes and the nucleic acids fixed as described above. The filters were prehybridized in 200 ml of the following solution at 65 ° C. for 3 hours: 5 × SSC (SSC is 0.15 M sodium chloride, 0.01 M sodium citrate, pH 7.2), a 10 × concentration of Denhardt's solution (10 × concentration) Denhart's solution is 0.2% w / v Ficoll 400, 0.2% bovine serum albumin, 0.2% polyvinyl chloride), 0.1% (w / v) sodium dodecyl sulfate, 50 mcg per ml of salmon / sperm DNA. Then, at 50 ° C. in 50 ml of a solution of the same composition as above, pre-warmed to 65 ° C. and added with a radioactive probe and a cold competition probe.
For 2.5 hours with gentle stirring. Oligonucleotides used as variant-specific probes were BC252 hybridizing to JL-5 variant and BC253 hybridizing to JL-2 variant. Oligonucleotides were labeled with gamma ³² P-ATP by transphosphorylation in a solution of the following composition: 100 ng of oligonucleotide to be labeled, 1
0x concentrated kinase buffer 3mcl (10X concentrated kinase buffer is
0.5 M Tris-HCl, pH 7.6, 0.1 M MgCl ₂ , 50 mM deionized water,
1mM spermidine and 1mM EDTA, including pH 8.0), ³² P
-ATP3mcl (Amersham International (Amers
Ham International) and 2 mcl of T4 polynucleotide kinase (Boehringer Mannheim) and 30 mcl with sterile water. The mixture was incubated at 37 ° C for 30 minutes and stopped by heating at 95 ° C for 5 minutes. The cold competition oligonucleotide was then added to 100
It was added at a ratio of 1 to 1 (w / w). That is, 10 mcg of cold competition oligonucleotide was added for every 100 ng of labeled probe before adding the mixture to the hybridization solution. After hybridization, in a 100 ml solution having the same composition as the hybridization solution, at 65 ° C. for 30 minutes (once), and then 100 ml of the following composition
The filters were washed at 65 ° C in a solution of
SC 5X, 0.1% sodium dodecyl sulfate 0.1%. Then
The filters were dried and exposed to X-ray film using an intensifying screen. When mumpsbacks were tested by this method, a large excess of plaques compared to BC253 resulted in JL-
It hybridized with the five variant specific oligonucleotide BC252. When lot MJ105 was tested, approximately the same number of plaques hybridized to both probes, but more plaques hybridized to BC253 than BC252.

6) Isolation of pure jeryl-phosphorus isolates To recover pure isolates of JL-5 and JL-2 variants, commercial mumpsbucks with a constant titer (4-6 log TCID50 infectious units) ( Lot 92A06, obtained from Merck Sharp and Dome, on November 5, 1993 under accession number Jeryl-Lyn Mumps strain: V93110585, Porton Down, Wiltshire, UK
orton Down) (deposited with Public Health Laboratory Services) and used to inoculate 0.1 infectious units per well and inoculate in 96-well microtiter plates. Infect chicken and embryo fibroblasts. Plates were incubated at 34 ° C. for 11 days to grow virus. Seventeen of the 192 inoculum showed cytopathic effects indicative of virus growth and were used to further inoculate CEF cell cultures. The identity of the isolated virus was determined based on this second passage. The virus preparation was filtered through a 0.8 μm filter,
Centrifuge at 2,000 rpm for 1 hour and remove the virus pellet to 100 mc
When resuspended in 1 liter of water, it had a titer of about 4.9 log TCID50. 1mcl RNase inhibitor (Boehringer
Mannheim, 40 units per mcl) was added and the mixture was incubated at 65 ° C. for 30 minutes, then 1/10
A volume of 3M sodium acetate (pH 4.5) and 2.5 volumes of ethanol were added. The material was precipitated overnight at -20 ° C or 1 hour at -70 ° C and then centrifuged at 4 ° C for 30 minutes in an Eppendorf bench-top centrifuge to dry the pellet.

