CN101291690A - Rotavirus vaccine inducing heterotypic cross protection - Google Patents

Abstract

The invention provides a method of inducing an immune response against rotavirus strain, the method comprising administering to a subject a composition comprising an attenuated rotavirus strain of a GxPy type, said composition generating an immune response against a rotavirus strain which is neither a Gx nor a Py type.

Description

The Rotavirus Vaccine of inducing heterotypic cross protection

Technical field

The present invention relates to the Rotavirus Vaccine preparation.The present invention relates to the purposes of attenuated rotavirus group in prevent disease from a rotavirus type, wherein said disease is with relevant from the rotavirus infection of another rotavirus type.

Technical background

Acute infection diarrhoea is disease and dead leading reason in numerous areas, the world.In developing country, the influence of diarrhoea property disease is serious.With regard to Asia, Africa and Latin America, have according to estimates that about 5,000,000 to 10,000,000 cases cause death (Walsh, J.A. etc.: N.Engl.J.Med., 301:967-974 (1979)) in hundred million of 30-40 diarrhoea case and these cases every year.

Rotavirus has been regarded as one of cacatory most important reason among baby and the child (Estes, M.K.Rotavirus and Their Replication in Fields Virology, the third edition, editors such as Fields, Raven Publishers, Philadelphia, 1996).Rotavirus causes above megadeath sick every year according to estimates.The inductive disease of rotavirus influences 6-24 monthly age child the most commonly, and the peak sickness rate of disease is usually during temperate climate comes across colder month, and occurs all the year round in the torrid areas.Rotavirus is propagated the pure man through fecal-oral route from the people usually, incubation period about 1 Ri-Yue 3 days.Different with the infection at 6 monthly age-24 monthly ages, neonate is normally asymptomatic or only have a minor ailment.Different with the serious disease that meets with usually among the child, most of adult was protected because of former rotavirus infection, was gentle or asymptomatic (Offit, Comp.Ther. such as P.A., 8 (8): 21-26,1982) so that most of adult infects.

It is spherical that rotavirus is usually, and their name derives from skin and the endothecium structure or the double capsid structure of its uniqueness.Usually, the double capsid structure of rotavirus is surrounded and is contained genomic internal protein shell or nuclear.The genome of rotavirus is made up of the proteinic 11 joint double-stranded RNAs of 11 kinds of different virus of coding at least.Two of called after VP4 and VP7 kinds of virus proteins are arranged in the outside of double capsid structure in these virus proteins.There is a kind of protein in the inside capsid of rotavirus, i.e. the rotavirus protein of called after VP6.These three kinds of specific rotavirus proteins excite the relative importance aspect the immunne response to be still unclear after rotavirus infection.But, VP6 protein decision group antigen and subgroup antigen, and VP4 protein and VP7 protein be serotype (by in and the type determined of algoscopy) the specific determiner of specificity and genotype (type of determining by non-determination of serology method).Genotypic name is identical with G to G serotype.And to P serotype and the specified numbering of P genotype be different (Santos N.et HoshinoY., 2005, Reviews in Medical Virology, 15,29-56).Therefore, with P serotype called after P, after wear specified numbering, and, be with specified numbering in the bracket of back with P genotype called after P.

So far, identified at least 14 rotavirus G serotypes and 14 rotavirus P serotype (Santos N.et Hoshino Y., 2005, Reviews in Medical Virology, 15,29-56).In these serotypes, 10 G (G1-6, G8-10 and G12) serotype and 9 P (P1, P2A, P3, P4, P5A, P7, P8, P11 and P12) serotype have obtained identifying in Human reoviruslike agent.23 P genotype have been described, wherein 10 P genotype from philtrum obtain (P[3]-[6], P[8]-[11], P[14] and P[19]).

VP7 protein is that this depends on Strain as the glycoprotein of the molecular weight 38,000 of the translation product of genome segment 7,8 or 9 (molecular weight 34,000 when non-glycosylated).Neutralizing antibody after this protein boost rotavirus infection forms.VP4 protein is the non-glycosylated protein matter of about molecular weight 88,000, and it is the translation product of genome segment 4.This protein also stimulates neutralizing antibody to produce after rotavirus infection.

Since VP4 and VP7 protein be neutralizing antibody institute at virus protein, are main candidates of exploitation Rotavirus Vaccine so it is believed that them, the protective effect of generation anti-rotavirus disease.

Knownly excite protective immunity in the early stage natural rotavirus infection of child.Therefore press for the rotavirus attenuated live vaccine.Suitably, this vaccine should be an oral vaccine, because this is the natural approach of rotavirus infection.

The early stage vaccine development of prevention rotavirus infection starts from the seventies in last century after this virus is found.At first, studied, but obtained delphic or disappointed result from animal and human's attenuated strain.Nearest work concentrates on more successful people-animal reprovision body (reassortant).

The rotavirus strain that is called 89-12 is described by Ward; See U.S. Patent number 5,474,773 and Bernstein, D.L. etc., Vaccine, 16 (4), 381-387,1998.Separate in the stool sample that the 89-12 strain is collected from 14 monthly age children of a natural rotavirus disease of trouble in 1988.According to U.S. Patent number 5,474,773, HRV 89-12 Human reoviruslike agent such as Ward are at J.Clin.Microbiol. subsequently, 19,748-753 describedly in 1984 is adapted to cultivation 4 times by going down to posterity 2 times and going down to posterity in the MA-104 cell in former generation African green monkey kidney (AGMK) cell.This virus is accepted 3 plaque purifications (to the 9th generation) and cultivates in these cells again to go down to posterity 2 times subsequently in the MA-104 cell.Once go down to posterity again (the 12nd generation), be used for the ATCC preservation, accession number ATCC VR 2272.The strain of preservation is called 89-12C2.

Hereinafter 1998 papers with Bernstein in " Vaccine " etc. are called " Vaccine (1998) " paper.This paper is described the safety and the immunogenicity of Orally administered Human reoviruslike agent candidate's live vaccine.This vaccine obtains from the 89-12 strain, obtains attenuation by mode like this, promptly in former generation AGMK cell, do not carry out plaque purification and go down to posterity 26 times and the AGMK cell line set up subsequently in repeated transmission 7 times (33 generations altogether) of generation.

After this this paper be called the described material of continuous passage 26 times P26 and the material of continuous passage 33 times be called P33.Usually, 89-12 is gone down to posterity by n time and deutero-rotavirus is called Pn.

In follow-up embodiment, the P33 material further goes down to posterity on the Vero cell 5 times.This material is called P38.

P26 and the P33 separated strain described in " Vaccine (1998) " paper are not preserved in the culture collection center, set up its heredity less than they are analyzed yet and characterize.

Have now found that the P26 group who describes in the document is made up of the variant mixture.This characterizes by heredity as mentioned below and obtains confirming (seeing embodiment).Therefore P26 is not the reliable homogeneity group who is used for further going down to posterity, and is like this for the production lot vaccine especially.Similarly, P33 comprises the variant mixture and the production lot vaccine is not had reliable homogeneity.

Found that the P26 material is the mixture of at least three kinds of VP4 genetic mutations.P33 and P38 are the mixture of two kinds of variants similarly.When the titre of the neutralizing antibody of anti-these variants in baby's the serum of P33 had been inoculated in assessment, with regard to neutralizing epitope, these variants showed different with the 89-12C2 strain antigenicity that is deposited in ATCC.

Have been found that in addition that when the P33 material is applied to the baby two kinds of variants of having identified are duplicated and discharge.Among 100 inoculation babies, only 2 babies show because of the gastroenteritis symptom due to the rotavirus infection, and 20% placebo group infections in infants.These discoveries show the variant identified and to avoid rotavirus sick relevant.

WO 01/12797 discloses the method for separation rotavirus variant and deutero-improvement rotavirus attenuated live vaccine from clone's's (homogeneous) Human reoviruslike agent strain.Also disclose attenuated rotavirus group (separated strain), it is characterized in that this group comprises single variant or single basically variant, wherein said prominent body is by the nucleotide sequence definition of the main diseases poisonous protein of at least a called after VP4 of coding and VP7.(42nd InterscienceConference on Antimicrobial Agents and Chemotherapy (ICAAC 2002) 27-302002 JIUYUE such as Perez-30 days on the 27th, San Diego) renderd a service and reported in the protection of the oral attenuated vaccine of Human reoviruslike agent of this anti-G9 allos strain in the baby of Latin America.WO 05/021033 discloses and can use a kind of rotavirus serotype to be protected from influence by another kind of serotype associated diseases.Especially WO 05/021033 discloses G1 rotavirus group and [for example has been preserved in European animal cell culture preservation center (ECACC) on August 13rd, 1999 with preserving number 99081301 according to the budapest treaty clause, vaccine research and production laboratory, public health laboratory service organization, applied microbiology and research center, Porton Down, Salisbury, Wiltshire, SP4 0JG, the United Kingdom, have another name called P43 or RIX4414] prevent purposes by G1 and at least one non-G1 rotavirus serotype associated diseases, wherein said non-G1 rotavirus serotype for example is but is not limited to G2, G3, G4 and G9 rotavirus serotype.

The complete content of WO 01/12797 and WO 05/021033 in this article by reference as a reference.

The accompanying drawing summary

Figure 1A(SEQ ID NO:1) is the nucleotide sequence of P43 (RIX4414) VP4 gene, comprises the proteinic sequence of VP4 of the P43 that encodes.

Figure 1B(SEQ ID NO:2) has from the extra nucleotide of two ends of VP4 gene and the definite nucleotide subsitution (capitalization-G is 18 alternative C in the position, cause TCG to substitute TCA, yet the coded protein that produces is not had influence) of sequencing technologies.Non-coding sequence shows with small letter.Figure 1B shows the correct sequence of P43 preservation thing.

Fig. 2 A(SEQ ID NO:3) is the nucleotide sequence of P43 (RIX4414) VP7 gene, comprises the proteinic sequence of VP7 of the P43 that encodes.

Fig. 2 B(SEQ ID NO:4) has the nucleotide subsitution determined from the extra nucleotide of two ends of VP7 gene and sequencing technologies (capitalization-A 58 substitutes C in the position, and the leucic ATT that causes encoding substitutes the CTT of the isoleucine of encoding).Non-coding sequence shows with small letter.Fig. 2 B shows the correct sequence of P43 preservation thing.

Fig. 3(SEQ ID NO:5) is the peptide sequence of RIX4414VP4.

Fig. 4(SEQ ID NO:6) is the peptide sequence of RIX4414VP7.

Fig. 5(SEQ ID NO:7) shows the proteinic peptide sequence of NSP4 of RIX4414.

Fig. 6The proteinic nucleotide sequence of NSP4 of (SEQ ID NO:8) code displaying RIX4414.Non-coding sequence shows with small letter.

Fig. 7(SEQ ID NO:9) shows the proteinic peptide sequence of VP6 of RIX4414.

Fig. 8The proteinic nucleotide sequence of VP6 of (SEQ ID NO:10) code displaying RIX4414.Non-coding sequence shows with small letter.

Detailed Description Of The Invention

In the present invention; we determine; the attenuated rotavirus group; an attenuated rotavirus group who in WO 01/12797, is characterized for example; can be as vaccine so that the cross protection of anti-disease to be provided, wherein said disease by with vaccine in due to the rotavirus infection of different shaped (serotype and/or genotype) of used rotavirus.VP7 protein regulation G type (serotype), and the P type (serotype or genotype) of VP4 protein regulation Strain.

Especially, the present invention relates to the purposes of attenuated rotavirus group in prevent disease from a P type, wherein said disease is correlated with the rotavirus infection from different P types, and be specifically related to attenuated rotavirus group or the purposes of attenuation in induce immune response and/or prevent disease from the GxPy type, wherein said disease with by neither the rotavirus infection that the rotavirus strain of the also non-Py type of Gx type causes is relevant.

Immunity can be by replying at the neutralizing antibody of vaccine or (promptly inoculating back serum antibody I gA level 〉=3 times of increases by serum rotavirus IgA antibody response such as seroconversion coefficient, as Ward etc., 1990, J.Infect.Disease, 161, describe in the 440-445) weighed.

In the context of the invention and consistent (Santos N.etHoshino Y. with the general knowledge of this area, 2005, Reviews in Medical Virology, 15,29-56), Gx refers to specific G type, i.e. G genotype or G serotype (two terms are identical), and term Py refers generally to specific P type, or P serotype (for example P8, P4) or P genotype (for example P[4], P[8]).When indicating specific P genotype, use the P that the numbering of specifying is arranged in its unquote; Otherwise the P type refers to serotype or genotype.

In this description in the whole text in the scope, be used for preventing purposes in the vaccine combination of rotavirus disease such as vaccine combination of the present invention in preparation, or will use interchangeably such as the term that comprises the Therapeutic Method that uses described vaccine combination.

We have determined GxP[8 now] rotavirus group [for example G1P[8], it is preserved in European animal cell culture preservation center (ECACC) on August 13rd, 1999 with preserving number 99081301 according to the budapest treaty clause, vaccine research and production laboratory, public health laboratory service organization, applied microbiology and research center, Porton Down, Salisbury, Wiltshire, SP40JG, the United Kingdom] can be used for prevention by GxP[8] (for example G1P[8]) and at least a neither the disease that the rotavirus strain of the also non-Py type of Gx type causes.Especially, we have determined G1P[8] the rotavirus group can be used for prevention by a G1P[8] genotype and at least one non-G1P[8] genotype (as G2P[4] the rotavirus gene type) caused disease.

Therefore, the present invention relates to the purposes of attenuated rotavirus group in prevent disease from a rotavirus type, wherein said disease is with relevant from the rotavirus infection of another rotavirus type, and wherein said type defines by the sequence with reference to colyliform virus VP 4 protein matter (P type) suitably.

The invention still further relates to the purposes of attenuated rotavirus group in prevent disease from a rotavirus strain (defining simultaneously) by specific G type and P type, wherein said disease is relevant with rotavirus infection from another rotavirus strain, and wherein said type defines by the sequence with reference to colyliform virus VP 4 protein matter (P type) and VP7 protein (G type) simultaneously suitably.Particularly, the present invention relates to be used for inducing purposes in the medicine of the immunne response that anti-rotavirus infects in preparation from the rotavirus attenuated strain of GxPy type, wherein said rotavirus infection by neither the rotavirus strain of the also non-Py type of Gx type cause.In other words, rotavirus strain of the present invention can be used for preventing the caused disease of infection by another rotavirus that is different from G type and P type.

Especially; claimed of the present invention aspect all in; described immunne response is a protective immune response, and suitably, the rotavirus group comprises VP4 virus protein and/or the VP7 virus protein from ECACC preservation thing 99081301 that is suitable for providing cross-protection.

In the whole text in the scope, the protective effect at the infection that is caused by the different shaped rotavirus that is provided by a rotavirus type is provided cross protection at presents.Cross protection can be isostructural or special-shaped.The homotype cross protection is to provide protective effect at strain of G type or the strain of P type, for example G1P[8 by a rotavirus type] strain passes through P[8] type gives at non-G1, P[8] cross protection of strain (for example G2P[8]).Another example of homotype cross protection is by G1P[8] strain provides the non-P[8 at G1 by the G1 type] cross protection of strain (for example G1P[4]).Heterotypic cross protection is to provide protective effect at different P types and G type rotavirus strain by a rotavirus strain, for example by G1P[8] the non-P[8 at non-G1 is provided] protective effect (the abnormal shape protection that is provided by G type and P type simultaneously) of strain (for example G2P[4]).

Suitably, the rotavirus serotype of attenuation be G1 and it can provide to by rotavirus G1 serotype and non-G1 serotype (as be selected from G2, G3, G4, G5, G6, G7, G8, G9, G10, G11, G12, G13 and G14 serotype) cause the cross protection of disease.

Especially, G1 attenuated rotavirus group [for example is preserved in European animal cell culture preservation center (ECACC) on August 13rd, 1999 with preserving number 99081301 according to the budapest treaty clause, vaccine research and production laboratory, public health laboratory service organization, applied microbiology and research center, Porton Down, Salisbury, Wiltshire, SP40JG, the purposes of the United Kingdom 1 can be used for preventing by rotavirus G1 serotype and be selected from G2, G3, G4, G5, G6, G7, G8, G9, G10, G11, G12, among G13 and the G14 at least 1, suitable at least 2, suitable at least 3, the caused disease of the non-G1 serotype of suitable at least 4 rotavirus.Therefore, provide from the rotavirus attenuated strain of G1 type to be used for inducing the purposes of the vaccine combination of the immunne response that anti-rotavirus infects in preparation, wherein said rotavirus infection is by being not that rotavirus strain from the G1 type causes.In particular aspects, the inductive immunne response of institute is at least 1, at least 2 or the non-G1 serotype of more a plurality of rotavirus, and is general at any serotype that is selected from G2, G3, G4, G5, G6, G7, G8, G9, G10, G11, G12, G13 and G14.Except that homotype (G1) protection, the inductive immunne response of institute is among G2, G3, G4 and the G9 at least 1, at least 2 suitably, at least 3 suitably at following non-G1 type also generally.Suitably, compositions comprises G1 rotavirus strain and said composition and is used for inducing immunne response at G1 type and G2 type.

Suitably, rotavirus attenuation plant type is P[8] and it can provide by rotavirus P [8] type with by the non-P[8 of rotavirus] type (as be selected from P[1], P[2], P[3], P[4], P[5], P[6], P[7], P[9], P[10], P[11], P[12], P[14] and P[19] any type) cause the cross protection of disease.

Especially, P[8] the attenuated rotavirus group, [for example be preserved in European animal cell culture preservation center (ECACC) on August 13rd, 1999 with preserving number 99081301 according to the budapest treaty clause, vaccine research and production laboratory, public health laboratory service organization, applied microbiology and research center, Porton Down, Salisbury, Wiltshire, SP40JG, the United Kingdom] purposes can be used for prevention by P[8] and at least a P[1 of being selected from], P[2], P[3], P[4], P[5], P[6], P[7], P[9], P[10], P[11], P[12], P[14] and P[19] the non-P[8 of rotavirus] the caused disease of type.Especially, except that at rotavirus P [8] type, anti-P[4 at least] immunne response of type also induced suitably.

Suitably, used vaccine combination comprises G1P[8 according to the present invention] rotavirus strain and said composition can induce at G2P[4] immunne response of rotavirus strain.

In particular aspects, the present invention relates to induce the method for the immunne response of anti-rotavirus strain, this method comprises to the experimenter uses the compositions that comprises rotavirus GxPy type attenuated strain, and said composition produces anti-neither the immunne response of the rotavirus strain of the also non-Py type of Gx type.

