CN101087886A - Synergistic attenuation of vesicular stomatitis virus, vectors thereof and immunogenic compositions thereof - Google Patents

Synergistic attenuation of vesicular stomatitis virus, vectors thereof and immunogenic compositions thereof Download PDF

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CN101087886A
CN101087886A CNA200580019072XA CN200580019072A CN101087886A CN 101087886 A CN101087886 A CN 101087886A CN A200580019072X A CNA200580019072X A CN A200580019072XA CN 200580019072 A CN200580019072 A CN 200580019072A CN 101087886 A CN101087886 A CN 101087886A
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gene
sudden change
virus
vsv
genome
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CN101087886B (en
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D·K·克拉克
R·M·亨德里
S·A·乌德姆
C·L·帕克斯
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Wyeth LLC
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Abstract

The present invention broadly relates to the synergistic attenuation of vesicular stornatitis virus (VSV). More particularly, the invention relates to the identification of combined mutation classes which synergistically attenuate the pathogenicitnof VSV vectors in mammals and imrPWnogenic compositions thereof.

Description

The Synergistic attenuation of stomatitis herpesvirus, its carrier and its immunogenic composition
Invention field
Present invention relates in general to virusology, microbiology, transmissible disease and field of immunology.More specifically, the present invention relates to the Synergistic attenuation of stomatitis herpesvirus and its carrier, this realizes by making up dissimilar sudden changes.
Background of invention
Stomatitis herpesvirus (VSV) is a member of Rhabdoviridae (Rhabdoviridae), and it has unsegmented, negative justice, single stranded RNA genome.Its 11 kb genomes have 5 genes, encode 5 kinds of structural protein of this virus of the latter: nucleocapsid protein (N) needs described albumen for the encapsidate of the RNA that duplicates with stoichiometric amount; Phosphorprotein (P), it is the cofactor of the RNA polymerase (L) that depends on RNA; Stromatin (M) and adhere to glycoprotein (G) (for example, see Gallione etc., 1981, Rose and Gallione, 1981; Rose and Schubert, 1987 and Schubert etc., 1985; United States Patent (USP) 6,033,886; United States Patent (USP) 6,168,943).
VSV is a kind of arthropod-borne virus, and it can pass to multiple mammalian hosts, the most common ox, horse, pig and rodent.It is rarer that VSV infects the mankind, and normally asymptomatic or be characterised in that gentle influenza-like symptom, and it can disappear at 3-8 days, and did not have complication.Because VSV is not considered human pathogen, and very rare in people colony to the pre-existing immunity of VSV, so the exploitation of VSV deutero-carrier has become the focus such as the field of immunogenic composition and gene therapy.For example, research determines that VSV can be as the efficient carrier of immunogenic composition, thereby expression of influenza viral hemagglutination element (Roberts etc., 1999), Measles virus H albumen (Schlereth etc., 2000) and HIV-1 env and gag albumen (Rose etc., 2001).The further feature that makes it become the VSV of attractive carrier comprises: the ability of (a) duplicating strongly in cell culture; (b) can not be integrated in the host cell DNA or the experience genetic recombination; (c) there is multiple serotype, thereby makes basis-booster immunization strategy become possibility; (d) the target alien gene can be inserted the VSV genome, and by virus transcription enzyme great expression; (e) be used for from exploitation (United States Patent (USP) 6,033,886 of the system of the eggcase of virus genomic cDNA copy rescue infective virus; United States Patent (USP) 6,168,943).
Although in natural infectious process, almost there is not the evidence of getting involved on the VSV neuroscience, the animal of the wild-type virus that wild-type virus that (and under the situation of rodent in the nose) inoculation wild-type virus, mouse brain go down to posterity in the brain or cell culture adapt to (for example, primate, rodent, livestock class animal), the clinical condition that can develop disease is levied, and death in 2-8 days after inoculation usually.Because these are observed, and production had the needs of the carrier that is used for human immunogenic composition of unusual security feature, so in the primate and animalcule neurovirulence model of strictness, the VSV carrier in the test development.These tests are used for detecting any remaining virulence of the VSV carrier of attenuation before considering further people's clinical trial.