The recovered viral RNA was reverse transcribed by adding 20 mcl of the following mixture to the pellet: 5 x core RT buffer, 4 mcl (Bethesda Research Laboratories), 2 ncl of a 10 nM deoxynucleotide triphosphate mixture. Perkin-Elmer-Cetus), random primer N6 1mcl (Biolabs, 100mcl / ml), water 11
mcl, 1mcl of MMLV reverse transcriptase (Bethesda Research Laboratories). The mixture is allowed to stand at 37 ° C. for 1 hour, then
Incubation at 5 ° C for 5 minutes inhibited reverse transcriptase. Then, 10 mcl of the heated mixture was added to a final volume of 100 mcl.
PCR amplification was carried out using NH30bis and NH31bis as follows, by performing the following heating program 30 times: 95 ° C 1
Min, 60 ° C for 1 minute, 72 ° C for 1 minute. The resulting fragment was purified by magic prep (Promega) according to the manufacturer's protocol.

Vero cells were inoculated with 6 isolates that only reacted with the JL-5 probe to obtain plaques. These were transferred to a nylon membrane, and the membrane was hybridized with both the oligonucleotide BC252 and the oligonucleotide BC253 labeled with 32 P by the above-described phosphoryl transfer. Hybridization was performed in a volume of 50 ml of 5XSSC with about 100 ng of labeled oligonucleotide and 10 mcg of cold competition oligonucleotide for 2.5 hours at 65 ° C. When about 200 plaques were tested for each isolate, none reacted with the JL-2 probe (oligonucleotide BC253). All plaques reacted with BC252.

One virus isolate from well 9H2A of the microtiter plate and further identified as strain JL-1 was passaged twice more in CEF cells. At the last passage (4 passages from the original mumpsbucks), the virus was used to infect Vero cells to obtain plaques. These were transferred to a nylon membrane and tested by hybridization with oligonucleotides BC252 and BC253, which were labeled with 32P by transphosphorylation. When more than 2000 plaques were tested for the JL-2 specific probe BC253, none reacted. When fewer plaques were tested for oligonucleotide BC252, all gave a positive response. The virus was centrifuged, precipitated with ethanol, and then reverse transcribed using the random primers described above, to directly sequence the virus pool of the JL-1 strain recovered at the fourth passage. Oligonucleotides NH14 and BC
Using 265 as a primer, the cDNA was amplified by PCR using the following temperature program: 94 ° C for 1 minute, 60 ° C for 1 minute.
Min, 30 cycles of 1 minute at 72 ° C. Purify the resulting DNA with a Magic Prep column (Promega Biotech) according to the supplier's instructions and then use the oligonucleotide NH1
4, sequenced on an Applied Biosystems 373A automated sequencer using BC265, NH30bis and NH31bis as primers according to the manufacturer's instructions. The sequence shown in FIG. 1 was obtained. Surprisingly, this sequence differs from the sequence obtained by Afzal et al. For JL-5 at six positions in the intergenic region between the SH and HN coding regions, as shown in FIG. Thus, the JL-1 isolate represents a further variant present in the Mumpsbucks preparation.

Vero cells were infected with the second generation virus, the plaques were transferred to a nylon membrane, sequenced after performing PCR amplification using NH14 and BC265 oligonucleotides as primers, with 10H5F that reacts only with JL-5. The second virus isolate identified was sequenced. It shows the same sequence as that for 9H2A above and differs from the published JL-5 isolate by 6 bases in the SH-HN intergenic region.

7) Immunogenicity The immunogenicity of the JL-1 strain obtained in Example 6 was tested in monkeys. A lyophilized JL-1 preparation called MJ11A42, passaged four times from Mumpsbucks and grown in CEF cells at 34 ° C. for 6 days, was injected subcutaneously into a group of four African green monkeys at a dose of 4.21 log TCID50. For immunization. In three additional groups of four monkeys, (a) a concentration of 4.
(B) Concentration of 4.3 logTCID derived from a virus harvested after 9 days at 32 ° C., which was directly passaged through CEF cells three times with mumps bucks.
50 (per single dose) freeze-dried preparation MJ21A42; (c)
A freeze-dried preparation MJ with a concentration of 4.2 logTCID50 (per dose) derived from a virus that was harvested after 7 days at 34 ° C and was directly passaged through mumps bucks three times with CEF cells.
05A42 (different from MJ21A42) was injected.

Blood samples were taken on day 0, day 28 and day 42 post-vaccination and were sent to Behring (Behringwerke AG, Marburg, Germany).
Germany)) and Enzygnost Anti-Parrotitis virus kit (Enzygnost Anti-
Parotitis Virus kit was used as described by the supplier to test for the presence of IgG antibodies to mumps virus. As shown in Table 1, the preparation derived from the pure JL-1 strain induces a higher titer of anti-mumps virus antibody in animals than other preparations including Mumpsbucks. These sera were further serially diluted 2-fold and tested in a plaque reduction assay using Mumpsbucks as the test virus. Sera from animals injected with lot MJ11A42, on average, reduced plaque numbers when compared to other sera.