Especially, the present invention relates to induce the method for the immunne response of anti-rotavirus G1 and non-G1 serotype, this method comprises to the experimenter uses the compositions that comprises rotavirus G1 serogroup vaccine.Suitably, non-G1 rotavirus serotype is selected from: G2, G3, G4, G5, G6, G7, G8, G9, G10, G11, G12, G13 and G14.Suitably, said composition comprises the immunne response that G1 rotavirus strain and said composition are used for inducing anti-G1 type and G2 type.

Suitably, used vaccine combination comprises G1P[8 according to the present invention] rotavirus strain and said composition can induce anti-G2P[4] immunne response of rotavirus strain.

Suitably, the rotavirus group in the vaccine combination has G1P1A (is G1P[8 according to present name]) strain specificity.Suitably, this rotavirus colony comprises VP4 virus protein and/or the VP7 virus protein from ECACC preservation thing 99081301 that is suitable for exciting immunne response and cross-protection is provided usually.Suitably, the present invention relates to G1P[8 in method or purposes as mentioned above] rotavirus strain.Usually this used Rotavirus Vaccine is an ECACC preservation thing 99081301, or derived from this preservation thing.

Aspect concrete, not inoculate individuality with (from placebo group) and compare, this vaccine is induced intersecting protective immunne response or the cross protection of anti-gastroenteritis in the inoculation individuality.Suitably, vaccine provides the cross protection of anti-rotavirus infection symptoms as diarrhoea or gastroenteritis.For example, gastroenteritis can be defined as the following diarrhoea of feature, and three times or water sample repeatedly or normal looser feces or the hyperemesis of ratio detect rotavirus simultaneously in promptly one day in being checked stool sample.

Understand disease seriousness and in the colony of the individual of inoculation or inoculation, induce the inoculation of protective immune response to render a service and to assess by several method as the technical staff.Protective immune response means immunne response like this, and it causes the seriousness minimizing of the clinical symptoms relevant with rotavirus infection or causes the susceptibility of rotavirus infection is reduced.Disease seriousness in not inoculating individual or inoculation individuality can be carried out classification according to disclosed marking system; (the Scand.J.Infect.Dis.1990 such as Ruuska T of improved form slightly as 20 Vesikari scales or the method; 22; 259-267); or according to report and any other appropriate system of classification rotavirus infection specific symptom (as at Clark HF; Borian EF, Bell LM.WC3 vaccine reported method in the protective effect of rotavirus diarrhea (Protective effect of WC3 vaccine against rotavirus diarrhea in infantsduring a predominantly serotype 1 rotavirus season.) the J Infect Dis.1988:570-86 among the anti-baby during 1 rotavirus season of advantage serotype).According to the Vesikari method, usually serious RVGE is defined as scoring 〉=11.

Protection can be assessed by vaccine potency (VE) at population level or group level.Vaccine potency calculates with following formula:

VE (%)=1-RR=1-(ARV/ARU), wherein

RR=relative risk=ARV/ARU

ARU=does not inoculate the interior experimenter's total number of persons of experimenter's number/matched group of affect rate (estimating)=at least the RV GE outbreak of report in the colony from placebo group

Experimenter's total number of persons in the number of subjects/HRV vaccine group of the affect rate in the ARV=inoculation group=at least RV GE outbreak of report

Therefore; in one aspect of the invention; as detailed above method or purposes are provided; the compositions that wherein comprises rotavirus GxPy type attenuated strain is induced the inductive gastroenteritis of anti-rotavirus, the intersecting protective immunne response and/or the protection of the inductive serious gastroenteritis of anti-rotavirus suitably, wherein said (seriously) gastroenteritis by neither the rotavirus virus strain infection of the also non-Py type of Gx type cause.In specific embodiments, as measuring according to any suitable marking system, described protective immune response can reduce severity of disease or eliminate the inductive disease of rotavirus.

In another embodiment, the method or the purposes of the present composition are provided, be intended to reduce the seriousness of disease (for example gastroenteritis) or eliminate the inductive disease of rotavirus, described disease seriousness or disease basis any suitable marking system are as mentioned above carried out record.

In specific embodiments; described compositions has nearly 60% diarrhea protective effect, nearly 81% diarrhea protective effect suitably in the groups of individuals in inoculation, and wherein said diarrhoea is infected by the rotavirus type different with the attenuated rotavirus type that exists in the compositions and causes.In another embodiment; at least 40% diarrhea protective effect, at least 45% diarrhea protective effect suitably, at least 50% diarrhea protective effect suitably, at least 60% diarrhea protective effect suitably that described compositions has in the groups of individuals in inoculation, wherein said diarrhoea by neither the rotavirus virus strain infection of the also non-Py type of Gx type cause.Aspect concrete, described compositions has the diarrhea protective effect of 40%-80%, the diarrhea protective effect of 50%-70% suitably, wherein said diarrhoea by neither the rotavirus virus strain infection of the also non-Py type of Gx type cause.Aspect concrete, described compositions comprises G1P[8] rotavirus strain, it provides by G2P[4] protective effect of the level as previously mentioned of the gastroenteritis of type rotavirus strain due to infecting.

Suitably; the protective rate of diarrhea of realizing in the individual colony of inoculation and/or gastroenteritis and/or serious gastroenteritis is 10-90%, 20-80%, 40%-80%, 45%-75% protective effect suitably suitably suitably, and wherein said inoculation individuality is by neither the rotavirus virus strain infection of the also non-Py type of Gx type.Usually, the level of anti-serious gastroenteritis protection is at least 40%, suitably at least 50%.

Aspect concrete; described compositions comprises G1P[8] the rotavirus strain; wherein as measured according to the Vesikari scale; described strain has in the groups of individuals at by rotavirus G2P[4 in inoculation] serotype infects the 40%-80% protection of the serious gastroenteritis that causes, 45%-75% protection suitably.

Suitably, vaccine is used for 2 dosage or 3 dosages.

The Rotavirus Vaccine that is used for producing cross protection has following suitable feature.

In one aspect, rotavirus in compositions used according to the invention has the VP4 gene that comprises nucleotide sequence like this, and wherein said nucleotide sequence comprises at least a of following base: apart from start codon in the position 788 adenine base (A), in the position 802 adenine base (A) and in the position 501 thymine alkali bases (T).

In yet another aspect, rotavirus in compositions used according to the invention has the VP7 gene that comprises nucleotide sequence like this, and wherein said nucleotide sequence comprises at least a of following base: from the thymus pyrimidine (T) of start codon position 605, the adenine (A) of position 897 or the guanine (G) of position 897.897 there is adenine (A) in the position suitably.

Aspect concrete, 788 and 802 the having adenine (A) and 501 having thymus pyrimidine (T) in the position in the VP4 gene order of the rotavirus in compositions used according to the invention in the position from start codon.

Another concrete aspect, the rotavirus in compositions used according to the invention in the VP7 gene order from start codon in the position 605 thymus pyrimidines that have (T) and in the position the 897 adenine/guanines that have (A/G).Most suitably in the VP7 sequence, 897 there is adenine (A) in the position.

Aspect specially suitable, rotavirus in compositions used according to the invention 788 and 802 having adenine (A) and having 5 thymus pyrimidine (T) in the position in the position in the VP4 gene order from start codon, and 897 there is adenine (A) in 605 having thymus pyrimidine (T) and 897 having adenine/guanine (A/G) in the position in the position from start codon in the VP7 sequence in the position suitably in the VP7 sequence.

In yet another aspect, rotavirus in compositions used according to the invention comprises the proteinic nucleotide sequence of coding VP4, wherein this nucleotide sequence be as shown in 1A figure (SEQ IDNO:1) or Figure 1B (SEQ ID NO:2) like that, and/or comprise coding VP7 proteinic nucleotide sequence, wherein said nucleotide sequence be as shown in 2A (SEQ ID NO:3) or Fig. 2 B (the SEQ ID NO:4) figure like that.In alternative embodiment, the rotavirus in compositions used according to the invention comprises as Fig. 3 (SEQ ID NO:5) described VP4 protein and/or as the described VP7 protein of Fig. 4 (SEQ ID NO:6).In another embodiment, used according to the invention described rotavirus colony also comprise as described in Fig. 5 (SEQ ID NO:7) or by as Fig. 6 (SEQ ID NO:8) in described nucleotide sequence coded NSP4 protein, and/or also comprise as described in Fig. 7 (SEQ ID NO:9) or by as the interior described nucleotide sequence coded VP6 protein of Fig. 8 (SEQ ID NO:10).

The suitable rotavirus group who uses among the present invention can be by obtaining as the method, and this method comprises:

The rotavirus prepared product goes down to posterity on suitable cell type;

Optional use following both one of step select the homogeneous culture:

A) limiting dilution; Or

B) single plaque separates; And

Check whether there is single basically variant by the suitable zone of VP4 gene order and/or VP7 gene order being carried out sequencing.

Suitably, the rotavirus group is derived from aforesaid P43 (RIX4414), P33 or P26 strain.

Sequencing can be implemented suitably by quantitative or sxemiquantitative hybridization technique such as slot blot hybridization or plaque hybridization.

Generation can obtain amplification by further going down to posterity on suitable cell line from clone's virus groups of the inventive method.

Be used for comprising it to be the cell line set up or African green monkey kidney (AGMK) cell of former generation AGMK cell at go down to posterity rotavirus group's suitable cell type of above method.Proper A GMK cell line comprises for example Vero (ATCC CCL-81), DBS-FRhL-2 (ATCCCL-160), BSC-1 (ECACC 85011422) and CV-1 (ATCC CCL-70).MA-104 (macaque (rhesus monkey)) and MRC-5 (people-ATCC CCL-171) cell line also are suitable.The Vero cell is particularly suitable for the purpose that increases.Going down to posterity on the Vero cell produces high viral yield.

The technology that whether has the technology of single variant in the virus groups that is used to check the inventive method to produce and be used for determining the character of this single variant relates to standard order-checking known in the art or hybridizing method and obtains hereinafter describing.

Aspect concrete, the inventive method is used suitable rotavirus, and the rotavirus that especially has the feature of 89-12 strain feature or its derivant that goes down to posterity is implemented.

Specially suitable single variant group is P43, wherein P43 clones step by continuous terminal point dilution from P33 (isolating Human reoviruslike agent is cultivated at suitable cell type and gone down to posterity P33 time), obtains by will clone property material going down to posterity to increase on the Vero cell subsequently.

P43 group is preserved in European animal cell culture preservation center (ECACC) on August 13rd, 1999 with preserving number 99081301 according to the budapest treaty clause, vaccine research and production laboratory, public health laboratory service organization, applied microbiology and research center, PortonDown, Salisbury, Wiltshire, SP4 0JG, the United Kingdom, and in WO 01/12797, obtain open.

Though this described public's availability is to obtain the simplest method of Human reoviruslike agent P43, however similar and can obtain by these methods or other method reading under the teachings of the present invention at essentially identical rotavirus on the function.This type of essentially identical rotavirus on function be considered to biologically be equal to Human reoviruslike agent P43 of the present invention and therefore belong to general range of the present invention.It is therefore to be understood that the present invention comprises and has the rotavirus group of the feature of P43 variant as described herein.

Be understood that, the present invention comprises by the P43 ECACC99081301 deutero-material of mode like this from preservation, wherein said method for example is further to handle P43 ECACC99081301, as by using live virus further to go down to posterity, clone or other method makes P43ECACC 99081301 propagation, or modify P43 by any way, comprise by gene engineering or reprovision technology.This type of step and technology are well-known in the art.

The deutero-material from the P43 of preservation that is comprised by the present invention comprises protein and genetic stocks.The special at least a antigen of P43 or the reassorted rotavirus of at least one sections of meaningfully comprising, the reprovision body that for example comprises the rotavirus virulent strain, wherein one of 11 genome segments in the rotavirus virulent strain or its part are replaced by genome segment or the part of P43.Particularly, encode the therein sections of NSP4 or sections that the part sections is P43 or the rotavirus reprovision body of part sections possesses useful characteristic.The technology that reassorted rotavirus and being used to prepares them is well-known (Foster, R.H. and Wagstaff, A.J. summarize, tetravalent rotavirus vaccine (Tetravalent Rotavirus Vaccine, a review) .ADIS drug evaluation, BioDrugs, Gev, 9 (2), 155-178,1998).

Filial generation that significant especially material is P43 and the immunocompetence derivant of P43.The immunocompetence derivant means the material that obtains from P43 virus, especially should the antigen of virus or obtain with them, and wherein said material excites the immunne response that rotavirus is responded in the time of can be in being injected to host animal.

In that rotavirus is adapted in the suitable cell line (for example Vero cell), may must handle virus so that remove any potential pollutant, as any adventitious agents that may exist and can pollute.In the example of the adventitious viruses of ether sensitivity, this can as mentioned belowly finish dealing with by ether.The invention still further relates to this ether handled as optional step and comprise in the holistic approach of the vaccine that into obtains the attenuated live rotavirus or prepared with this attenuated live rotavirus.

Intersecting protective rotavirus strain of the present invention can make up with other rotavirus strain, so that the Additional Protection or the cross protection of anti-rotavirus infection or disease to be provided.

The present invention also provides and suitable adjuvant or the blended rotavirus attenuated live vaccine of pharmaceutical carrier, and it can provide the cross protection defined above as this paper.

In one embodiment, Rotavirus Vaccine used according to the invention is to contain single rotavirus strain such as G1P[8] the unit price Rotavirus Vaccine of strain.

The present invention is particularly advantageous in provides free wheel shape viral lived vaccine, and wherein this attenuated rotavirus is Human reoviruslike agent and does not cause intussusception.

Be suitable for comprising known in the art as being suitable for Orally administeredly, be particularly useful for those pharmaceutical carriers of baby with the pharmaceutical carrier that rotavirus attenuated strain of the present invention is used.Examples of such carriers comprises and is not limited to saccharide, polyhydric alcohol, aminoacid, aluminium hydroxide, magnesium hydroxide, hydroxyapatite, Talcum, titanium oxide, ferrum oxide, magnesium stearate, carboxymethyl cellulose, hydroxypropyl emthylcellulose, microcrystalline Cellulose, gelatin, phytone, xanthane, caraghenane, Radix Acaciae senegalis, beta-schardinger dextrin-.

The present invention also is provided for preparing the technology of Rotavirus Vaccine, and is for example viral or virus of the present invention mixed with suitable adjuvant or pharmaceutical carrier by lyophilizing in the presence of suitable stabilizers.

Can be advantageously with virus of the present invention based on the carrier of lipid such as viroplast or liposome, preparation or prepare in oil in water emulsion with carrier particle.Alternatively or extraly, can in preparation, comprise immunopotentiating agent (those immunopotentiating agents that are used for oral vaccine as known in the art).This type of immunopotentiating agent comprises bacteriotoxin, especially is in holotoxin (complete molecule) form or the only cholera toxin (CT) and escherichia coli (E.coli) heat labile enterotoxins (LT) of B chain (CTB) form.Than the more impossible sudden change LT (mLT) that is transformed into its activity form of natural LT WO96/06627 description is arranged among WO 93/13202 and the US 5,182,109.

Other immunopotentiating agent that can advantageously comprise into is saponin derivative such as QS21 and monophosphoryl lipid A, especially 3-deoxidation-O-acetylation monophosphoryl lipid A (3D-MPL).Purification saponin as oral adjuvant is described in WO 98/56415.Saponin and monophosphoryl lipid A can distinguish or use (for example WO 94/00153) and can be formulated together with other medicament in adjuvant system in combination.3D-MPL is by Ribi Immunochem, the well-known adjuvant that Montana makes, and its manufacturing is described in GB 2122204.

To the Vaccine Design that the general discussion of the carrier that is used for oral immunity and adjuvant can be edited at Powell and Newman, The Subunit and Adjuvant Approach, PlenumPress, New York finds in 1995.

The present invention also is provided for by inoculating especially baby's method of people experimenter to the vaccine combination of the present invention that has the experimenter who needs to use effective dose.Suitably, attenuated live vaccine is by Orally administered.

Aspect concrete, with the preparation of rotavirus attenuated strain of the present invention and antacid so that the acid in the stomach is minimized the inactivation of vaccine.Suitable antacid composition comprises inorganic antacid, for example aluminium hydroxide Al (OH) ₃With magnesium hydroxide Mg (OH) ₂Be suitable for to comprise the Mylanta (trade mark) that contains aluminium hydroxide and magnesium hydroxide by the commercial antacid that obtains among the present invention.These antacids are water insoluble, give with suspensoid liquid form.

Aluminium hydroxide is a particularly suitable composition in the vaccine combination of the present invention, because it not only can provide the antacid effect, can also provide adjuvant effect.

What also be suitable for using as antacid in the vaccine of the present invention is organic antacid such as organic acid carboxylate.Suitable antacid in vaccine combination of the present invention contains the organic acid carboxylate, specifically is citrate, as sodium citrate or potassium citrate.

The specially suitable antacid that can use in vaccine combination of the present invention is that insoluble inorganic salt is calcium carbonate (CaCO ₃).Calcium carbonate also combine with rotavirus and the rotavirus activity be maintained during calcium carbonate combines.

Be the sedimentation of prevention calcium carbonate during filling step, thickening agent is present in the preparation suitably.

Operable possibility thickening agent comprise the pseudoplastic behavior excipient.Pseudoplastic behavior solution solution doubly is defined as with the viscosity under its stirring and compares, and has more full-bodied solution when leaving standstill.The excipient of this type is a natural polymer, as Radix Acaciae senegalis, gum tragacanth, agar, alginate, pectin or narrow and for example become second nature polymer: carboxymethyl cellulose (Tyloses

), methylcellulose (Methocels

Viscotrans

Tylose

With

), hydroxypropyl cellulose And hydroxypropyl emthylcellulose (Methocels

With

Viscontrans ).Usually, these pseudoplastic behavior excipient are used with thixotropic agent.Operable alternative thickening agent is the pseudoplastic behavior excipient with low fluid ability.These polymer produce structural fluid and arrange (structural fluid arrangement) when sufficiently high concentration, be formed on the high viscosity solution that has low fluid ability when leaving standstill.Need give certain quantity of energy to allow mobile and transmission (transfer) to this system.Need external energy (stirring) to destroy structural fluid arranges so that obtain fluid solution temporarily.

Examples of such polymers is

And xanthane gum.

The thixotroping excipient becomes gel structure when leaving standstill, under agitation then form fluid solution.

The example of thixotroping excipient is:

(zeopan) and Avicel (about 89% microcrystalline Cellulose and 11% sodium carboxymethyl cellulose).