Prototype-attenuation of VSV carrier is derived from the synthetic and assembling process of continuous subculture in vitro separately and genome cDNA and runs through the accumulation that a plurality of Nucleotide in the viral genome replace.These sudden changes have the multiple-effect effect, and described effect makes the virus of viral laboratory-adaptation of originating than it in mouse have lower pathogenic (for example, seeing Roberts etc., 1998).By the proteic cytoplasmic tail of brachymemma virus G, thereby cause producing the VSV mutant of existence, also developed the VSV carrier (Schnell etc., 1998) of the further attenuation of prototype from the defective of the plasma membrane budding of infected cell.
When testing in animalcule and inhuman primate neurovirulence model, the known VSV carrier of inferring attenuation or not having attenuation has now had the remaining virulence of unacceptable level.As the exploitation of the VSV carrier of the carrier of immunogenic composition for example, gene therapy vector etc., need in animal nerve virulence model, have be low to moderate most can not detection level pathogenic VSV carrier.Thereby this area needs virus vector at present, (or elimination) pathogenic VSV mutant that has remarkable minimizing in Mammals that modify with sldh gene, attenuation.
Summary of the invention
The present invention relates to the Synergistic attenuation of stomatitis herpesvirus (VSV) widely.More specifically, the present invention relates to the collaborative discriminating that weakens the pathogenic combinatorial mutagenesis type of VSV carrier in the Mammals and its immunogenic composition.
Thereby, in certain embodiments, the present invention relates to genetic modification VSV, it comprises at least 2 kinds of dissimilar sudden changes in its genome, and wherein collaborative to weaken VSV pathogenic for these two kinds sudden changes.In a specific embodiment, with the pathogenic neurovirulence that is further defined as of VSV.In another embodiment, the type of sudden change is temperature sensitive (ts) sudden change, point mutation, gene reorganization sudden change, the change of G-belemnoid, non-cytopathogenic M transgenation, ambisense RNA sudden change, the G transgenation of brachymemma, the G gene inserts sudden change and genetically deficient is suddenlyd change.
In a specific embodiment, two kinds of VSV sudden changes are G transgenation (" G hereinafter, of brachymemma (ct)") and N gene reorganization sudden change (that is to say that the N gene moves to the position in distally in the gene order of VSV from first position of its wild-type 3 ' promotor-nearside).In another embodiment, the VSV G albumen by the G genes encoding of brachymemma has last 20 the amino acid whose disappearance of C-terminal (" G hereinafter, (ct-9)").In another embodiment, the VSV G albumen by the G genes encoding of brachymemma has last 28 the amino acid whose disappearance of C-terminal (" G hereinafter, (ct-1)").In another embodiment, with wild-type VSV genome 3 '-NPMGL-5 ' compares, VSV N gene reorganizes into 3 '-PNMGL-5 ' or 3 '-PMNGL-5, wherein N is the gene of coding nucleocapsid protein, P is the proteic gene of coding phosphor, M is the gene of coding stromatin, and G is the gene that coding adheres to glycoprotein, and L is the proteic gene of RNA polymerase that coding depends on RNA.In certain embodiments, VSV comprise 3 '-PNMG (ct-1)L-5 ', 3 '-PNMG (ct-9)) L-5 ', 3 '-PMNG (ct-1)L-5 ' or 3 '-PMNG (ct-9)The genome of the sudden change of L-5 ', wherein N is the gene of coding nucleocapsid protein, and P is the proteic gene of coding phosphor, and M is the gene of coding stromatin, G (ct-1)Be the gene that coding adheres to glycoprotein, the described glycoprotein that adheres to has the cytoplasmic tail of being made up of an amino acid, G (ct-9)Be the gene that coding adheres to glycoprotein, the described glycoprotein that adheres to has the cytoplasmic tail of being made up of 9 amino acid, and L is the proteic gene of RNA polymerase that coding depends on RNA.In a specific embodiment, the VSV genome of sudden change is 3 '-PMNG (ct-1)L-5 '.In another specific embodiment, the VSV genome of sudden change is 3 '-PNMG (ct-1)L-5 '.In another embodiment, VSV also comprises the sudden change of the 3rd class in its genome, and wherein this sudden change is ts sudden change, point mutation, ambisense RNA sudden change, the change of G-belemnoid, the insertion of G gene, genetically deficient or non-cytopathogenic M transgenation.