8) Clinical study The JL-1 strain was further tested on seronegative 15-month-old children. Trivalent measles using either the pure JL-1 strain as a mumps component or using the mumps virus obtained by direct passage of mumps backs in CEF cells as in Example 6 above. , Mumps and rubella vaccines were prescribed and lyophilized.
M-M-R ^R II vaccine manufactured by Merck and Co Inc and commercially available from Merck Frossr Inc, Kirkland, Quebec, Canada Was also included in the trial. It contains a jeryl-phosphorus strain (B-level) as a mumps component.

The mumps virus titer in the three vaccine preparations was determined by lot number MJR111D42 containing the pure JL-1 strain.
4.5 vaccine TCID dose for the vaccine, lot number MJR1 containing the passaged mumpsbacks virus
Vaccine dose of 4.7logTCID for the 21C42, for the commercial ^M-M-R R II vaccine lot number 80391OU 4.
It was determined to be a dose of 5 log TCID.

Blood samples were obtained from children before vaccination and 42 days after vaccination.

The presence of IgG antibodies against mumps virus was tested using the same commercial kit as described in Example 6. As shown in Table 3, the MMR vaccine using the pure JL-1 strain induced the highest seroconversion rate and showed the highest geometric mean titer among the three preparations.

Sequence Listing (1) General Information: (i) Applicant: SmithKline Beecham Biologicals Societe Anonym (ii) Title of Invention: Vaccine (iii) Number of Sequences: 13 (iv) Contact: ( A) Address: SmithKline Beecham Corporate Intellectual Property (B) Street: SB House El / 5 Great West Road (C) City: Brenford (D) State: Middle Sex (E) Country: United Kingdom (F) ZIP: TW8 9BD (v) Computer readable form: (A) Media type: Floppy disk (B) Computer: IBM PC compatible (C) Operating system: PC-DOS / MS-DOS (D) Software: PatentIn Release # 1.0, Version # 1.25 (vi) Current application data: (A) Application number: (B) Application Date: (C) Classification: (viii) Information of agents, etc .: (A) Name: Dalton, Marcus J. D. Brux (ix) Telecommunications information: (A) Phone: 44 181 975 4093 (B) Fax: 44 181 975 3688 (2) Information on SEQ ID NO: 1 (i) Sequence characteristics: (A) Sequence length: 393 base pairs (B) Sequence type: nucleic acid (C) Number of strands: 1 strand (D) Topology: linear (ii) Sequence type: DNA (genome) (iii) Hypothetical: None (iv) Antisense: None (vi) Origin: (A) Organism: Mumps virus (B) Strain : Description of the sequence of Jeryl-Lin-1 (xi): SEQ ID NO: 1: (2) Information on SEQ ID NO: 2: (i) Sequence characteristics: (A) Sequence length: 15 base pairs (B) Sequence type: nucleic acid (C) Number of strands: 1 strand (D) Topology : Linear (ii) Type of sequence: DNA (genome) (iii) Hypothetical: None (iv) Antisense: None (vi) Origin: (A) Organism: nh2 (xi) Description of sequence: SEQ ID NO: : 2: (2) Information on SEQ ID NO: 3: (i) Sequence characteristics: (A) Sequence length: 20 base pairs (B) Sequence type: nucleic acid (C) Number of strands: 1 strand (D) Topology : Linear (ii) Sequence type: DNA (genome) (vi) Origin: (A) Organism: NH8 (xi) Description of sequence: SEQ ID NO: 3: (2) Information on SEQ ID NO: 4: (i) Sequence characteristics: (A) Sequence length: 21 base pairs (B) Sequence type: Nucleic acid (C) Number of strands: Single strand (D) Topology : Linear (ii) Sequence type: DNA (genome) (vi) Origin: (A) Organism: NH14 (xi) Description of sequence: SEQ ID NO: 4: (2) Information on SEQ ID NO: 5: (i) Sequence characteristics: (A) Sequence length: 33 base pairs (B) Sequence type: Nucleic acid (C) Number of strands: Single strand (D) Topology : Linear (ii) Sequence type: DNA (genome) (vi) Origin: (A) Organism: NH22 (xi) Description of sequence: SEQ ID NO: 5: (2) Information on SEQ ID NO: 6: (i) Sequence characteristics: (A) Sequence length: 32 base pairs (B) Sequence type: nucleic acid (C) Number of strands: 1 strand (D) Topology : Linear (ii) Sequence type: DNA (genome) (vi) Origin: (A) Organism: NH23 (xi) Description of sequence: SEQ ID NO: 6: (2) Information on SEQ ID NO: 7: (i) Sequence characteristics: (A) Sequence length: 18 base pairs (B) Sequence type: Nucleic acid (C) Number of strands: Single strand (D) Topology : Linear (ii) Sequence type: DNA (genome) (vi) Origin: (A) Organism: NH23 (xi) Description of sequence: SEQ ID NO: 7: (2) Information on SEQ ID NO: 8: (i) Sequence characteristics: (A) Sequence length: 18 base pairs (B) Sequence type: Nucleic acid (C) Number of strands: 1 strand (D) Topology : Linear (ii) Type of sequence: DNA (genome) (vi) Origin: (A) Organism: NH31 (xi) Description of sequence: SEQ ID NO: 8: (2) Information on SEQ ID NO: 9: (i) Sequence characteristics: (A) Sequence length: 28 base pairs (B) Sequence type: Nucleic acid (C) Number of strands: Single strand (D) Topology : Linear (ii) Type of sequence: DNA (genome) (vi) Origin: (A) Organism: NH30BIS (xi) Description of sequence: SEQ ID NO: 9: (2) Information on SEQ ID NO: 10: (i) Sequence characteristics: (A) Sequence length: 24 base pairs (B) Sequence type: Nucleic acid (C) Number of strands: Single strand (D) Topology : Linear (ii) Type of sequence: DNA (genome) (vi) Origin: (A) Organism: NH31BIS (xi) Description of sequence: SEQ ID NO: 10: (2) Information on SEQ ID NO: 11: (i) Sequence characteristics: (A) Sequence length: 29 base pairs (B) Sequence type: Nucleic acid (C) Number of strands: Single strand (D) Topology : Linear (ii) Sequence type: DNA (genome) (vi) Origin: (A) Organism: BC265 (xi) Sequence description: SEQ ID NO: 11: (2) Information on SEQ ID NO: 12: (i) Sequence characteristics: (A) Sequence length: 31 base pairs (B) Sequence type: nucleic acid (C) Number of strands: 1 strand (D) Topology : Linear (ii) Sequence type: DNA (genome) (vi) Origin: (A) Organism: bc252 (xi) Description of sequence: SEQ ID NO: 12: (2) Information on SEQ ID NO: 13: (i) Sequence characteristics: (A) Sequence length: 31 base pairs (B) Sequence type: Nucleic acid (C) Number of strands: Single strand (D) Topology : Linear (ii) Sequence type: DNA (genome) (vi) Origin: (A) Organism: Bc253 (xi) Description of sequence: SEQ ID NO: 13:

────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Corot, Bridget Desiree Albert to 1330 Rixenzart, Rue du Lansitet 89th Belgium, Miscline Beecham Biology Callus (Societe Anonym) (72) ) Inventor Didre, Jean Bécé-1330 Rixenzart, Rue du Lansituit 89th Miss Klein Beecham Biological Callus (Societe Anonym) (56) References Gen. Virol. Vol. 74, No. part5 (1993. May) p. 917-920 (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 7/00 A61K 39/165 BIOSIS (DIALOG) MEDLINE (STN) WPI / L (DIALOG)

Claims (6)

(57) [Claims] 1. An attenuated jeryl-phosphorus mumps virus isolate characterized by the N-terminus of the SH and HN genes comprising the nucleic acid sequence shown in FIG. 2. A mumps vaccine comprising the substantially uniform immunogenic geryl-phosphorus isolate of claim 1. 3. The method of claim 2, wherein the isolate comprises a homogeneous isolate of claim 1.
The mumps vaccine described. 4. The substantially uniform immunogenic geryl-phosphorus isolate of claim 1 and one or more attenuated measles virus, or attenuated rubella virus or killed measles or rubella. A virus or combination vaccine comprising subunits of such a virus. 5. The vaccine according to claim 3, further comprising an agent for protecting against chickenpox or shingles infection. 6. A method of substituting a jeryl-phosphorus preparation in a suitable cell line, selecting a pure culture using any of the following steps: a) limiting dilution; or b) isolating individual plaques. A method for the preparation of a geryl-phosphorus isolate of claim 1 which is substantially homogeneous and immunogenic.