Vaccine combination of the present invention comprises the thickening agent that is selected from xanthane gum or starch suitably.

Therefore, vaccine combination of the present invention is prepared with the combination of calcium carbonate and xanthane gum usually.

Other composition of the compositions of Shi Yonging comprises sugar for example sucrose and/or lactose suitably in the present invention.

Vaccine combination of the present invention can contain extra composition, comprises for example fumet (especially for oral vaccine) and antibacterial.

Expect that vaccine combination of the present invention has different expression form.

In a suitable embodiment, vaccine is used as liquid preparation.Suitably, this liquid preparation obtains reconstruct from following two components at least before using:

I) virus composition

Ii) liquid component

In this embodiment, virus composition and liquid component are present in the container separately usually, the wherein said container that separates can be the compartment that separates in the single vessel expediently, or in the vessel that separate, exist, the wherein said vessel that separate can according to mode like this connect so that final vaccine combination not ingress of air promptly be able to reconstruct.

Before reconstruct, virus can be dried forms or liquid form.Suitably, virus composition is by lyophilizing.Freeze dried virus is more stable than the virus in the aqueous solution.Freeze dried virus can use liquid antiacid compositions suitably in addition reconstruct to produce liquid vaccine preparation.Alternatively, freeze dried virus can water or aqueous solution be reconstructed, freeze dried in this case viral compositions contains the antacid composition suitably.

Suitably, the virus composition and this bacterin preparation that are included in a compartment or the vessel with calcium carbonate and xanthane gum preparation of bacterin preparation is reconstructed with water or the aqueous solution that is present in second compartment or the vessel.

In another embodiment, vaccine combination is a solid preparation, is applicable to dissolved immediately lyophilizing block when being placed in the mouth suitably.Lyophilized formulations can provide with the tablet form in the medicinal blister expediently.

In yet another aspect, the invention provides and be in the Rotavirus Vaccine that is used for Orally administered dissolving tablet form.

In yet another aspect, the invention provides the compositions of the attenuation Human reoviruslike agent strain that comprises attenuated rotavirus strain alive, especially lives, wherein said compositions is a dissolved immediately lyophilized solid in the time of can being placed in the mouth.

Suitably, dissolving tablet of the present invention enough promptly dissolves to prevent to swallow undissolved tablet in experimenter's mouth.This method is favourable to the department of pediatrics Rotavirus Vaccine especially.

Suitably, virus is the attenuation Human reoviruslike agent with the work of inorganic antacid such as calcium carbonate and thickening agent such as xanthane gum preparation.

Another aspect of the present invention provides such lyophilized formulations, and wherein virus composition is any rotavirus strain of preparing with calcium carbonate and xanthane gum.

Vaccine of the present invention can use the live virus of appropriate amount to prepare and use by known technology, with the effective protection that provides anti-rotavirus to infect, and does not have obvious deleterious side effect in the general vaccine.The normally every dosage 10 of the appropriate amount of live virus ⁴-10 ⁷Individual focus form unit (focus formingunits, ffu).The general dosage of vaccine can comprise every dosage 10 ⁵-10 ⁶Individual ffu and can give several dosage in a period of time scope for example gave two dosage at interval in two months.Yet, can be by surpassing 2 dosage, for example 3 dosage or 4 dosages and obtain an advantage are especially in developing country.Can be greater or less than two months the interval that gives between the dosage.Be used for single dosage or be used for the best live virus amount of a plurality of dosages and be used to give the Best Times of dosage can be by comprising that antibody titer and other research on standard of replying of observing the experimenter are determined.

Vaccine of the present invention also can comprise other the suitable live virus that is used for anti-other disease, for example poliovirus.Alternatively, the suitable live-virus vaccine of Orally administered other can separate administration, but with Rotavirus Vaccine compositions of the present invention simultaneously.

Test from 4-6 monthly age baby (through as " Vaccine (1998) " paper in as described in the P33 material inoculate) serum to the neutralization of P33, P38, P43 and 89-12C2.

For P33, P38 and P43, be similar with the titre scope in the serum of one-hundred-percent inspection.Statistical analysis does not show significant difference at the integral body of whole 3 kinds of viruses He on the titre.This shows that the conformation neutralizing epitope of P33, P38 and P43 and non-conformation neutralizing epitope are equal fully by the baby's who is inoculated by P33 anti-P33 serum identification.This observation shows that indirectly the neutralizing epitope that discloses does not change between P33, P38 and P43 in this analyzed in vitro method.

Yet, obviously be different from P33, P38 and P43 with the titre scope among the P89-12C2.This observation shows that the conformation neutralizing epitope of P33, P38 and P43 and non-conformation neutralizing epitope do not have to be discerned by the baby's who is inoculated by P33 anti-P33 serum on an equal basis fully.This observation shows that indirectly the neutralizing epitope that discloses changes between 89-12C2 and P33, P38 and P43 in this analyzed in vitro method.

The specially suitable embodiment of the present invention comprises:

1. be used for inducing the purposes of vaccine combination of the immunne response of anti-rotavirus in preparation from the rotavirus attenuated strain of P type, wherein said rotavirus is the P type different with the rotavirus in the described vaccine combination.

2. from P[8] the rotavirus attenuated strain of type is used for inducing anti-non-P[8 in preparation] purposes of the vaccine combination of the immunne response of rotavirus.

3. from G1P[8] the rotavirus attenuated strain of type is used for inducing anti-non-G1P[8 in preparation] purposes of the vaccine combination of the immunne response of rotavirus.

4. according to the purposes of 1-3 item, wherein anti-G1P[8] immunne response of the viral infection that causes of type also induced.

5. according to the purposes of 1-4 item, wherein induced at the immunne response of two or more rotavirus serotypes, these serotypes are by being defined with reference to G type or P type.

6. according to the purposes of 1-5 item, wherein the serotype of vaccine strain is that G1 serotype and non-G1 serotype are selected from G2, G3, G4, G5, G6, G7, G8, G10, G11, G12, G13 and G14.

7. according to 6 purposes, wherein induced at the immunne response of G1 type and G2 type simultaneously.

8. according to 1-5 purposes arbitrarily, wherein the type of vaccine strain is P[8] type and non-P[8] type is selected from P[1], P[2], P[3], P[4], P[5], P[6], P[7], P[9] and P[11] type.

9. according to 8 purposes, wherein simultaneously at P[8] type and P[4] immunne response of type induced.

10. according to 1-9 purposes arbitrarily, wherein compositions comprises the rotavirus with VP4 gene like this, and this VP4 gene comprises at least a of following base in nucleotide sequence: from start codon in the position 788 adenine base (A), in the position 802 adenine base (A) and in the position 501 thymine alkali bases (T).

11. according to 10 purposes, wherein the VP4 gene comprises nucleotide sequence like this, this nucleotide sequence comprise from start codon in the position 788 and 802 adenine base (A) and in the position 501 thymine alkali bases (T).

12. purposes according to 11, wherein compositions comprises the rotavirus with VP7 gene like this, and this VP7 gene comprises at least a of following base in nucleotide sequence: from start codon in the position 605 thymus pyrimidine (T), in the position 897 adenine (A) and in the position 897 guanine (G).

13. according to 12 purposes, wherein the VP7 gene comprises nucleotide sequence like this, this nucleotide sequence comprise from start codon in the position 605 thymus pyrimidine (T) and in the position 897 adenine (A) or guanine (G).

14. according to 1-13 purposes arbitrarily, wherein compositions comprises the rotavirus with VP4 gene like this, this VP4 gene in nucleotide sequence, comprise from start codon in the position 788 and 802 adenine (A) and in the position 501 thymus pyrimidine (T); Wherein the VP7 gene in nucleotide sequence, comprise from start codon in the position 605 thymus pyrimidine (T) and in the position 897 adenine (A).

15. purposes according to any of 1-14, wherein compositions can weaken or prevent gastroenteritis and/or diarrhoea, wherein said gastroenteritis and/or diarrhoea are caused that by the infection of veriform rotavirus wherein said veriform rotavirus is by being defined with reference to the G type and/or the P type attenuated rotavirus that are present in the compositions.

16. purposes according to 15; wherein compositions has at least 40% protection of anti-serious gastroenteritis in the individual colony of inoculation; wherein said serious gastroenteritis causes by the infection of the 2 strain rotavirus that obtain defining by reference G type and/or P type at least, these types be present in compositions in the G1P[8 of attenuated rotavirus] type is different.

17. according to 16 purposes, wherein serious gastroenteritis is caused by the infection of the rotavirus of 3,4 non-G1 serotypes at least at least.

18. according to 17 purposes, wherein non-G1 serotype is G2, G3, G4 and G9 serotype arbitrarily.

19. according to 18 purposes, wherein serious gastroenteritis is by at least 2 non-P[8] rotavirus infection of type causes.

20. according to 9 purposes, wherein serious gastroenteritis is by P[4] the type rotavirus infection causes.

21. according to 1-20 purposes arbitrarily, wherein the rotavirus strain is an ECACC preservation thing 99081301, or from ECACC preservation thing 99081301 is available maybe can be from wherein deutero-.

23. according to the purposes of any of 1-20, wherein vaccine is used in 2 dosages.

In yet another aspect, the invention still further relates to the method for inducing at from the immunne response of the rotavirus infection of rotavirus strain, this method comprises using to the experimenter and comprises from the compositions of the rotavirus attenuated vaccine of homophyletic not.Particularly, the present invention relates to be used to induce at from the abductive approach of the immunne response of the rotavirus of a P type and/or be used to prevent and method from the rotavirus infection diseases associated of a P type, this method comprises to the patient that needs are arranged uses attenuated rotavirus group from different P types.

Of the present invention concrete aspect, provide and induce at rotavirus P [8] type and be selected from P[1], P[2], P[3], P[4], P[5], P[6], P[7], P[9], P[11], P[12], P[14] and P[19] at least a non--P[8 in the type] type, suitably at P[4] method of the immunne response of rotavirus type, this method comprises to the experimenter uses the compositions that comprises rotavirus P [8] type vaccine.

Aspect another, provide i on the present invention ground) isolating nonstructural proteins 4 (NSP4) or its immunogenic fragments that has as described protein sequence in Fig. 5 (SEQ ID NO:7); Ii) isolating polynucleotide sequence, it comprises the nucleotide sequence of the described NSP4 polypeptide of coding or its immunogenic fragments; Iii) isolating polynucleotide sequence, it comprises as described nucleotide sequence in Fig. 6 (SEQ ID NO:8).

Aspect another, provide i of the present invention) isolating rotavirus protein 6 (VP6) or its immunogenic fragments that has as described protein sequence in Fig. 7 (SEQ ID NO:9); Ii) isolating polynucleotide sequence, it comprises the nucleotide sequence of the described VP6 polypeptide of coding or its immunogenic fragments; Iii) isolating polynucleotide sequence, it comprises as described nucleotide sequence in Fig. 8 (SEQ ID NO:10).

Immunogenic fragments can be defined as the fragment when the protective immune response that excites anti-rotavirus to infect with effective dose (individually or as the hapten that is connected with carrier) when using in the context of the present invention.

Following non-limiting example explanation the present invention.

Embodiment

Embodiment 1: the 89-12 strain that confirms the 26th generation (P26) is the variant mixture

Order-checking to the VP4 gene and the VP7 gene of the lot number that goes down to posterity from difference

VP4 gene and VP7 gene from P26 generation (former generation AGMK cell), P33 generation ((with respect to former generation) AGMK cell line of having set up), P41 generation and P43 generation are checked order.Total RNA extract is able to reverse transcription and amplification by PCR in single tube/single stage.

Primer Rota5bis and the complete VP4 gene of Rota29bis amplification, primer Rota1 and the complete VP7 gene of Rota2bis amplification.The PCR material has used different primers check order (seeing Table 1).

The P26 generation sequence has 3 bases different (at position 501,788 and the 802bp from start codon) and 3 base differences (from the position 108,605 and the 897bp of start codon) is arranged in VP7 with the P33 generation sequence in VP4.

The P26 generation sequence scintigram of VP4 and VP7 has shown at sudden change position existence P33 generation sequence as a setting.Therefore, can see P26 generation is the mixture of at least 2 kinds of variants.

P33 generation sequence scintigram as if in VP4 be homogeneous and be heterogeneous (seeing Table 2) for VP7.

In P38 generation (derived from P33 generation), go down to posterity on the Vero cell 5 times, and show as P33 for identical VP4 sequence and VP7 sequence set (AGMK cell line).Therefore between P33 and P38 group, there is not great change.

Table 1: the oligonucleotide that is used for RT-PCR and order-checking

Table 2: used oligonucleotide in the hybridization

	Title	Sequence	The position
				VP7	Rota41 Rota42	AGT ATT TTA TAC TAT AGT AGA TTA TAT TAA TC(SEQ ID NO：37) AGT ATT TTA TAC TAT GGT AGA TTA TAT TAA TC(SEQ ID NO：38)	882-913 882-913
VP4	Rota15 Rota16 Rota35 Rota36	ATC CCC ATT ATA CTG CAT TCC TTT C(SEQ ID NO：39) ATC CCT ATT ATA CTG CAT TTC TTT C(SEQ ID NO：40) ATC CCC ATT ATA CTG CAT TTC TTT C(SEQ ID NO：41) ATC CCT ATT ATA CTG CAT TCC TTT C(SEQ ID NO：42)	807-783 807-783 807-783 807-783

The base that shows with capitalization in the table 2 is the position of the concrete sequence variations among VP4 and the VP7.

The sequence variations of table 3:VP4 gene and VP7 gene

Table 3.1

Attention: in from second clone who is developed to 3 clones that produce batch level, the nucleotide of VP7 897bp position is G, rather than as the A among the selected P43 clone.This causes the isoleucine in the methionine replacement aminoacid sequence.So variant is excreted in the baby's who uses the inoculation of P33 material the feces, and wherein said variant is with selected P43 clone and wherein exist the clone of G corresponding from start codon at 897bp in VP7.

In table 3.1, wherein in ad-hoc location, exist under the situation of two alternative bases, first base representative in two bases comes across the base in most of colony, and second base is to come across the intragroup base of fraction.Main variation colony and secondary variance colony are judged by the intensity of signal in the order-checking.

Table 3.2

Table 3.2 shows between variant because of the amino acid change due to the nucleotide difference.

Table 4

Slot blot hybridization

Variation on the AGMK cell in the colony of P26-P33 between generation is further confirmed by slot blot hybridization.VP4 genetic fragment that produces by RT/PCR and VP7 genetic fragment with to the special oligonucleotide probe hybridization of every kind of variant (seeing Table 3.1 and 3.2).Be different from Rota16, Rota35 and Rota36 hybridization not the P26 with Rota15 hybridization, the VP4 PCR fragment of P33 material in the position 788 and 802 only with Rota16 hybridization not with Rota15 or Rota35 or Rota36 hybridization.These results establish and have at least 3 kinds of variants (seeing Table 4) in the P26.

For the VP7 PCR fragment of P33 material, position 897 and Rota41 and Rota42 hybridization.These results establish and have at least 2 kinds of variants in the P33.

Embodiment 2: to P43 clone's separation and sign

For separating P33 composition, on the Vero cell, carry out 3 times of terminal point dilutions of P33/AGMK and use the virus that obtains to come vero cells infection as the homogeneous virus groups.

As conventional institute does, use the positive hole of two Standard Selection: separate best positive hole by growth and dull and stereotyped going up that the kitchen range speckle of detected maximum number in the hole is showed.In 96 hole microtitration plates behind 3 times of terminal point diluted passages, successfully increased on the Vero cell and assessed with regard to its output in 10 positive holes.

Based on output, 3 clones are developed to the level that goes down to posterity of producing batch (production lot).Show that polyclonal antibody is being similar to immunity identification between the P33 between these 3 clones and described clone.Clone's homogeneity is by the assessment of slot blot hybridization method.Single clone's final selection is based on output and sequence.

Selected clone obtains increasing by continuous passage on the Vero cell and criticizes to produce main seed, work seed and final production.

The selected difference that is cloned in goes down to posterity on the level by carrying out the heredity sign to VP4 and VP7 order-checking (identity) and by the specificity slot blot hybridization to VP4 in the material that PCR increases and VP7.The sequence of VP4 gene and VP7 gene provides in Fig. 1 and Fig. 2 respectively and identical with P41 in the P43 material.

Selected clone's homogeneity is assessed by the selective cross that uses oligonucleotide probe, and wherein said oligonucleotide probe is distinguished every kind of variant (seeing Table 4) of being identified during the former generation AGMK order-checking of P26/ VP4 district and/or the nucleotide in the VP7 district change.

VP4 fragment and Rota16 are hybridized and are not hybridized with Rota15, Rota35 or Rota36.VP7 fragment and Rota41 are hybridized and are not hybridized with Rota42.

These results confirm that P43 is a homogeneous colony.

Embodiment 3: remove potential adventitious viruses

In (the AGMK growth) P33, add ether, continue 1 hour to final concentration 20%.Make ether with the N that is blown into subsequently ₂Discharge, continue 35 minutes.Do not observe influence to P33 seed titre.

Embodiment 4: the preparation of attenuated live vaccine

Aforesaid production is criticized by following method and is obtained preparation so that use to baby oral by following method.

1. Freeze dried virus

Use standard technique to prepare viral agent.Until required standard virus concentration, is 10 with the virus stock solution used thawing of refrigerated purification and with suitable culture based composition and use thereof in packaging dilution (being Dulbecco ' s improvement Eagle culture medium in this example) in this example ^6.2Ffu/ml.The virus of dilution further uses lyophilizing stabilizing agent (sucrose 4%, dextran 8%, sorbitol 6%, aminoacid 4%) dilution until the purpose virus titer subsequently, is 10 in this example ^5.6The ffu/ agent.The aseptic 3ml bottle that is transferred to of 0.5ml aliquot with the viral compositions of stabilisation.Every bottle partly seals with rubber closure subsequently, and sample is lyophilization under vacuum subsequently, subsequently bottle is sealed fully and aluminum medicated cap turnup around the bottle is put in place so that rubber closure is in place.

For use, virus is with the in addition reconstruct of one of following antiacid reconstructors:

(a) citrate reconstructors

Sodium citrate is dissolved in the water, carries out filtration sterilization and with amount (every 1.5ml dosage 544mg trisodium citrate 2H of 1.5ml ₂The concentration of O) aseptic being transferred in the reconstruct container.The reconstruct container can for example be 3ml bottle or 4ml bottle or 2ml syringe or be used for Orally administered flexible plastic and can push capsule.Be in substituting under the aseptic condition as keeping aseptic composition, last container can be through autoclaving eventually.