In certain embodiments, the LD of the VSV of the modification that encephalic ground is injected in age in 4-week female Swiss-Webster mouse 50100 times of the wild-type VSV that injects for encephalic ground in age in 4-week female Swiss-Webster mouse.At some in other the embodiment, the LD of the VSV that encephalic ground is injected in 4-age in week female Swiss-Webster mouse 501,000 times of the wild-type VSV that injects for encephalic ground in age in 4-week female Swiss-Webster mouse.In other embodiments, the LD of the VSV that encephalic ground is injected in age in 4-week female Swiss-Webster mouse 5010,000 times of the wild-type VSV that injects for encephalic ground in age in 4-week female Swiss-Webster mouse.In other embodiments, the LD of the VSV that encephalic ground is injected in age in 4-week female Swiss-Webster mouse 50100,000 times of the wild-type VSV that injects for encephalic ground in age in 4-week female Swiss-Webster mouse.
In other embodiments of the present invention, two kinds of VSV sudden changes are the G transgenation and the non-cytopathogenic M transgenation of brachymemma.In certain embodiments, the G albumen by the G genes encoding of brachymemma has the kytoplasm stern construction territory (G that is made up of an amino acid (ct-1)) or the kytoplasm stern construction territory (G that forms by 9 amino acid (ct-9)).In other embodiments, non-cytopathogenic the sudden change (" M hereinafter, of M gene (ncp)") be the sudden change (M51A) of methionine(Met) to the methionine(Met) at the sudden change (M33A) of L-Ala and 51 places, position to L-Ala at 33 places, the proteic position of M.In a specific embodiment, VSV comprises 3 '-NPM (ncp)G (ct-1)L-5 ' or 3 '-NPM (ncp)G (ct-9)The genome of the sudden change of L-5 '.In another embodiment, VSV also comprises the 3rd class sudden change in its genome, wherein this sudden change is that ts sudden change, ambisense RNA sudden change, gene reorganization sudden change, genetically deficient sudden change, gene insert that sudden change is inserted in sudden change, G gene, the G-belemnoid becomes or point mutation.
As described in following A.3 part, the ts of any in VSV G, M, N, P or L gene sudden change is " mutation type " of the present invention separately.Thereby in certain embodiments of the invention, two kinds of VSV sudden changes are ts N transgenation (" N hereinafter, (ts)") and ts L transgenation (" L hereinafter, (ts)").In a specific embodiment, VSV comprises 3 '-N (ts)PMGL (ts)-5 ' the genome of sudden change.At some in other the embodiment, VSV also comprises the sudden change of the 3rd class in its genome, wherein this sudden change is G transgenation, G gene insertion sudden change or the genetically deficient sudden change of point mutation, gene reorganization sudden change, the change of G-belemnoid, non-cytopathogenic M transgenation, ambisense RNA sudden change, brachymemma.
In certain embodiments, two kinds of VSV sudden changes are that the G-belemnoid becomes (" G hereinafter, (stem)") and gene reorganization sudden change.In other embodiments, VSV also comprises the sudden change of the 3rd class in its genome, wherein this sudden change is G transgenation, G gene insertion sudden change and the genetically deficient sudden change of point mutation, ts sudden change, gene reorganization sudden change, non-cytopathogenic M transgenation, ambisense RNA sudden change, brachymemma.
In another embodiment, the present invention relates to genetic modification VSV carrier, it comprises at least 2 kinds of dissimilar sudden changes in its genome, with at least one foreign rna sequence as independent transcription unit, the non-essential VSV genome area that duplicates is inserted or replaced to described foreign rna sequence, and wherein collaborative to weaken VSV pathogenic for these two kinds sudden changes.As defined hereinafter, " foreign rna " sequence is non-endogenous any polynucleotide sequence for the genome of wild-type VSV.In a specific embodiment, with the pathogenic neurovirulence that is further defined as of carrier.In other the embodiment, foreign rna is defined as open reading-frame (ORF) (ORF) at some.In other the embodiment, mutation type is selected from some: ts sudden change, point mutation, gene reorganization sudden change, the change of G-belemnoid, non-cytopathogenic M transgenation, ambisense RNA sudden change, the G transgenation of brachymemma, G gene insert sudden change and genetically deficient sudden change.