(b) Al (OH) ₃ Reconstructors

Aseptic Actal Suspension (Mylanta-trade mark) carries out aseptic dilution in sterilized water, with the aseptic reconstructors container (for example 2ml syringe, or flexible plastic can push capsule) that is transferred to of the amount of 2ml, wherein said each amount contains 48mgAl (OH) ₃Use substituting of aseptic composition down as aseptic condition, Actal Suspension (preferably under the stage of dilution) is carried out gamma-radiation.

Comprise standard ingredient to prevent this suspension precipitation.This type of standard ingredient comprises for example magnesium stearate, carboxymethyl cellulose, hydroxypropyl emthylcellulose, microcrystalline Cellulose and siloxane polymer.Can comprise that also for example butyl hydroxybenzoate, propyl hydroxybenzoate or other are used for the standard antibacterial and the fumet of food to antibacterial.

2. contain Al (OH) in the liquid preparation ₃ Lyophilizing virus

Use standard technique to prepare viral agent.Until required standard virus concentration, is 10 with the virus stock solution used thawing of refrigerated purification and with suitable culture based composition and use thereof in packaging dilution (being Dulbecco ' s improvement Eagle culture medium in this example) in this example ^6.2Ffu/ml.Add Actal Suspension reach end amount 48mg/ agent eventually and viral compositions with lyophilizing stabilizing agent (can be sucrose, dextran or aminoacid 4%, or gelatin, or phytone, or xanthane) dilution until the purpose virus titer, be 10 in this example ^5.6The ffu/ agent.The aseptic 3ml bottle that is transferred to of 0.5ml aliquot with the viral compositions of stabilisation.Subsequently described in part 1 to bottle lyophilizing and sealing.

3. What be used for blister contains Al (OH) ₃ Lyophilizing virus

Use standard technique to prepare viral agent.Until required standard virus concentration, is 10 with the virus stock solution used thawing of refrigerated purification and with suitable culture based composition and use thereof in packaging dilution (being Dulbecco ' s improvement Eagle culture medium in this example) in this example ^6.2Ffu/ml.Add Actal Suspension reaching end amount 48mg/ agent eventually and viral compositions with can being sucrose, dextran or aminoacid 4%, or gelatin, or phytone, or the lyophilizing stabilizing agent of xanthane dilutes until purpose virus titer 10 ^5.6Individual ffu/ agent.Adopt the sterile filling operation with 0.5ml dosage or preferably still less dosage be transferred in the blister cavity.The lyophilizing said composition, and by heat sealing method sealing blister cavity.

Randomly comprise standard ingredient to prevent this suspension precipitation.This type of standard ingredient comprises for example magnesium stearate, carboxymethyl cellulose, hydroxypropyl emthylcellulose, microcrystalline Cellulose, and siloxane polymer.Also can comprise fumet.

Embodiment 5: the titration of virus that several formulations is carried out rotavirus

5.1: reach based on the comparison between the preparation of sucrose based on lactose:

Table 5

Lot number	Preparation compositions	Virus titer before the lyophilizing	Lyophilizing and the virus titer after 37 ℃ of following 1 weeks
				98G06/01	Lactose: 2%; Dextran: 4%; Sorbitol: 3%; Aminoacid: 2%	10 5.22	10 4.67
98G06/03	Sucrose: 2%; Dextran: 4%; Sorbitol: 3%; Aminoacid: 2%	10 5.28	10 4.92

P43 rotavirus and sucrose and prepared shown in as above showing with lactose.

Titration of virus before the lyophilizing is in finishing the liquid of preparation (containing sucrose, dextran, sorbitol and aminoacid) and the virus titer when not having step of freeze drying.

Good results is result like this, is wherein realizing on the step of freeze drying that＜0.5 logarithm descends and realizing that＜0.5 logarithm descends 1 week during 37 ℃ (stability test of acceleration).

The accuracy of titration of virus is+or-0.2 logarithm.

This result shows that sucrose can be used to replace lactose.

5.2: arginine and hydrogenated maltose are replaced the effect of sorbitol:

Table 6

Lot number	Preparation compositions	After the lyophilizing at the virus titer of zero-time	Lyophilizing and the virus titer after 37 ℃ of following 1 weeks
				98L16/01	Lactose: 2%; Dextran: 4%; Sorbitol: 3%; Aminoacid: 2%	10 4.8	10 4.8
98L16/02	Lactose: 2%; Dextran: 4%; Sorbitol: 3%; Aminoacid: 2% arginine: 3%	10 4.8	10 4.9
				98L16/04	Lactose: 2%; Dextran: 4%; Maltose alcohol: 3%; Aminoacid: 2% arginine: 3%	10 4.7	10 5

This result confirms that arginic interpolation (known this improves the stability of virus during lyophilizing and provide alkaline medium so that offset the acidity of stomach) keeps virus titer.

Sorbitol always reduces the glass transition temperature of lyophilizing block significantly.As implied above, this can substitute by the use maltose alcohol, and sorbitol is overcome and virus titer is still kept.

5.3: the several formulations compositions

This test confirms that several formulations is feasible.

Table 7

Lot number	Preparation compositions	Virus titer before the lyophilizing	Lyophilizing and the virus titer after 37 ℃ of following 1 weeks
				99C11/01	Sucrose: 2%; Dextran: 4%; Sorbitol: 3%; Aminoacid: 2%	10 5.24	10 5.07
99C11/02	Sucrose: 2%; Dextran: 4%; Maltose alcohol: 3%; Aminoacid: 2%	10 5.09	10 4.92
				99C11/04	Dextran: 4%; Maltose alcohol: 3%; Aminoacid: 2%	10 4.89	10 5.06

Lot number	Preparation compositions	After the lyophilizing at the virus titer of zero-time	Sick lyophilizing and the virus titer after 37 ℃ of following 1 weeks
				99C17/01	Sucrose: 2%; Dextran: 4%; Sorbitol: 3%; Aminoacid: 2%	10 5.40	10 5.41
99C17/02	Sucrose: 2%; Dextran: 4%; Sorbitol: 1.5%; Aminoacid: 2%	10 5.30	10 4.93
				99C17/03	Sucrose: 2%; Dextran: 4%; Aminoacid: 2%	10 5.31	10 5.24
99C17/04	Sucrose: 2%; Dextran: 4%; Hydrogenated maltose: 3%; Aminoacid: 2%	10 4.42	10 4.45

99C17/05	Sucrose: 2%; Dextran: 4%; Maltose alcohol: 1.5%; Aminoacid: 2%	10 4.39	10 4.40
				99C17/06	Sucrose: 2%; Dextran: 4%; Sorbitol: 3%;	10 5.44	10 4.97
99C17/07	Sucrose: 2%; Dextran: 4%; Sorbitol: 1.5%;	10 5.11	10 4.89

5.4: rotavirus and Al (OH) ₃Combination between antacid:

Table 8

Rotavirus	Al(OH) 3	H 2O	In the time of contact of room temperature	Centrifugal	The supernatant virus titer, the ffu/ml of unit	Virus titer in the precipitation, the ffu/ml of unit
							10 5.6ffu/ml	48mg in 0.240ml	0.76ml	30 minutes	8000 rev/mins, 10 minutes	10 3.66
10 5.6ffu/ml	0.48mg in 0.240ml	0.76ml	30 minutes	8000 rev/mins, 10 minutes	10 4.41
							10 5.6ffu/ml		1ml	30 minutes	8000 rev/mins, 10 minutes	10 5.68
Rotavirus in the lyophilizing block	12mg in 0.120ml	1.380ml	30 minutes	8000 rev/mins, 10 minutes	Be lower than detectability	10 4.7

Al (OH) ₃Be used as antacid.This shows rotavirus and insoluble inorganic salt (Al (OH) ₃) combination, because rotavirus is with Al (OH) ₃Obtain centrifugal (virus activity in the supernatant reduces) together.

5.5: dissolve Al (OH) by sodium citrate before the titration of virus ₃Antacid

Table 9

Virus sample	Dissolving	Condition	Virus titer ffu/ml
				Liquid preparation before the 99B10/06 lyophilizing; 10 5.43	1.5ml trisodium citrate	At room temperature 24 hours	10 5.11
99B10/06: lyophilizing 10 5.43	1.5ml trisodium citrate	At room temperature 24 hours	10 4.53

As rotavirus and Al (OH) ₃In conjunction with the time, might (comprise Al (OH) by the lyophilizing total material ₃).After the lyophilizing, might be by in sodium citrate, dissolving Al (OH) ₃And the recovery rotavirus.This step is not destroyed rotavirus and keep the rotavirus activity behind this dissolving step.

5.6: removing Al (OH) ₃Combine the infectivity of back rotavirus with rotavirus:

(by the dissolving carrier) viral releasing mechanism (in vivo) in vivo takes place well.In fact below pH6, aluminium hydroxide is soluble fully, so rotavirus is released at gastric.

Al (OH) ₃+ 3H ⁺The Al of---→ ⁺⁺⁺(water solublity)+3H ₂O

Under one's belt, Al ⁺⁺⁺Ion is not absorbed (J.J.Powell, R.Jugdaohsingh and R.P.H.Thompson, regulating action (The regulation of mineraladsorption in the gastrointestinal track) to the absorption of gastrointestinal tract mineral, Proceedings of the NutritionSociety (1999), 58,147-153).

In small intestinal, because of pH increases, the aluminum of insoluble form (Al (OH) ₃Or AlPO ₄) separate out, and be eliminated by natural approach.

Al (OH) for new formation ₃(or AlPO ₄) whether precipitate can combine with free rotavirus again is unknown.This has proposed Al (OH) ₃The infectious problem of-rotavirus coalition itself.

Also might make rotavirus from Al (OH) by other mechanism ₃Discharge in-rotavirus the coalition.For example, the lysine viral interference is at Al (OH) ₃On absorption.

Known other anion such as borate, sulfate, carbonate and phosphate are adsorbed on the aluminium hydroxide specifically, therefore, in theory, should might (by the competitive Adsorption site) from Al (OH) ₃Displace rotavirus in the-rotavirus coalition.

Therefore, rotavirus can be from rotavirus-Al (OH) ₃It is active that the rotavirus that is discharged in the coalition and discharge keeps.

This release can by gastric by HCl or external by trisodium citrate) dissolving Al (OH) ₃Or by finishing by basic amino acid (lysine) displacement rotavirus.

5.7:Al (OH) ₃The infectivity of-rotavirus coalition

The single dose water of lyophilizing rotavirus is reconstructed and separated into two parts.The first that regards reference as accepts the water of additional volumes.Second portion accepts to be suspended in the 24mgAl (OH) in the 0.240ml water ₃(preclinical titration of virus).

As Al (OH) ₃When existing, rotavirus is activated and the titration of virus value is compared higher with reference sample.

Repeat this experiment and do not tell freeze dried dosage, and add 12mg Al (OH) ₃Or 24mgAl (OH) ₃

Here, sample is the sample with citrate-bicarbonate buffer reconstruct.Therefore, virus titer is at Al (OH) ₃Exist still higher down.

DRVC003A46 DRVC003A46 DRVC003A46

+ + +

1.5ml WL at 0.120ml in 0.240ml

12mg Al (OH) in the buffer ₃24mg Al (OH) ₃

+ +

1.380ml H ₂O 1.260ml H ₂O

5.34 6.24 6.05

5.32 5.95 6.26

As among the above embodiment, rotavirus and Al (OH) ₃The particle combination is because virus can be precipitated by centrifugal.DRVC003A46 is the rotavirus (sucrose: 2% of lyophilizing preparation; Dextran: 4%; Sorbitol: 3%; Aminoacid: 2%).

SDSAA=sucrose 2%, dextran 4%, sorbitol 3%, aminoacid 2%.

According to the titration of virus of on supernatant, implementing, the absorption required Al of rotavirus (OH) ₃Seemingly low (the beginning) of amount with freeze dried dosage 5.7 logarithms amplify titration of virus in proportion):

Table 10

Al(OH)3	Adsorption time	Titre in supernatant
12mg	Room temperature	1 hour				2.7
			24mg	Room temperature	1 hour		3.4
48mg	Room temperature	1 hour				3.4
			72mg	Room temperature	1 hour		2.0
96mg	Room temperature	1 hour				Be lower than detectability
12mg	Spend the night	2.7
			24mg	Spend the night	Be lower than detectability
48mg	Spend the night	2.5
12mg	Immediately	Be lower than detectability
			24mg	Immediately	2.0
48mg	Immediately	Be lower than detectability

Rotavirus is adsorbed on Al (OH) ₃The upward required time is seemingly of short duration:

With a freeze dried rotavirus of dosage at 24mg Al (OH) ₃Be reconstructed under existing, and centrifugal after 0 minute, 15 minutes, 60 minutes and 24 hours." culot " is resuspended in the SDSAA before titration of virus:

Table 11

Time	Culot	Supernatant
			0 minute	5.26	3.17
15 minutes	5.34	＜1.44
			60 minutes	5.96	＜1.44
24 hours	6.13	＜1.44

5.8: use CaCO ₃As antacid

For fear of in vaccine, using aluminum, antacid Al (OH) ₃With another insoluble inorganic salt CaCO ₃(calcium carbonate) replaced.

To CaCO ₃Observed phenomenon with to Al (OH) ₃The phenomenon unanimity of describing:

-rotavirus combines with inorganic salt;

-when combining with inorganic salt, the rotavirus activity is kept;

-might dissolve inorganic base and from coalition, discharge rotavirus by acid;

-might make antacid and the common lyophilizing of rotavirus.

CaCO ₃ Combine with rotavirus

In first test, freeze dried rotavirus (virus titer 5.7) is used CaCO ₃Suspension in water (50mg among the 1.5ml) reconstruct; And centrifugal subsequently, and the virus titer and the sedimentary virus titer of supernatant compared.

This shows rotavirus and CaCO above 90% ₃In conjunction with.

When virus in conjunction with the time, also might discharge this titre and recover primary virus quantity.

In addition, virus titer is slightly higher than no CaCO ₃Following resulting virus titer.

CaCO ₃ With the bonded amount of rotavirus

Freeze dried rotavirus is used in the CaCO in the water (1.5ml) ₃Suspension reconstruct:

10mg

50mg

100mg

And centrifugal subsequently, and the virus titer of supernatant and the virus titer of culot compared.

Table 12

Obviously, more CaCO ₃Combine with how viral, and virus still less is present in the supernatant.

Yet, all dosage thoroughly be not restored (expection amount at least 5.3 or as previous resulting even 5.8-see above).

CaCO during the titration of baby Rossett-Rice antacid ₃ Protection to rotavirus

Use 10 freeze dried rotavirus of dosage (DRVC003A46) and 50mg CaCO ₃, implement two types baby Rossett-Rice titration:

In traditional Rossett-Rice titration, antacid mixes with rotavirus and HCl is toppled over so far in the medium.

In " reverse " baby Rossett-Rice, situation is just in time opposite: antacid is splashed into (as taking place in vivo) in the HCl pond.

Table 13

Therefore, in this experiment in vitro, calcium carbonate can protect about 20% rotavirus to avoid the influence that HCl exists, and aluminium hydroxide can not be accomplished this point.

5.9: at CaCO ₃There is the lyophilizing of rotavirus down in antacid:

Table 14

Lot number	Compositions	After the lyophilizing at the virus titer of zero-time	Lyophilizing and the virus titer after 37 ℃ of following 1 weeks
				99K08/01	Sucrose: 2% dextran: 4% sorbitol: 3% aminoacid: 2% CaCO 3：50mg	10 5.28	10 5.10
99K08/02	Sucrose: 2% dextran: 4% sorbitol: 3% aminoacid: 2% CaCO 3：60mg	10 5.16	10 5.15
				00C24/01	Sucrose: 2% dextran: 4% sorbitol: 3% aminoacid: 2% CaCO 3: 60mg xanthane 0.3%	10 5.07	10 4.69
00C24/03	Sucrose: 2% dextran: 4% sorbitol: 3% aminoacid: 2% CaCO 3: 60mg xanthane 0.3%	10 5.07	10 4.85
				00E09/25	Sucrose: 2% dextran: 4% sorbitol: 3% aminoacid: 2% CaCO 3: 60mg xanthane 0.25%	10 5.03	10 4.91

00E09/30	Sucrose: 2% dextran: 4% sorbitol: 3% aminoacid: 2% CaCO 3: 60mg xanthane 0.30%	10 5.01	10 4.87
				00F26/06	Sucrose: 2% dextran: 4% sorbitol: 3% aminoacid: 2% CaCO 3: 60mg starch: 2%	10 4.50	10 4.70

This is " whole preparation " i.e. lyophilizing rotavirus and antacid (CaCO together in same bottle ₃).For preventing CaCO during the fill step ₃Sedimentation, need thickening agent.The example of this type of thickening agent comprises xanthane gum and starch.Rotavirus is active even be maintained in the presence of xanthane gum and starch.

5.10 the lyophilizing tablet of rapid disintegrate when being used for being placed on mouth:

Following series preparation confirms instant (" lyoc ") notion.Lyophilizing block dissolving rapidly in mouth just.

Table 15

Lot number	Preparation compositions	After the lyophilizing at the virus titer of zero-time	Lyophilizing and the virus titer after 37 ° of following 1 weeks
				00C24/05	Sucrose: 2% dextran: 4% sorbitol: 3% aminoacid: 2% CaCO 3: 60mg xanthane 0.3%	10 5.02	10 4.54
00C24/06	Sucrose: 2% dextran: 4% sorbitol: 3% aminoacid: 2% CaCO 3: 60mg xanthane 0.3%	10 4.86	10 4.56
				00F26/11	Sucrose: 1% dextran: 2% sorbitol: 1.5% aminoacid: 1% CaCO 3: 60mg starch: 2%	10 4.70	10 4.40

In " instant notion ", xanthane and starch all can obtain using (keeping the instant characteristic of separating of lyophilizing block).

Embodiment 6: calcium carbonate is used for the Rotavirus Vaccine compositions as antacid

Work as CaCO ₃When the suspension in water is used for rotavirus as antacid, have such problem, rapid sedimentation when promptly calcium carbonate particle is statically placed in water, reason be the powder density value near 2.6 and particle mean size be 30 μ m.

This sedimentation can delay in the following way:

1 increases the density of surrounding medium

2 increase the viscosity of surrounding medium

3 dwindle particle diameter

4 keep particle to be separated from each other

6.1: the density that increases surrounding medium:

Work as CaCO ₃-aqueous suspension (in placing syringe) (contains sucrose 2% at the lyophilizing block; Dextran 4%; Sorbitol 3%; Aminoacid 2%) go up when placing, increase the density of surrounding medium, but CaCO ₃The unobvious and CaCO of settled speed ₃-aqueous suspension difference.

6.2 increase the viscosity of surrounding medium:

The pseudoplastic behavior excipient

Pseudoplastic behavior solution is defined as with the viscosity under its stirring compares, when leaving standstill, have more full-bodied solution

The usual excipients of the type is:

Natural polymer is for example:

Radix Acaciae senegalis

Gum tragacanth

Agar

Alginate

Pectin

Narrow and for example become second nature polymer:

Carboxymethyl cellulose (Tyloses

)

Methylcellulose (Methocels

Viscotrans

Tylose With )

Hydroxypropyl cellulose

Hydroxypropyl emthylcellulose (Methocels

With

Viscontrans

)

Usually, these pseudoplastic behavior excipient use with thixotropic agent.

Pseudoplastic behavior excipient with low fluid ability

These polymer When sufficiently high concentrationProduce structural fluid and arrange, be formed on the high viscosity solution that has low fluid ability when leaving standstill.Need give certain quantity of energy to allow mobile and to shift to this system.Need external energy (stirring) to destroy structural fluid arranges so that obtain fluid solution temporarily.

Examples of such polymers is

And xanthane gum

The thixotroping excipient

By these excipient, when leaving standstill, obtain gel structure; And under agitation obtain fluid solution.

The example of thixotroping excipient is:

(zeopan) and Avicel

(about 89% microcrystalline Cellulose and 11% sodium carboxymethyl cellulose).

6.3 reduce particle diameter

CaCO ₃The decline that reduces to cause the antiacid capacity of this chemical compound of particle diameter.

6.4 keep particle to be separated from each other

This

With

In be exactly so, just will compare CaCO ₃The insoluble particle that particle is littler (about 1 μ m) places CaCO ₃Between the particle, to prevent gathering.

Embodiment 7: product design

The product design example that following schematic diagram shows is possible.

7.1 the CaCO in syringe ₃

After allowing the rotavirus of clinical batch (clinical batche) be positioned at the lyophilizing bottle, antacid can be placed in the reconstituted liquid that is contained in syringe.

In this product form, CaCO ₃Be deposited in during the fill step and the whole storage life of product between (at least 2 years) must be controlled.

7.2 the CaCO in the lyophilizing bottle ₃

7.3. the lyophilizing in blister

In the present example, rotavirus, CaCO ₃Directly in blister, carry out lyophilizing together with xanthane gum.

Embodiment 8: to the not lyophilizing of homophyletic of rotavirus

Table 16

Lot number	Rotavirus strain	Preparation compositions	Virus titer after the lyophilizing during t=0	Lyophilizing and the virus titer after 37 ° of following 1 weeks
					00F26/01	N ° of 61 PRO/0232 of G1 SB purity	Sucrose: 2% dextran: 4% sorbitol: 3% aminoacid: 2%	10 4.6	10 4.7
00F26/02	G2(DS-1)	Sucrose: 2% dextran: 4% sorbitol: 3% aminoacid: 2%	10 4.4	10 4.4
					00F26/03	G3(P)	Sucrose: 2% dextran: 4% sorbitol: 3% aminoacid: 2%	10 4.6	10 4.5
00F26/04	G4(VA-70)	Sucrose: 2% dextran: 4% sorbitol: 3% aminoacid: 2%	10 4.8	10 4.8
					00F26/05	G9(W161)	Sucrose: 2% dextran: 4% sorbitol: 3% aminoacid: 2%	10 4.6	10 4.5

DS-1 strain, P strain and VA70 strain in the 361st page of nineteen ninety Raven publishing house " Fields " second edition respectively the Human reoviruslike agent as serotype G2, G3 and G4 obtain describing with reference to strain.

In this experiment, make different rotavirus strain lyophilizing.

For whole rotavirus poison, not only virus titer is kept during lyophilizing, and accelerated stability (37 ℃ of following 1 weeks) is confirmed.

Embodiment 9: the I phase safety research in the adult of an Orally administered Rotavirus Vaccine

Implementing the I phase studies to assess the single oral dose 10 of P43 vaccine ^6.0Safety and the reactionogenicity of ffu in age 18-45 year health adult.

Clinical research is double blinding and randomized.It is that placebo-contrast and oneself comprise (self-contained).The research is implemented at Belgian one single center.

9.1. research colony

Recruited and amounted to 33 experimenters, wherein 11 in placebo group, and 22 in vaccine group, and all the experimenter finishes research.All the volunteer is white people.They are 35.3 years old in the mean age in when inoculation, and scope is in 18-44 year.Test began and only carries out one-month period in the January.

9.2. material

Vaccine

Clinical batch of the P43 vaccine according to good manufacturing practice (Good Manufacturing Practices) produce, purification, preparation and lyophilizing.Each batch is according to quality control and quality assurance (QualityControl and Quality Assurance) distribution.Every bottle vaccine contains following ingredients:

Active component:

P43 strain minimum 10 ^5.8Ffu

Excipient, stabilizing agent:

Sucrose 9mg

Dextran 1 8mg

Sorbitol 13.5mg

Aminoacid 9mg

Placebo

The placebo of preparation and distribution bottle.Every bottle placebo contains following ingredients:

Excipient, stabilizing agent:

Sucrose 9mg

Dextran 1 8mg

Sorbitol 13.5mg

Aminoacid 9mg

Diluent

Use water for injection as diluent with reconstruct vaccine and placebo.

9.3. use

Before using vaccine or placebo approximately 10-15 minute, the experimenter 10ml's of two groups of orally give

It is the antacid of registration.This antacid increases gastric pH, prevents that rotavirus from passing through inactivation between gastric phase.

For the preparation vaccine, every bottle is contained 10 ^5.8The freeze dried P43 of two bottles of ffu is water for injection reconstruct with the 1.5ml diluent.This realizes every dose of virus titer that is calculated 10 ^6.1Ffu.The vaccine of reconstruct is used rapidly as single oral dose.

Be the preparation placebo, be water for injection reconstruct with the freeze dried placebo of two bottles with the 1.5ml diluent and use as single oral dose.

9.4. safety and reactionogenicity

Adopt the standard of following safety and reactionogenicity:

Desired general symptom is heating, diarrhoea, vomiting, feel sick, stomachache and inappetence.These symptoms were carried out record in back 8 days using.

Do not make the symptom that requires and carried out record in back 30 days using.

Serious harmful incident obtains record during whole research.

The diarrhoea sample is being used collection in back 8 days.

The result is:

Desired symptom, symptom and the seriously harmful incident not doing to require are not all being reported during observing time separately.

There is not diarrhoea case report.

9.5. conclusion

With 10 ^6.1Ffu dosage as single dose in the double blinding mode when age 18-44 year, health adult was Orally administered, SB P43 biology vaccine is safe with respect to placebo.

People's unit price Rotavirus Vaccine that two doses of embodiment 10-contain RIX 4414 in prevention because of the effectiveness in the gastroenteritis due to G1 and non-G1 (G9) rotavirus

10.1. method

The II phase that randomization, double blinding, placebo compare test implement in Latin America with assessment be used for infant immunization derived from G1P[8] protection of the vaccine of people 89-12 strain (RIX4414 Human reoviruslike agent strain) renders a service.The RIX4414 vaccine comprises the attenuation G1P[8 as the rotavirus composition] people's strain, its as ECACC preservation thing 99081301 (WO 01/12797) by preservation.

Vaccine combination(table 17)

The entire contents of the pre-filled syringe by will containing the calcium carbonate buffer agent is injected in the bottle of lyophilized products (vaccine or placebo), and preparation HRV vaccine or placebo are to use.The jolting bottle is so that vaccine/placebo is resuspended.With the same syringe of resuspended product suction of whole volumes, discard syringe needle and resuspended product and used (approximately 1.0ml) rapidly as single oral dose.

Table 17-RIX4414 Rotavirus Vaccine compositions

Become component (every nominal standard dose: 1ml)
	Active component RIX4414 10 5.8Ffu/ dosage
Liquid diluent in the pre-filling injection agent of vaccine dextran 1 8mg sorbitol 13.5mg aminoacid 9mg Dulbecco ' the s improvement Eagle 2.25mg culture medium (DMEM) of excipient s lyophilizing sucrose 9mg in vial is (with CaCO 3Being the basis) calcium carbonate 60mg xanthane 2.5mg water for injection is added into 1ml in right amount

Vaccine administration

Healthy babies (493) is accepted (every dose 10 of two dosage at 2 monthly ages and 4 monthly ages ^5.8The virus concentration of ffu) RIX4414-Rotavirus Vaccine or placebo (504) accompany by DPT vaccine-hepatitis B vaccine (DTPw-HBV vaccine) and Hib vaccine.Apart from research 2 weeks of vaccine, give the OPV (oral polio virus vaccine) of three dosage, promptly OPV will not use at the time durations that began extremely every dose of 2 week of research vaccine back end before 2 weeks from every dose of research vaccine.It is 10 that two other windings are subjected to the different virus concentration that has of two dosage ^4.7Ffu and 10 ^5.2The RIX4414 Rotavirus Vaccine of ffu.Detect the diarrhoea sample and whether have rotavirus (ELISA), and in positive, determine serotype (RT-PCR).Second back diarrhoea outbreak of being reported of 2 weeks of dosage is considered for Validity Analysis.Use 20 point scales (Ruuska and Vesikari, 1990) to determine seriousness.20 marking systems that are used for assessing each diarrhoea outbreak seriousness in this research show in following table 18.Scoring 〉=11 definition serious diseases.

Table 18

Disadvantageous experience	Count
		Feces is than normal looser persistent period (day) 1-4 5 〉=6	1 2 3
Feces is than normal looser maximum times 1-3 4-5 〉=6 in 24 hours	1 2 3
		The persistent period (day) 12 〉=3 of vomiting	1 2 3
Maximum times 1 2-4 〉=5 of vomiting outbreak in 24 hours	1 2 3
		Heating (measurement of rectum measurement/axillary fossa) * 37.1-38.4 ℃/36.6-37.9 ℃ 38.5-38.9 ℃/38.0-38.4 ℃ 〉=39 ℃/〉=38.5 ℃	1 2 3
The treatment moisturizing is in hospital	1 2
		Dehydration 1-5% 〉=6%	2 3

* to the maximum temperature scoring of stage of attack interocclusal record

10.2. result

Carry out the interim analysis of effectiveness in above-mentioned group, isolating serotype mainly is G1 and G9, almost is evenly distributed.Integral body invasion and attack rate in the placebo group was that 4.8%-is 3.6% for G9 for G1 during 6 months observing times.Two dose 10 ^5.8The RIX4414 Rotavirus Vaccine of ffu is renderd a service [95%CI:50.4-95.7] anti-diarrhoea by all types due to the G1 with 83%, and renders a service [95%CI:47.6-99.8] anti-serious gastroenteritis with 92.1%.If diarrhoea is caused that by G9 then the diarrhoea of anti-all types is 60.2%[95%CI:0.2-86.0], anti-serious gastroenteritis is 80.8%[95%CI:33.0-96.4].For these each (concerning G1 and G9 arbitrarily with serious) that renders a service terminal points, compare with placebo group, in the HRV group, there is the diarrhoea outbreak of statistically significant descend (p＜0.05, bilateral Fisher Precision Test).

The result who obtains in other 2 vaccine group (the concentration difference of rotavirus) is consistent with institute's results reported among the embodiment, is presenting (embodiment 11) in the analysis of end eventually.Also analyze the efficacy data of G2, G3 and G4.From this research, do not draw conclusion about G2, G3 and G4 cross protection, reason be the report case load very little.Yet, present (embodiment 11) at the end eventually on the large sample amount more in analyzing at the effectiveness number of G2, G3 and G4.

10.3. conclusion

These results support the unit price HRV vaccine of 2 dosage to be the RIX4414 Rotavirus Vaccine to heavens and resist effectiveness aspect G1 strain and the cross protection opposing G9 strain the young baby of protection.

Because of the effectiveness in the gastroenteritis due to G1 and non-G1 (G2, G3, G4, the G9) rotavirus, use on 3 different virus concentrations by wherein said Human reoviruslike agent univalent vaccine in prevention for people's unit price Rotavirus Vaccine that two doses of embodiment 11-contain RIX 4414 strains

11.1. method

The II phase that randomization, double blinding, placebo compare test implement in Latin America with assessment be used for infant immunization derived from G1P[8] protection of the vaccine of people 89-12 strain (RIX4414 Human reoviruslike agent strain) renders a service and exempts to be in hospital and render a service.Particularly, the vaccine of use is called the RIX4414 vaccine, and comprises the attenuation G1 people strain as the rotavirus composition, its as ECACC preservation thing 99081301 by preservation.

Healthy babies is accepted two doses of RIX4414-Rotavirus Vaccines on 3 different virus concentration.Validity Analysis is made up of 1846 experimenters at 2 monthly ages and 4 monthly ages with formation that (468 experimenters are 10 ^4.7Ffu HRV vaccine group, 460 experimenters are 10 ^5.2Ffu HRV vaccine group, 464 experimenters are 10 ^5.8Ffu HRV vaccine group and 454 experimenters are in placebo group), accompany by DTPw-HBV and Hib vaccine.Distance is studied 2 weeks of vaccine, gives the OPV of three dosage, and promptly OPV will not use at the time durations that each dosage from the research vaccine began before 2 weeks and finishes in each dosage 2 all back of research vaccine.Whether check diarrhoea sample exists rotavirus (ELISA) and determine serotype (RT-PCR) in positive.From second dosage 2 week the back be considered for Validity Analysis to the full diarrhoea outbreak of being reported when one-year-old of experimenter.Use 20 point scales (Ruuska and Vesikari, 1990) to determine seriousness.Scoring 〉=11 definition serious diseases (seeing the description of 10 pairs of 20 marking systems of embodiment).

11.2. result

In following table, show the result who analyzes as to the end eventually of mentioned data in the embodiment 10.Baby in the vaccine group has remarkable rotavirus gastroenteritis outbreak (p＜0.001, bilateral Fisher Precision Test) (table 19) still less than the child in the placebo group.Depend on dosage, the protection of anti-serious rotavirus gastroenteritis is renderd a service the protection effectiveness that reaches 85.6% (95%CI:63.0%-95.6%) and anti-any rotavirus gastroenteritis and is reached 70% (95%CI, 45.7%-84.4%) (table 20).Render a service in terminal points each for these, compare, in the HRV group, have the diarrhoea outbreak of statistically significant descend (p＜0.05, bilateral Fisher Precision Test) with placebo group.(ELISA and RT-PCR) identifies a plurality of rotavirus serotypes (G1, G2, G3, G4 and G9) from gastroenteritis feces, allows also to calculate the vaccine potency of anti-non-G1 serotype.Especially as can from table 21, seeing; for non-G1 serotype (G2, G3, G4 and G9) and depend on dosage; the effectiveness of anti-serious rotavirus gastroenteritis reaches 82.7% (95%CI:40.3%-96.8%); this provides evidence, promptly monovalent G1P1A P[8 based on G1 for like this notion] the Human reoviruslike agent vaccine excites the cross protection of anti-special-shaped (be non-G1 and non-P[8]) strain.

Table 19: the feature of the rotavirus gastroenteritis outbreak of being reported during the research

Table 20: the protection of two doses of RIX4414 Human reoviruslike agent vaccine anti-rotavirus gastroenteritiss is renderd a service

* for every kind of comparison between vaccine group and placebo group, by bilateral Fisher Precision Test p＜0.001 (significant level of α=0.05)

For the comparison between vaccine group and placebo group, by bilateral Fisher Precision Test p=0.037 (significant level of α=0.05)

For the comparison between vaccine group and placebo group, by bilateral Fisher Precision Test p=0.007 (significant level of α=0.05)

N=experimenter's number

N/%=reports experimenter's number/percent of at least a described rotavirus gastroenteritis outbreak.

Show accurate 95% confidence interval

Table 21: the protection of the serious rotavirus gastroenteritis of antiserum type specificity of two doses RIX4414 Human reoviruslike agent vaccine is renderd a service

* the bilateral Fisher Precision Test (significant level of α=0.05) that is used for every kind of comparison between vaccine group and placebo group

N=experimenter's number

N/%=reports experimenter's number/percent of at least a described serious rotavirus gastroenteritis outbreak

Show accurate 95% confidence interval

11.3. conclusion

These as a result height support the effectiveness of the unit price HRV vaccine that contains RIX4414 of 2 dosage, wherein this effectiveness is embodied in the extensive cross protection aspect that the young baby's opposing of protection is G2, G3, G4 and G9 by any rotavirus gastroenteritis due to the G1 strain and serious rotavirus gastroenteritis and anti-other RV G type.

Two doses of Human reoviruslike agent attenuated vaccines of embodiment 12-RIX4414 vaccine shows special-shaped protection in Latin America and Europe

2 doses of oral Human reoviruslike agent (RV) attenuation G1P[8 that contain the RIX4414 strain] effectiveness of live vaccine analyzed in the II/III clinical trial phase in Finland and Latin America.RIX 4414 Rotavirus Vaccines comprise the attenuation G1 Human virus strain as the rotavirus composition, its as ECACC preservation thing 99081301 by preservation.

12.1. method

The partial results of embodiment 12 shows in embodiment 10 and 11.Compile one and study (embodiment 10 and 11) and III phase of carrying out the II phase that (Brazil, Mexico and Venezuela) carries out in Latin America Finland and one in 11 Latin American countries and study the data of (embodiment 13), these study use same procedure and effect standards.Followed up a case by regular visits to 2 doses of RIX 4414 vaccines or placebo 20081 healthy babies of total (being used for the formation of Validity Analysis) in 2 monthly ages and the inoculation of 4 monthly ages, until an one full year of life, observation has the (Scand.J.Infect.Dis.1990 such as Ruuska T at Vesikari, 22,259-267) the serious gastroenteritis (GE) of scoring 〉=11 on the seriousness scale.To GE sample (passing through ELISA) check rotavirus and by RT-PCR check rotavirus type.

Implementing Meta in three researchs of mentioning analyzes.The effectiveness of compiling to serious RV GE (being defined as Vesikari seriousness scoring 〉=11) is calculated (using the Mantel-Haenszel method of approximation to adjust research effect) from giving 22 all backs to 1 one full year of life of dosage.

12.2. result

In this renders a service formation, in vaccine group, detect 5 routine G2P[4 with Vesikari scoring 〉=11] the serious rotavirus GE outbreak of type and in placebo group, detect 13 example outbreaks.Anti-G2P[4] vaccine potency of type is 67.2% (95%CI:14.8; 87.1), this show except that anti-homotype strain (G1P[8], G3P[8] and G4P[8]) protection, the RIX4414 vaccine also is protected from by the non-P[8 of abnormal shape] G2P[4 of non-G1] influence of the serious rotavirus GE that causes of strain.

The type specificity of crossing between different researchs is renderd a service following provide (table 22).

Table 22

* VE adjusts research effect with the Mantel-Haenszel method of approximation

Only 3 G4 cases appear in whole 3 tests, and 1 G4 case appears among the vaccine receptor and appears among the placebo receptor with 2 G4 cases.

12.3. conclusion

This analysis shows except that the high level protection that produces anti-homology G1 rotavirus strain (this strain has at two kinds of outer capsid albumen (VP4 and VP7) and a kind of inner capsid albumen (VP6) similar to vaccine on the antigenicity); the RIX4414 vaccine also provides the height protection at other strain; wherein said other strain has different G type (for example G3, G9), different P type (for example P[4]) or simultaneously different G type and P types, as to G2P[4] effectiveness shown in.

Embodiment 13-Meta the analysis showed that the Human reoviruslike agent attenuated vaccine RIX 4414 of two dosage shows special-shaped protection

Along with multidata more can obtain from Singapore and from European Studies (embodiment 15), implement extra Meta and analyze, so that the research of in embodiment 12, mentioning, also comprise into these researchs.

13.1. method

3 II phases are studied (Finland and Latin America and Singapore) and 2 III phases and study in (Latin America and Europe) comprised that into meta-analyzes.Twice oral administration according to according to 0, the plan in January to February is applied to when the giving dosage 1 6-14 healthy babies in age week in week.In full-fledged research, serious RVGE is defined as scoring 〉=11 in 20 Vesikari scales.The diarrhoea sample is analyzed by ELISA with regard to the existence of RV and is carried out typing by the method based on RT-PCR.Also only in III phase European Studies, obtain assessment during the effectiveness of anti-any RV GE was studied 3 II phases, as in the research of III phase Latin America, only write down serious RV GE.

Adopt this to study stratified accurate Poisson rate than (exact Poisson rate ratio) (ProcStatXact4 for SAS Users, 1999, cytel software corporation, exactConfidence Interval for common relative risk, the 298th page), VE and 95%CI thereof are assessed as RVGE 1-rate with respect to placebo

13.2. result

In 8221 babies altogether with the inoculation of two doses of RIX4414 or placebo, in RIX4414 (N=5783), detect arbitrarily G2P[4 of 4 examples] the RVGE outbreak and in placebo (N=2438) group, detect 9 examples G2P[4 arbitrarily] the RVGE outbreak, this shows for because of G2P[4] RVGE due to the strain with any seriousness, VE is 81.0% (95%CI:31.6; 95.8).

In 26088 healthy babies altogether with the inoculation of two doses of RIX4414 or placebo, in RIX4414 (N=14792), detect 6 examples because of G2P[4] serious G2P[4 due to the type] the RVGE outbreak and in placebo (N=11296) group, detect 15 examples because of G2P[4] serious G2P[4 due to the type] the RVGE outbreak, show for because of G2P[4] serious RVGE due to the strain, VE is 71.4% (95%CI:20.1; 91.1).The result reports in table 23.

Table 23-report is by G2P[4] any RV GE outbreak due to the RV type or experimenter's number of serious RV GE outbreak and the vaccine potency percent during the first effectiveness phase-(Meta analysiss), effectiveness formation

Table 23

The experimenter's number that comprises in each group of N=;

At least a described RV G2P[4 of report in each group of n/%=] experimenter's number/percent of GE outbreak;

The observed vaccine potency of %VE=,

The 95%CI=95% confidence interval

* 2 among 13 G2 do not carry out the P typing.

13.3. conclusion:

At anti-RV G2P[4] this Meta on the vaccine potency of type the analysis showed that anti-because of G2P[4] vaccine potency of any RV GE due to the type is 81.0% (95%CI:31.6%; 95.8%) and anti-G2P[4] the vaccine potency of serious RV GE due to the type is 71.4% (95%CI:20.1%; 91.1%).

Embodiment 14-Human reoviruslike agent attenuated vaccine Rotarix ^TMEffectiveness in testing in the III phase of a plurality of countries

14.1. method

Will be from 20169 healthy babies of 11 Latin American countries at the HRV vaccine (10159) or the placebo (10010) of 2 doses of about 2 monthly ages and the oral acceptance of 4 monthly ages.Fecal specimens check rotavirus (RV) by ELISA and adopt suitable primer and the type particular probe by the RT-PCR typing.To the clinical case definition of catching the outbreak of serious gastroenteritis be with or not have the diarrhoea outbreak (in 24 hours continuous 3 times or more frequently than normal looser feces or watery stools) of vomiting, this diarrhoea outbreak need spend the night in hospital and/or in medical institutions such as hospital, clinic or the rural care centre that is subjected to supervision, carry out with WHO plan B (oral moisturizing therapy) or WHO plan C (intravenous moisturizing therapy) ( Http: //www.who.int/child-adolescent-health/New Publications/CHILDHEALTH/textrev4.htm) the moisturizing therapy that is equal to.Disease seriousness uses 20 Vesikari scales to carry out classification; Serious RVGE is defined as scoring 〉=11.The Vesikari scoring is improved: because dehydration does not obtain record in eCRF, following rule obtains using: the experimenter that will have serious GE outbreak is considered as the 1%-5% that dewaters, if this experimenter accepts oral moisturizing.The experimenter is considered as dehydration 〉=6%, if this experimenter is admitted to hospital and/or accepts intravenous (IV) moisturizing.

14.2. vaccine potency

The vaccine potency of anti-serious rotavirus gastroenteritis(table 24)

Rendeing a service formation is made up of with 8858 experimenters that accept placebo 9009 experimenters with HRV vaccination.In vaccine group, exist 12 to suffer from the child of serious rotavirus gastroenteritis and in placebo group, have 77 children that suffer from serious rotavirus gastroenteritis that (2.0 children are to 13.3 children according to clinical definition, time outbreak of per 1,000 child-year 〉=1; P＜0.001, bilateral Fisher Precision Test), produce vaccine potency until the 84.7% anti-serious rotavirus gastroenteritis of 1 one full year of life (table 24, showing) on the 15th after giving 2 dosage.Analog result is used from giving 1 the whole inoculation formations of dosage until 1 one full year of life and is obtained (vaccine potency 81.1%; 95%C.I.68.5-89.3; P＜0.001, bilateral Fisher Precision Test).In the vaccine group in 9 children and the placebo group 59 children need be in hospital 1 evening (per 1 at least, 000 child-year is respectively that 1.5 examples are in hospital to 10.2 examples in hospital), the vaccine potency that avoids being in hospital because of serious rotavirus gastroenteritis is 85% (p＜0.001, bilateral Fisher Precision Test) (table 24).

Table 24-after giving the 2nd dose 2 weeks until 1 one full year of life time durations, the serious gastroenteritis of anti-rotavirus, the serious gastroenteritis of specificity rotavirus G type and complete because of (all-cause) the serious vaccine potency of gastroenteritis

The explanation of his-and-hers watches 24:

Outbreak is arranged and carry the participant's counting in each classification of detected rotavirus type classification that has separated G type rotavirus more than a kind of

N=reports baby's number of at least a described outbreak

The ratio of experimenter's sickness rate of report at least one outbreak in experimenter's sickness rate of report at least one outbreak and the placebo group in RR=relative risk=vaccine group.

The CI=confidence interval

1000 babies-year ratio is baby's number of the described outbreak of every baby-year performance＞=1

* according to the case definition of project: with or the moisturizing therapy that do not have the diarrhoea outbreak (feces of continuous 3 times or looser than normal feces more frequently or water sample in 1 day) of vomiting, this diarrhoea outbreak need spend the night and/or in medical institutions such as hospital, clinic or the rural care centre that is subjected to supervision, carry out being equal in hospital with WHO plan B (oral moisturizing therapy) or WHO plan C (intravenous moisturizing therapy)

^#Isolating whole G1 type is the wild type rotavirus; G1P[8] with G9P[8] from 1 baby, separate

^aG1P[8] the type list separates from 2 babies; G1P[8] with G9P[8] from 1 baby, separate

^bG1P[8] the type list separates from 34 babies; G1P[8] with G9P[8] from 1 baby, separate; G1, G2, G9 type separate from 1 baby

^cG3P[8] the type list separates G4P[8 from 1 baby] the type list obtains and G9P[8 from 1 baby] singly from 1 baby, separate; G1P[8] with G9P[8] from 1 baby, separate

^dG3P[8] the type list separates G4P[8 from 8 babies] the type list separates and G9P[8 from 2 babies] singly from 19 babies, separate; G1P[8] with G9P[8] from 1 baby, separate and G1P[8] and with G2P[4] and G9P[8] from 1 baby, separate

^eG2P[4] single from 9 babies, separate and G1P[8], G2P[4] with G9P[8] from 1 baby, separate.

P_ value=bilateral Fisher Precision Test (significant level of α=0.05)

Vaccine potency according to the Vesikari scoring

Have in the vaccine group among 12 children of serious rotavirus outbreak that 71 children have Vesikari scoring 〉=11 among 77 children that have serious rotavirus outbreak in 11 children and the placebo group, obtain 84.7% vaccine potency (P＜0.001, bilateral Fisher Precision Test).For the disease seriousness with 11-20 scoring that rises, vaccine potency is higher, reaches the more serious rotavirus gastroenteritis of 100% opposing.16 serious rotavirus gastroenteritises outbreaks altogether of Vesikari scoring 〉=11 obtain report from giving first dosage during giving second dosage, 6 in vaccine group, and 10 in placebo group.

The vaccine potency according to the Vesikari scoring of each rotavirus type

The type specificity vaccine potency of anti-wild type strain shows in table 24.The vaccine potency of the serious rotavirus outbreak of anti-Vesikari scoring 〉=11 is 91.8% (P＜0.001, bilateral Fisher Precision Test), wherein said serious rotavirus outbreak by with the homologous G1P[8 of vaccine strain] the type strain causes.To total P[8] antigen (G3P[8], G4P[8] and G9P[8]) the vaccine potency of strain be 86.9% (P＜0.001, bilateral Fisher Precision Test).With total G antigen of vaccine strain and the antigenic rotavirus G2P[4 of P] in 5 outbreaks of type in vaccine group and obtain detecting in 9 outbreaks in placebo group, cause 45% effectiveness (P=0.298, bilateral Fisher Precision Test).Because the number of G2 outbreak is few in this research, thus to 5 researchs (embodiment 13) carry out the meta analysis and compile these 5 researchs (embodiment 13) as a result the time, viewed trend becomes a remarkable value in this research.

Vaccine potency aspect the burden of diarrhea disease

Need to be in hospital and/or in vaccine group, to have sickness rate 30.9/1 according to the child who suffers from any cause of disease gastroenteritis of WHO plan B/C moisturizing, 000 child-year, by comparison, sickness rate in placebo group is 51.7/1,000 child-year, thereby in the vaccine receptor entirely because of property whole 40% (P＜0.001, the bilateral Fisher Precision Test) that reduce of outbreak of seriously suffering from diarrhoea.Similarly, because of 42% (P＜0.001, bilateral Fisher Precision Test) (table 24 is entirely because of GE) that significantly descend that be in hospital due to any cause of disease diarrhoea).

14.3. the result sums up

Anti-serious rotavirus gastroenteritis (RV GE) and to avoid the vaccine potency that the rotavirus dependency is in hospital be 85% (P＜0.001, bilateral Fisher Precision Test) reaches 100% in the colony of the RV GE that suffers from Vesikari scoring＞=19.At G1P[8] effectiveness and at the only total P[8 of HRV] effectiveness of strain of epi-position is respectively 92% (95%C.I.74,98) and 87% (95%C.I.64,97) (P＜0.001, bilateral Fisher Precision Test), because of entirely because of reducing by 42% (95%C.I.29,53 in hospital due to the property diarrhoea; P＜0.001, bilateral Fisher Precision Test).

Embodiment 15-Human reoviruslike agent attenuated vaccine Rotarix ^TMEffectiveness 6 European countries

15.1. method

Will be in 3,994 child's random packet of 6 European countries, so that when using jointly, accept 10 with conventional child's inoculation ^6.5CCID ₅₀HRV (Human reoviruslike agent) vaccine Rotarix ^TM(seeing compositions) or placebo.First renders a service follow-up period started from behind the 2nd dosage for 2 weeks and ends in June, 2005-July.3874 experimenters are parts of first year rendeing a service formation altogether.

Vaccine combination:(table 25)

Vaccine	Prescription
		The HRV vaccine of GSK Biologicals	RIX4414 HRV strain 106.5 half cell culture infective dose (CCID50) Dulbecco ' s improvement Eagle culture medium (DMEM) 3.7mg sucrose 9mg dextran 1 8mg sorbitol 13.5mg aminoacid 9mg derived from the 89-12HRV vaccine strain
The diluent of GSK Biologicals	Calcium carbonate 80mg xanthane 3.25mg water for injection is an amount of, is added into 1.3ml

15.2. vaccine potency

During the first effectiveness phase, the HRV vaccine is being highly effective aspect the anti-RV GE protection.The vaccine potency of anti-any RV GE outbreak is 87.1% (95%CI:79.6%; 92.1%) and the vaccine potency of anti-serious RV GE outbreak be 95.8% (95%CI:89.6%; 98.7%).For the disease seriousness (Vesikari marks between 11 and 20) that rises, vaccine potency is higher, reaches 100% in the colony of the RV GE that suffers from 〉=17 of Vesikari scorings.Avoid because of the vaccine potency of being in hospital due to the RV GE be 100% (95%CI:81.8%; 100%) and needing to avoid the vaccine potency of the RV GE outbreak of medical nursing be 91.8% (95%CI:84.0%; 96.3%) (table 26 and table 27).

Table 26-reports experimenter's percent, experimenter's percent and the vaccine potency during the first effectiveness phase-effectiveness formation because of being in hospital due to the RV GE outbreak of any RV GE and serious RV GE

The experimenter's number that comprises in each group of N=

Report has the experimenter's number and the percent of at least a described RV GE outbreak in each group of N%=

P-value=bilateral Fisher Precision Test (significant level of α=0.05)

The observed vaccine potency of %VE=

The 95%CI=95% confidence interval

Scoring 〉=11 is defined as serious in 20 Vesikari scales

Table 27-report needs the experimenter's percent and the vaccine potency during the first effectiveness phase-effectiveness formation of the RV GE outbreak of medical nursing

The experimenter's number that comprises in each group of N=;

Report has at least a experimenter's number/percent that needs the RV GE outbreak of medical nursing in each group of N/%=;

P-value=bilateral Fisher Precision Test (significant level of α=0.05);

The observed vaccine potency of %VE=,

The 95%CI=95% confidence interval

This HRV vaccine height is protected from effectively by G1P[8], G3P[8], G4P[8] and G9P[8] any RV GE and serious RV GE (table 28) due to the strain.

In this research at the total antigenic G2P[4 of any outer capsid of HRV vaccine] protection of RV type is lower, yet the Meta analysis result that II and III phase effect research are included consideration in is shown at by G2P[4] due to any GE and effective protectiveness of serious GE render a service (seeing embodiment 13).

Table 28-report experimenter's percent of any RV GE outbreak or serious RV GE outbreak and during the first effectiveness phase at the vaccine potency-effectiveness formation of serotype

The experimenter's number that comprises in each group of N=;

Report has experimenter's number/percent of at least a RV GE outbreak in each group of n/%=;

P-value=bilateral Fisher Precision Test (significant level of α=0.05);

The observed vaccine potency of %VE=,

The 95%CI=95% confidence interval

Scoring 〉=11 is defined as serious in 20 Vesikari scales

Experimenter from placebo group is counted in G1 and G4 classification, because these two serotypes all obtain separating.

15.3. the result sums up

The HRV Rotarix of 2 oral doses using jointly with child's inoculation ^TMVaccine was compared with placebo during the first effectiveness phase, in protection baby opposing by G1P[8] wild type RV and by non-G1P[8] be highly effective aspect any RV GE due to the RV type, vaccine potency is respectively 95.6% (95%CI:87.9%; 98.8%) and 79.3% (95%CI:64.6%; 88.4%).Opposing is by G1P[8] wild type RV and by non-G1P[8] effectiveness of serious RV GE due to the RV type is respectively 96.4% (95%CI:85.7%; 99.6%) and 95.4% (95%CI:85.3%; 99.1%).

These results greatly support conclusion like this, promptly the HRV vaccine provide anti-circulation RV strain extensive spreadability (see Table 28:G1P[8], G2P[4], G3P[8], G4P[8], G9P[8]).Carried out special at anti-G2P[4] the Meta of vaccine potency analyze, please refer to embodiment 13.

Overall conclusion

The RIX4414 Rotavirus Vaccine proves the influence that highly is protected from the rotavirus gastroenteritis outbreak; the outbreak of wherein said rotavirus gastroenteritis is by being used for to be in hospital and moisturizing is that the clinical definition that the case of core is caught is weighed; and by effective Vesikari scale measurement, wherein said Vesikari scale comprises the quantitatively property sickness rate consequence that relates to diarrhoea, vomits, generates heat, dewaters and be in hospital.Two doses of oral HRV vaccines are being protected the baby to resist by any RV GE due to the multiple cyclicity rotavirus strain and serious RVGE and are being highly effective aspect being in hospital.

At having the two kinds of outer capsid albumen (VP4 and VP7) similar to the HRV vaccine on antigen and the homology G1P[8 of a kind of inner capsid albumen (VP6)] rotavirus shown high-caliber protective effect.This protective effect is also to only having genotype P[8] antigen (VP4 antigen) and the antigenic strain of VP6.In analyzing, Meta also shows at the not antigenic protection of any outer capsid of total HRV vaccine; wherein said Meta analyzes and comprises from Finland; Singapore is studied with 3 II phases of Latin America (all using identical methodology and effect standard) and from the result that studies of 2 III phases in Latin America and Europe; and as report in embodiment 13; anti-G2P[4] vaccine potency of any seriousness GE of type) be 81% (95%C.I.31.6-95.8); anti-because of G2P[4] vaccine potency of the serious GE of type is 71.4% (95%C.I.20.1-91.1), shows that this vaccine also can be to providing protection with total identical G albumen of vaccine strain or the proteic strain of P.

Sequence table

<110>GlaxoSmithKline Biologicals s.a.

＜120〉vaccine

<130>VB61582

<160>42

<170>FastSEQ for Windows Version 4.0

<210>1

<211>2350

<212>DNA

＜213〉rotavirus

<400>1

atggcttcac tcatttatag acaacttctc actaattcat attcagtaga tttacatgat 60

gaaatagagc aaattggatc agaaaaaact cagaatgtaa ctataaatcc gggtccattt 120

gcacagacta gatatgctcc agtcaattgg gatcatggag agataaatga ttcgactaca 180

gtagaaccaa ttttagatgg tccttatcag ccaactacat ttactccacc taatgattat 240

tggatactta ttaattcaaa tacaaatgga gtagtatatg aaagtacaaa taatagtgac 300

ttttggactg cagtcgttgc tattgaaccg cacgtcaacc cagtagatag acaatatatg 360

atatttggtg aaagcaagca atttaatgtg agtaacgatt caaataaatg gaagttttta 420

gaaatgttta gaagcagtag tcaaaatgaa ttttataata gacgtacatt aacttctgat 480

accagacttg taggaatatt taaatatggt ggaagagtat ggacatttca tggtgaaaca 540

ccgagagcta ctactgacag ttcaagtact gcaaatttaa ataatatatc aattacaatt 600

cattcagaat tttacattat tccaaggtcc caggaatcta aatgtaatga atatattaat 660

aatggtctgc caccaattca aaatactaga aatgtagttc cattgccatt atcatctaga 720

tcgatacagt ataagagagc acaagttaat gaagacatta tagtttcaaa aacttcatta 780

tggaaagaaa tgcagtataa tagggatatt ataattagat ttaaatttgg taatagtatt 840

gtaaagatgg gaggactagg ttataaatgg tctgaaatat catataaggc agcaaattat 900

caatataatt acttacgtga cggtgaacaa gtaaccgcac acaccacttg ttcagtaaat 960

ggagtgaaca attttagcta taatggaggg tttctaccca ctgattttgg tatttcaagg 1020

tatgaagtta ttaaagagaa ttcttatgta tatgtagact attgggatga ttcaaaagca 1080

tttagaaata tggtatatgt tagatcatta gcagctaatt taaattcagt gaaatgtaca 1140

ggtggaagtt attatttcag tataccagta ggtgcatggc cagtaatgaa tggtggcgct 1200

gtttcgttgc attttgccgg agttacatta tccacgcaat ttactgattt tgtatcatta 1260

aattcactac gatttagatt tagtttgaca gttgatgaac cacctttctc aatactgaga 1320

acacgtacag tgaatttgta tggattacca gccgctaatc caaataatgg aaatgaatac 1380

tacgaaatat caggaaggtt ttcactcatt tctttagttc caactaatga tgattatcag 1440

actccaatta tgaattcagt gacggtaaga caagatttag agcgccaact tactgattta 1500

cgagaagaat ttaactcatt gtcacaagaa atagctatgg cacaattgat tgatttagca 1560

ctgttgcctc tagatatgtt ttccatgttt tcaggaatta aaagtacaat tgatttaact 1620

aaatcaatgg cgactagtgt aatgaagaaa tttagaaaat caaaattagc tacatcaatt 1680

tcagaaatga ctaattcatt gtcagatgct gcttcatcag catcaagaaa cgtttctatt 1740

agatcgaatt tatctgcgat ttcaaattgg actaatgttt caaatgatgt gtcaaacgta 1800

actaattcat tgaacgatat ttcaacacaa acatctacaa ttagtaagaa acttagatta 1860

aaagaaatga ttactcaaac tgaaggaatg agctttgacg acatttcagc agctgtacta 1920

aaaacaaaaa tagatatgtc tactcaaatt ggaaaaaata ctttacctga tatagttaca 1980

gaagcatctg agaaatttat tccaaaacga tcatatcgaa tattaaagga tgatgaagta 2040

atggaaatta atactgaagg aaaattcttt gcatacaaaa ttaatacatt tgatgaagtg 2100

ccattcgatg taaataaatt cgctgaacta gtaacagatt ctccagttat atcagcgata 2160

atcgatttta agacattgaa aaatttaaat gataattatg gaatcactcg tacagaagcg 2220

ttaaatttaa ttaaatcgaa tccaaatatg ttacgtaatt tcattaatca aaataatcca 2280

attataagga atagaattga acagttaata ctacaatgta aattgtgaga acgctattga 2340

ggatgtgacc 2350

<210>2

<211>2359

<212>DNA

＜213〉rotavirus

<400>2

ggctataaaa tggcttcgct catttataga caacttctca ctaattcata ttcagtagat 60

ttacatgatg aaatagagca aattggatca gaaaaaactc agaatgtaac tataaatccg 120

ggtccatttg cacagactag atatgctcca gtcaattggg atcatggaga gataaatgat 180

tcgactacag tagaaccaat tttagatggt ccttatcagc caactacatt tactccacct 240

aatgattatt ggatacttat taattcaaat acaaatggag tagtatatga aagtacaaat 300

aatagtgact tttggactgc agtcgttgct attgaaccgc acgtcaaccc agtagataga 360

caatatatga tatttggtga aagcaagcaa tttaatgtga gtaacgattc aaataaatgg 420

aagtttttag aaatgtttag aagcagtagt caaaatgaat tttataatag acgtacatta 480

acttctgata ccagacttgt aggaatattt aaatatggtg gaagagtatg gacatttcat 540

ggtgaaacac cgagagctac tactgacagt tcaagtactg caaatttaaa taatatatca 600

attacaattc attcagaatt ttacattatt ccaaggtccc aggaatctaa atgtaatgaa 660

tatattaata atggtctgcc accaattcaa aatactagaa atgtagttcc attgccatta 720

tcatctagat cgatacagta taagagagca caagttaatg aagacattat agtttcaaaa 780

acttcattat ggaaagaaat gcagtataat agggatatta taattagatt taaatttggt 840

aatagtattg taaagatggg aggactaggt tataaatggt ctgaaatatc atataaggca 900

gcaaattatc aatataatta cttacgtgac ggtgaacaag taaccgcaca caccacttgt 960

tcagtaaatg gagtgaacaa ttttagctat aatggagggt ttctacccac tgattttggt 1020

atttcaaggt atgaagttat taaagagaat tcttatgtat atgtagacta ttgggatgat 1080

tcaaaagcat ttagaaatat ggtatatgtt agatcattag cagctaattt aaattcagtg 1140

aaatgtacag gtggaagtta ttatttcagt ataccagtag gtgcatggcc agtaatgaat 1200

ggtggcgctg tttcgttgca ttttgccgga gttacattat ccacgcaatt tactgatttt 1260

gtatcattaa attcactacg atttagattt agtttgacag ttgatgaacc acctttctca 1320

atactgagaa cacgtacagt gaatttgtat ggattaccag ccgctaatcc aaataatgga 1380

aatgaatact acgaaatatc aggaaggttt tcactcattt ctttagttcc aactaatgat 1440

gattatcaga ctccaattat gaattcagtg acggtaagac aagatttaga gcgccaactt 1500

actgatttac gagaagaatt taactcattg tcacaagaaa tagctatggc acaattgatt 1560

gatttagcac tgttgcctct agatatgttt tccatgtttt caggaattaa aagtacaatt 1620

gatttaacta aatcaatggc gactagtgta atgaagaaat ttagaaaatc aaaattagct 1680

acatcaattt cagaaatgac taattcattg tcagatgctg cttcatcagc atcaagaaac 1740

gtttctatta gatcgaattt atctgcgatt tcaaattgga ctaatgtttc aaatgatgtg 1800

tcaaacgtaa ctaattcatt gaacgatatt tcaacacaaa catctacaat tagtaagaaa 1860

cttagattaa aagaaatgat tactcaaact gaaggaatga gctttgacga catttcagca 1920

gctgtactaa aaacaaaaat agatatgtct actcaaattg gaaaaaatac tttacctgat 1980

atagttacag aagcatctga gaaatttatt ccaaaacgat catatcgaat attaaaggat 2040

gatgaagtaa tggaaattaa tactgaagga aaattctttg catacaaaat taatacattt 2100

gatgaagtgc cattcgatgt aaataaattc gctgaactag taacagattc tccagttata 2160

tcagcgataa tcgattttaa gacattgaaa aatttaaatg ataattatgg aatcactcgt 2220

acagaagcgt taaatttaat taaatcgaat ccaaatatgt tacgtaattt cattaatcaa 2280

aataatccaa ttataaggaa tagaattgaa cagttaatac tacaatgtaa attgtgagaa 2340

cgctattgag gatgtgacc 2359

<210>3

<211>1009

<212>DNA

＜213〉rotavirus

<400>3

atgtatggtc ttgaatatac cacaattcta atctttctga tatcaattat tctactcaac 60

tatatattaa aatcagtaac tcgaataatg gactacatta tatatagatc tttgttgatt 120

tatgtagcat tatttgcctt gacaagagct cagaattatg ggcttaactt accaataaca 180

ggatcaatgg acactgtata cgctaactct actcaagaag gaatatttct aacatccaca 240

ttatgtttgt attatccaac tgaagcaagt actcaaatta atgatggtga atggaaagac 300

tcattgtcac aaatgtttct cacaaaaggt tggccaacag gatcagtcta ttttaaagag 360

tattcaagta ttgttgattt ttctgtcgat ccacaattat attgtgatta taacttagta 420

ctaatgaaat atgatcaaaa tcttgaatta gatatgtcag agttagctga tttaatattg 480

aatgaatggt tatgtaatcc aatggatata acattatatt attatcaaca atcgggagaa 540

tcaaataagt ggatatcaat gggatcatca tgtactgtga aagtgtgtcc actgaatacg 600

caaatgttag gaataggttg tcaaacaaca aatgtagact cgtttgaaat ggttgctgag 660

aatgagaaat tagctatagt ggatgtcgtt gatgggataa atcataaaat aaatttgaca 720

actacgacat gtactattcg aaattgtaag aagttaggtc caagagagaa tgtagctgta 780

atacaagttg gtggctctaa tgtattagac ataacagcag atccaacgac taatccacaa 840

actgagagaa tgatgagagt gaattggaaa aaatggtggc aagtatttta tactatagta 900

gattatatta accaaatcgt gcaggtaatg tccaaaagat caagatcatt aaattctgca 960

gctttttatt atagagtata gatatatctt agattagatc gatgtgacc 1009

<210>4

<211>1046

<212>DNA

＜213〉rotavirus

<400>4

ggctttaaaa gagagaattt ccgtctggct aacggttagc tccttttaat gtatggtatt 60

gaatatacca caattctaat ctttctgata tcaattattc tactcaacta tatattaaaa 120

tcagtaactc gaataatgga ctacattata tatagatctt tgttgattta tgtagcatta 180

tttgccttga caagagctca gaattatggg cttaacttac caataacagg atcaatggac 240

actgtatacg ctaactctac tcaagaagga atatttctaa catccacatt atgtttgtat 300

tatccaactg aagcaagtac tcaaattaat gatggtgaat ggaaagactc attgtcacaa 360

atgtttctca caaaaggttg gccaacagga tcagtctatt ttaaagagta ttcaagtatt 420

gttgattttt ctgtcgatcc acaattatat tgtgattata acttagtact aatgaaatat 480

gatcaaaatc ttgaattaga tatgtcagag ttagctgatt taatattgaa tgaatggtta 540

tgtaatccaa tggatataac attatattat tatcaacaat cgggagaatc aaataagtgg 600

atatcaatgg gatcatcatg tactgtgaaa gtgtgtccac tgaatacgca aatgttagga 660

ataggttgtc aaacaacaaa tgtagactcg tttgaaatgg ttgctgagaa tgagaaatta 720

gctatagtgg atgtcgttga tgggataaat cataaaataa atttgacaac tacgacatgt 780

actattcgaa attgtaagaa gttaggtcca agagagaatg tagctgtaat acaagttggt 840

ggctctaatg tattagacat aacagcagat ccaacgacta atccacaaac tgagagaatg 900

atgagagtga attggaaaaa atggtggcaa gtattttata ctatagtaga ttatattaac 960

caaatcgtgc aggtaatgtc caaaagatca agatcattaa attctgcagc tttttattat 1020

agagtataga tatatcttag attaga 1046

<210>5

<211>775

<212>PRT

＜213〉rotavirus

<400>5

Met Ala Ser Leu Ile Tyr Arg Gln Leu Leu Thr Asn Ser Tyr Ser Val

1 5 10 15

Asp Leu His Asp Glu Ile Glu Gln Ile Gly Ser Glu Lys Thr Gln Asn

20 25 30

Val Thr Ile Asn Pro Gly Pro Phe Ala Gln Thr Arg Tyr Ala Pro Val

35 40 45

Asn Trp Asp His Gly Glu Ile Asn Asp Ser Thr Thr Val Glu Pro Ile

50 55 60

Leu Asp Gly Pro Tyr Gln Pro Thr Thr Phe Thr Pro Pro Asn Asp Tyr

65 70 75 80

Trp Ile Leu Ile Asn Ser Asn Thr Asn Gly Val Val Tyr Glu Ser Thr

85 90 95

Asn Asn Ser Asp Phe Trp Thr Ala Val Val Ala Ile Glu Pro His Val

100 105 110

Asn Pro Val Asp Arg Gln Tyr Met Ile Phe Gly Glu Ser Lys Gln Phe

115 120 125

Asn Val Ser Asn Asp Ser Asn Lys Trp Lys Phe Leu Glu Met Phe Arg

130 135 140

Ser Ser Ser Gln Asn Glu Phe Tyr Asn Arg Arg Thr Leu Thr Ser Asp

145 150 155 160

Thr Arg Leu Val Gly Ile Phe Lys Tyr Gly Gly Arg Val Trp Thr Phe

165 170 175

His Gly Glu Thr Pro Arg Ala Thr Thr Asp Ser Ser Ser Thr Ala Asn

180 185 190

Leu Asn Asn Ile Ser Ile Thr Ile His Ser Glu Phe Tyr Ile Ile Pro

195 200 205

Arg Ser Gln Glu Ser Lys Cys Asn Glu Tyr Ile Asn Asn Gly Leu Pro

210 215 220

Pro Ile Gln Asn Thr Arg Asn Val Val Pro Leu Pro Leu Ser Ser Arg

225 230 235 240

Ser Ile Gln Tyr Lys Arg Ala Gln Val Asn Glu Asp Ile Ile Val Ser

245 250 255

Lys Thr Ser Leu Trp Lys Glu Met Gln Tyr Asn Arg Asp Ile Ile Ile

260 265 270

Arg Phe Lys Phe Gly Asn Ser Ile Val Lys Met Gly Gly Leu Gly Tyr

275 280 285

Lys Trp Ser Glu Ile Ser Tyr Lys Ala Ala Asn Tyr Gln Tyr Asn Tyr

290 295 300

Leu Arg Asp Gly Glu Gln Val Thr Ala His Thr Thr Cys Ser Val Asn

305 310 315 320

Gly Val Asn Asn Phe Ser Tyr Asn Gly Gly Phe Leu Pro Thr Asp Phe

325 330 335

Gly Ile Ser Arg Tyr Glu Val Ile Lys Glu Asn Ser Tyr Val Tyr Val

340 345 350

Asp Tyr Trp Asp Asp Ser Lys Ala Phe Arg Asn Met Val Tyr Val Arg

355 360 365

Ser Leu Ala Ala Asn Leu Asn Ser Val Lys Cys Thr Gly Gly Ser Tyr

370 375 380

Tyr Phe Ser Ile Pro Val Gly Ala Trp Pro Val Met Asn Gly Gly Ala

385 390 395 400

Val Ser Leu His Phe Ala Gly Val Thr Leu Ser Thr Gln Phe Thr Asp

405 410 415

Phe Val Ser Leu Asn Ser Leu Arg Phe Arg Phe Ser Leu Thr Val Asp

420 425 430

Glu Pro Pro Phe Ser Ile Leu Arg Thr Arg Thr Val Asn Leu Tyr Gly

435 440 445

Leu Pro Ala Ala Asn Pro Asn Asn Gly Asn Glu Tyr Tyr Glu Ile Ser

450 455 460

Gly Arg Phe Ser Leu Ile Ser Leu Val Pro Thr Asn Asp Asp Tyr Gln

465 470 475 480

Thr Pro Ile Met Asn Ser Val Thr Val Arg Gln Asp Leu Glu Arg Gln

485 490 495

Leu Thr Asp Leu Arg Glu Glu Phe Asn Ser Leu Ser Gln Glu Ile Ala

500 505 510

Met Ala Gln Leu Ile Asp Leu Ala Leu Leu Pro Leu Asp Met Phe Ser

515 520 525

Met Phe Ser Gly Ile Lys Ser Thr Ile Asp Leu Thr Lys Ser Met Ala

530 535 540

Thr Ser Val Met Lys Lys Phe Arg Lys Ser Lys Leu Ala Thr Ser Ile

545 550 555 560

Ser Glu Met Thr Asn Ser Leu Ser Asp Ala Ala Ser Ser Ala Ser Arg

565 570 575

Asn Val Ser Ile Arg Ser Asn Leu Ser Ala Ile Ser Asn Trp Thr Asn

580 585 590

Val Ser Asn Asp Val Ser Asn Val Thr Asn Ser Leu Asn Asp Ile Ser

595 600 605

Thr Gln Thr Ser Thr Ile Ser Lys Lys Leu Arg Leu Lys Glu Met Ile

610 615 620

Thr Gln Thr Glu Gly Met Ser Phe Asp Asp Ile Ser Ala Ala Val Leu

625 630 635 640

Lys Thr Lys Ile Asp Met Ser Thr Gln Ile Gly Lys Asn Thr Leu Pro

645 650 655

Asp Ile Val Thr Glu Ala Ser Glu Lys Phe Ile Pro Lys Arg Ser Tyr

660 665 670

Arg Ile Leu Lys Asp Asp Glu Val Met Glu Ile Asn Thr Glu Gly Lys

675 680 685

Phe Phe Ala Tyr Lys Ile Asn Thr Phe Asp Glu Val Pro Phe Asp Val

690 695 700

Asn Lys Phe Ala Glu Leu Val Thr Asp Ser Pro Val Ile Ser Ala Ile

705 710 715 720

Ile Asp Phe Lys Thr Leu Lys Asn Leu Asn Asp Asn Tyr Gly Ile Thr

725 730 735

Arg Thr Glu Ala Leu Asn Leu Ile Lys Ser Asn Pro Asn Met Leu Arg

740 745 750

Asn Phe Ile Asn Gln Asn Asn Pro Ile Ile Arg Asn Arg Ile Glu Gln

755 760 765

Leu Ile Leu Gln Cys Lys Leu

770 775

<210>6

<211>326

<212>PRT

＜213〉rotavirus

<400>6

Met Tyr Gly Ile Glu Tyr Thr Thr Ile Leu Ile Phe Leu Ile Ser Ile

1 5 10 15

Ile Leu Leu Asn Tyr Ile Leu Lys Ser Val Thr Arg Ile Met Asp Tyr

20 25 30

Ile Ile Tyr Arg Ser Leu Leu Ile Tyr Val Ala Leu Phe Ala Leu Thr

35 40 45

Arg Ala Gln Asn Tyr Gly Leu Asn Leu Pro Ile Thr Gly Ser Met Asp

50 55 60

Thr Val Tyr Ala Asn Ser Thr Gln Glu Gly Ile Phe Leu Thr Ser Thr

65 70 75 80

Leu Cys Leu Tyr Tyr Pro Thr Glu Ala Ser Thr Gln Ile Asn Asp Gly

85 90 95

Glu Trp Lys Asp Ser Leu Ser Gln Met Phe Leu Thr Lys Gly Trp Pro

100 105 110

Thr Gly Ser Val Tyr Phe Lys Glu Tyr Ser Ser Ile Val Asp Phe Ser

115 120 125

Val Asp Pro Gln Leu Tyr Cys Asp Tyr Asn Leu Val Leu Met Lys Tyr

130 135 140

Asp Gln Asn Leu Glu Leu Asp Met Ser Glu Leu Ala Asp Leu Ile Leu

145 150 155 160

Asn Glu Trp Leu Cys Asn Pro Met Asp Ile Thr Leu Tyr Tyr Tyr Gln

165 170 175

Gln Ser Gly Glu Ser Asn Lys Trp Ile Ser Met Gly Ser Ser Cys Thr

180 185 190

Val Lys Val Cys Pro Leu Asn Thr Gln Met Leu Gly Ile Gly Cys Gln

195 200 205

Thr Thr Asn Val Asp Ser Phe Glu Met Val Ala Glu Asn Glu Lys Leu

210 215 220

Ala Ile Val Asp Val Val Asp Gly Ile Asn His Lys Ile Asn Leu Thr

225 230 235 240

Thr Thr Thr Cys Thr Ile Arg Asn Cys Lys Lys Leu Gly Pro Arg Glu

245 250 255

Asn Val Ala Val Ile Gln Val Gly Gly Ser Asn Val Leu Asp Ile Thr

260 265 270

Ala Asp Pro Thr Thr Asn Pro Gln Thr Glu Arg Met Met Arg Val Asn

275 280 285

Trp Lys Lys Trp Trp Gln Val Phe Tyr Thr Ile Val Asp Tyr Ile Asn

290 295 300

Gln Ile Val Gln Val Met Ser Lys Arg Ser Arg Ser Leu Asn Ser Ala

305 310 315 320

Ala Phe Tyr Tyr Arg Val

325

<210>7

<211>175

<212>PRT

＜213〉rotavirus

<400>7

Met Asp Lys Leu Ala Asp Leu Asn Tyr Thr Leu Ser Val Ile Thr Leu

1 5 10 15

Met Asn Asp Thr Leu His Ser Ile Ile Gln Asp Pro Gly Met Ala Tyr

20 25 30

Phe Ser Tyr Ile Ala Ser Val Leu Thr Val Leu Phe Ile Leu His Lys

35 40 45

Ala Ser Ile Pro Thr Met Lys Ile Ala Leu Lys Thr Ser Lys Cys Ser

50 55 60

Tyr Lys Val Ile Lys Tyr Cys Ile Val Thr Ile Ile Asn Thr Leu Leu

65 70 75 80

Lys Leu Ala Gly Tyr Lys Glu Gln Val Thr Thr Lys Asp Glu Ile Glu

85 90 95

Gln Gln Met Asp Arg Ile Val Lys Glu Met Arg Arg Gln Leu Asp Met

100 105 110

Ile Asp Lys Leu Thr Thr Arg Glu Ile Glu Gln Val Glu Leu Leu Lys

115 120 125

Arg Ile His Asp Asn Leu Ile Thr Arg Pro Val Asp Val Ile Asp Met

130 135 140

Ser Lys Glu Phe Asn Gln Lys Asn Ile Lys Thr Leu Asp Glu Trp Glu

145 150 155 160

Ser Gly Lys Asn Pro Tyr Glu Pro Ser Glu Val Thr Ala Ser Met

165 170 175

<210>8

<211>750

<212>DNA

＜213〉rotavirus

<400>8

ggcttttaaa agttctgttc cgagagagcg cgtgcggaaa gatggataag cttgccgacc 60

tcaactacac attgagtgta atcactttaa tgaatgacac attgcattet ataattcaag 120

atcctggaat ggcgtatttt tcatatattg catctgttct aacagtttta ttcatattac 180

ataaagcttc aattccaacc atgaaaatag cattgaaaac atcaaaatgt tcatataaag 240

tgattaaata ttgtatagtc acgatcatta atactctttt aaaattggct ggatataaag 300

agcaggttac tacaaaagac gaaattgagc aacagatgga cagaattgtt aaagagatga 360

gacgtcagct ggatatgatt gataaattaa ctactcgtga aattgaacag gttgaattgc 420

ttaaacgtat acatgacaac ctgataacta gaccagttga cgtcatagat atgtcgaagg 480

aattcaatca gaaaaacatc aaaacgctag atgaatggga gagtggaaaa aatccatatg 540

aaccgtcaga agtgactgca tccatgtgag aggttgagtt accgtcgtct gtcttcggaa 600

gcggcggaac tcttcaccgc aagccccatt agacctgatg attgactgag aagccacagt 660

caatcatatc gcgtgtggct cagccttaat cccgtttaac caatccagcg agtgttggac 720

gttaatggaa ggaatggtct tagtgtgacc 750

<210>9

<211>397

<212>PRT

＜213〉rotavirus

<400>9

Met Glu Val Leu Tyr Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp

1 5 10 15

Lys Ile Val Glu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu Ile Gln

20 25 30

Gln Phe Asn Gln Met Ile Val Thr Met Asn Gly Asn Asp Phe Gln Thr

35 40 45

Gly Gly lle Gly Asn Leu Pro Val Arg Asn Trp Thr Phe Asp Phe Gly

50 55 60

Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val Glu Asn

65 70 75 80

Ala Arg Thr Thr Ile Glu Tyr Phe Ile Asp Phe Ile Asp Asn Val Cys

85 90 95

Met Asp Glu Met Ala Arg Glu Ser Gln Arg Asn Gly Val Ser Pro Gln

100 105 110

Ser Glu Ala Leu Arg Lys Leu Ala Gly Ile Lys Phe Lys Arg Ile Asn

115 120 125

Phe Asp Asn Ser Ser Glu Tyr Ile Glu Asn Trp Asn Leu Gln Asn Arg

130 135 140

Arg Gln Arg Thr Gly Phe Val Phe His Lys Pro Asn Ile Phe Pro Tyr

145 150 155 160

Ser Ala Ser Phe Thr Leu Asn Arg Ser Gln Pro Met His Asp Asn Leu

165 170 175

Met Gly Thr Met Trp Leu Asn Ala Gly Ser Glu Ile Gln Val Ala Gly

180 185 190

Phe Asp Tyr Ser Cys Ala Ile Asn Ala Pro Ala Asn Ile Gln Gln Phe

195 200 205

Glu His Ile Val Gln Leu Arg Arg Ala Leu Thr Thr Ala Thr Ile Thr

210 215 220

Leu Leu Pro Asp Ala Glu Arg Phe Ser Phe Pro Arg Val Ile Asn Ser

225 230 235 240

Ala Asp Gly Ala Thr Thr Trp Phe Phe Asn Pro Val Ile Leu Arg Pro

245 250 255

Asn Asn Val Glu Val Glu Phe Leu Leu Asn Gly Gln Ile Ile Asn Thr

260 265 270

Tyr Gln Ala Arg Phe Gly Thr Ile Ile Ala Arg Asn Phe Asp Thr Ile

275 280 285

Arg Leu Leu Phe Gln Leu Met Arg Pro Pro Asn Met Thr Pro Ala Val

290 295 300

Asn Ala Leu Phe Pro Gln Ala Gln Pro Phe Gln His His Ala Thr Val

305 310 315 320

Gly Leu Thr Leu Arg Ile Glu Ser Ala Val Cys Glu Ser Val Leu Ala

325 330 335

Asp Ala Asn Glu Thr Leu Leu Ala Asn Val Thr Ala Val Arg Gln Glu

340 345 350

Tyr Ala Ile Pro Val Gly Pro Val Phe Pro Pro Gly Met Asn Trp Thr

355 360 365

Glu Leu Ile Thr Asn Tyr Ser Pro Ser Arg Glu Asp Asn Leu Gln Arg

370 375 380

Val Phe Thr Val Ala Ser Ile Arg Ser Met Leu Ile Lys

385 390 395

<210>10

<211>1356

<212>DNA

＜213〉rotavirus

<400>10

ggctttaaaa cgaagtcttc gacatggagg ttctgtactc actgtcaaaa actcttaaag 60

atgctaggga caaaattgtt gaaggtacat tatattctaa cgttagcgat cttattcagc 120

aattcaatca aatgatagta actatgaatg gaaatgactt tcagactgga ggaattggta 180

atttacctgt tagaaattgg actttcgatt ttggtctatt aggtacaaca cttttgaact 240

tggatgctaa ttatgttgag aatgcaagaa ctacaattga atattttatt gactttattg 300

ataatgtatg tatggatgaa atggcaagag aatctcaaag aaatggagta tcgccacaat 360

ctgaagcgtt aagaaagcta gcgggaatta aatttaagag aataaatttc gataattcat 420

cagaatacat agaaaattgg aacttacaaa atagaagaca gcgcaccgga tttgtttttc 480

ataaacctaa catttttcca tactcagctt catttactct aaatagatct caaccaatgc 540

atgataattt aatgggaacc atgtggctta atgctggatc agaaattcaa gtggctggat 600

ttgattactc atgcgccata aatgcaccag cgaacataca gcaatttgag catatcgttc 660

agcttaggcg cgcactgact acagctacta taactttatt acctgatgca gagagattta 720

gttttccaag agtaattaat tcagctgatg gcgcgactac atggttcttt aatccagtta 780

ttctaagacc aaacaatgta gaggtagaat ttttattgaa tggacaaatt attaatacat 840

atcaggctag atttggtact atcatcgcaa gaaattttga tacaattcgt ttattatttc 900

agttgatgcg tccacctaat atgacaccag ctgttaatgc actatttcca caagcacaac 960

cttttcagca ccatgcaaca gttggactta cattacgtat tgaatctgcg gtttgtgaat 1020

cagtgcttgc ggacgcgaat gaaactctgt tagcaaatgt gaccgcggtg cgtcaagaat 1080

atgccatacc agttggaccg gtatttccac caggcatgaa ttggactgaa ttgattacta 1140

actattcgcc atctagagaa gataacttgc aacgcgtttt cacggtagct tccattagaa 1200

gcatgttgat taagtgagga ccagactaaa catctggtat ccaatcttag ttagcatgta 1260

gctacatcaa gtcattcaga ctcttcaagt aaggacatga tttcatgttc gctacgtaga 1320

gtaactgtct gaatgatgta gtgagaggat gtgacc 1356

<210>11

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>11

ggctttaaaa gagagaattt ccgtctgg 28

<210>12

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>12

ggttagctcc ttttaatgta tggta 25

<210>13

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>13

ggtcacatcg aacaattcta atctaag 27

<210>14

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>14

caagtactca aatcaatgat gg 22

<210>15

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>15

tgttgatttt tctgtcgatc cac 23

<210>16

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>16

ggttgctgag aatgagaaat tagctatagt gg 32

<210>17

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>17

ccactatagc taatttctca ttctcagcaa cc 32

<210>18

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>18

tggcttcgcc attttataga ca 22

<210>19

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>19

atttcggacc atttataacc 20

<210>20

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>20

tggcttcact catttataga ca 22

<210>21

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>21

atttcagacc atttataacc tag 23

<210>22

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>22

ggagtagtat atgaaagtac aaataatag 29

<210>23

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>23

ctattatttg tactttcata tactactcc 29

<210>24

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>24

tcgatacagt ataagagagc acaag 25

<210>25

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>25

ttcattaact tgtgctctct tatactg 27

<210>26

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>26

gtatatgtag actattggga tg 22

<210>27

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>27

catcccaata gtctacatat ac 22

<210>28

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>28

tgtaactccg gcaaaatgca acg 23

<210>29

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>29

cgttgcattt tgccggagtt aca 23

<210>30

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>30

gtaagacaag atttagagcg cca 23

<210>31

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>31

tggcgctcta aatcttgtct tac 23

<210>32

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>32

cttgatgctg atgaagcagc atctg 25

<210>33

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>33

cagatgctgc ttcatcagca tcaag 25

<210>34

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>34

cgatcatatc gaatattaaa ggatg 25

<210>35

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>35

catcctttaa tattcgatat gatcg 25

<210>36

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>36

agcgttcaca caatttacat tgtag 25

<210>37

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>37

agtattttat actatagtag attatattaa tc 32

<210>38

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>38

agtattttat actatggtag attatattaa tc 32

<210>39

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>39

atccccatta tactgcattc ctttc 25

<210>40

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>40

atccctatta tactgcattt ctttc 25

<210>41

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>41

atccccatta tactgcattt ctttc 25

<210>42

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉with the oligonucleotide of the total homology of rotavirus

Nucleotide sequence

<400>42

atccctatta tactgcattc ctttc 25

Claims (22)

1. induce the method at the immunne response of rotavirus strain for one kind, this method comprises to the experimenter uses the compositions that comprises attenuation GxPy type rotavirus strain, and described compositions produces at neither the immunne response of the rotavirus strain of the also non-Py type of Gx type.

2. be used for inducing at the purposes in the medicine of the immunne response of rotavirus infection in preparation from the attenuated rotavirus strain of GxPy type, wherein said rotavirus infection by neither the rotavirus strain of the also non-Py type of Gx type cause.

3. according to the method or the purposes of arbitrary claim formerly, wherein said compositions comprises the rotavirus with VP4 gene like this, and described VP4 gene comprises at least a of following base in nucleotide sequence: from start codon in the position 788 adenine base (A), in the position 802 adenine base (A) and in the position 501 thymine alkali bases (T).

4. according to the method or the purposes of claim 3, wherein said VP4 gene comprises nucleotide sequence like this, and it comprises adenine base (A) and 501 comprises thymine alkali bases (T) in the position in the position 788 and 802 from start codon.

5. according to method or the purposes of claim 1-4 in each, wherein said compositions comprises the rotavirus with VP7 gene like this, and described VP7 gene comprises at least a of following base in nucleotide sequence: from start codon in the position 605 thymus pyrimidine (T), in the position 897 adenine (A) and in the position 897 guanine (G).

6. according to the method or the purposes of claim 5, wherein the VP7 gene comprises nucleotide sequence like this, and it comprises thymus pyrimidine (T) and 897 comprises adenine (A) or guanine (G) in the position in the position 605 from start codon.

7. according to method or the purposes of claim 1-6 in each, wherein said compositions comprises the rotavirus with VP4 gene like this, and described VP4 gene comprises adenine (A) and 501 comprises thymus pyrimidine (T) in the position in the position 788 and 802 from start codon in nucleotide sequence; And wherein the VP7 gene comprises thymus pyrimidine (T) and 897 comprises adenine (A) in the position in the position 605 from start codon in nucleotide sequence.

8. according to method or the purposes of claim 1-7 in each, wherein said compositions comprises the G1 rotavirus strain and is used to induce immunne response at the non-G1 serotype of G1 serotype and at least a G2 of being selected from, G3, G4, G5, G6, G7, G8, G9, G10, G11, G12, G13 and G14 serotype.

9. method according to Claim 8 or purposes, wherein said compositions comprise the G1 rotavirus strain and are used to induce immunne response at G1 serotype and G2 serotype.

10. according to the method or the purposes of claim 9, wherein said compositions can also be induced the immunne response at non--G1 serotype of at least a G3 of being selected from, G4 and G9.

11. according to method or the purposes of claim 1-10 in each, wherein said compositions comprises P[8] rotavirus strain and be used to induce at P[8] and at least a P[1 of being selected from], P[2], P[3], P[4], P[5], P[6], P[7], P[9], P[11], P[12], P[14] and P[19] the non-P[8 of type] immunne response of type.

12. according to the method or the purposes of claim 11, wherein said compositions is used to induce at P[4] immunne response of type.

13. according to method or the purposes of claim 1-12 in each, wherein said compositions comprises G1P[8] rotavirus strain and induce at G2P[4] immunne response of rotavirus strain.

14. method or purposes according to arbitrary claim formerly; the wherein said compositions that comprises attenuation GxPy type rotavirus strain provides the protection at the inductive serious gastroenteritis of rotavirus, the inductive serious gastroenteritis of wherein said rotavirus by neither the infection of the rotavirus strain of the also non-Py type of Gx type cause.

15 according to method or the purposes of claim 1-14 in each, and the wherein said compositions that comprises attenuation GxPy type rotavirus strain provides in the inoculation groups of individuals at by neither diarrhoea 40% protection due to the rotavirus strain of the also non-Py type of Gx type at least.

16. according to the method or the purposes of claim 15, wherein said compositions provides at least 50% protection.

17. according to the method or the purposes of arbitrary claim formerly, wherein said compositions provides at least 60% protection.

18. according to method or the purposes of claim 14-17 in each, wherein said compositions provides 40%-80% protection.

19. according to the method or the purposes of claim 18, wherein said compositions provides 50%-70% protection.

20. according to method or the purposes of claim 1-19 in each; wherein said compositions comprises G1P[8] rotavirus strain, it provides in the inoculation groups of individuals at the serious gastroenteritis 40%-75% protection that is caused by the rotavirus infection with G2P4 serotype.

21. according to method or the purposes of claim 1-20 in each, wherein the rotavirus strain in described compositions is an ECACC preservation thing 99081301, or available or can be deutero-from ECACC preservation thing 99081301.

22. according to method or purposes in any item of claim 1-21, wherein said compositions is used with 2 doses of schemes.

23. according to method or purposes in any item of claim 1-21, wherein said attenuated rotavirus strain is prepared with suitable pharmaceutical carrier or with antiacid buffer agent or with both.

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