In a specific embodiment, two kinds of G transgenation and N gene reorganization sudden changes that the sudden change of VSV carrier is brachymemma.In another embodiment, the G albumen by the G genes encoding of brachymemma has last 20 amino acid whose disappearances of C-terminal or last 28 amino acid whose disappearances of C-terminal.At some in other the embodiment, with wild-type VSV genome 3 '-NPMGL-5 ' compares, N gene VSV carrier reorganizes into 3 '-PNMGL-5 ' or 3 '-PMNGL-5 '.In a specific embodiment, the VSV carrier comprises 3 '-PNMG (ct-1)L-5 ', 3 '-PNMG (ct-9)L-5 ', 3 '-PMNG (ct-1)L-5 ' or 3 '-PMNG (ct-9)The genome of the sudden change of L-5 '.In a specific embodiment, the vector gene group of sudden change is 3 '-PMNG (ct-1)L-5 '.In another embodiment, the vector gene group of sudden change be 3 '-PNMG (ct-1)L-5 '.
In other embodiments, the VSV carrier also comprises the sudden change of the 3rd class in its genome, wherein this sudden change is ts sudden change, point mutation, ambisense RNA sudden change, the change of G-belemnoid, G gene insertion sudden change, genetically deficient sudden change or non-cytopathogenic M transgenation.At some in other the embodiment, the LD of the VSV that encephalic ground is injected in 4-age in week female Swiss-Webster mouse 50100 times of the wild-type VSV that injects for encephalic ground in age in 4-week female Swiss-Webster mouse.In other embodiments, the LD of the VSV of the modification that encephalic ground is injected in age in 4-week female Swiss-Webster mouse 501,000 times of the wild-type VSV that injects for encephalic ground in age in 4-week female Swiss-Webster mouse.In other embodiments, the LD of the VSV that encephalic ground is injected in age in 4-week female Swiss-Webster mouse 5010,000 times of the wild-type VSV that injects for encephalic ground in age in 4-week female Swiss-Webster mouse.In another embodiment, the LD of the VSV that encephalic ground is injected in age in 4-week female Swiss-Webster mouse 50100,000 times of the wild-type VSV that injects for encephalic ground in age in 4-week female Swiss-Webster mouse.
, insert or replace the non-foreign rna that duplicates essential VSV genome area and be selected from: the HIV gene in other the embodiment at some, the HTLV gene, the SIV gene, the RSV gene, the PIV gene, the HSV gene, the CMV gene, the Epstein-Barr virus gene, the varicella zoster virus gene, the mumps virus gene, the Measles virus gene, influenza virus gene, virogene of poliomyelitis, the rhinovirus gene, hepatitis A virus genes, hepatitis B virogene, hepatitis c virus gene, the Norwalk virus gene, the togavirus gene, the Alphavirus gene, rubella virus genome, the rabies virus gene, the Marburg virus gene, the Ou Baola virogene, the papillomatosis virus gene, the polyomavirus gene, between matter pneumonitis virus (metapneumovirus) gene, the coronavirus gene, vibrio cholerae (Vibrio cholera) gene, streptococcus pneumoniae (Streptococcus pneumoniae) gene, streptococcus pyogenes (Streptococcus pyogenes) gene, streptococcus agalactiae (Streptococcus agalactiae) gene, meningitis naphthalene Se Shi coccus (Neisseria meningitidis) gene, gonorrhoea naphthalene Se Shi coccus (Neisseria gonorrheae) gene, corynebacterium diphtheriae (Corynebacteria diphtheria) gene, clostridium tetani (Clostridium tetani) gene, Bordetella pertussis (Bordetella pertussis) gene, helicobacter pylori (Helicobacter pylori) gene, Haemophilus spp (Haemophilus) gene, chlamydiaceae (Chlamydia) gene, intestinal bacteria (Escherichia coli) gene, cytokine gene, T-assists epi-position, the CTL epi-position, adjuvant gene and cofactor gene.In a specific embodiment, foreign rna is to be selected from following HIV gene: gag, env, pol, vif, nef, tat, vpr, rev and vpu.In a specific embodiment, the HIV gene is gag, and wherein this gag gene is inserted in genomic position 1 of VSV or position 5.In specific embodiment, the genome of the VSV carrier of sudden change is 3 '-gag 1-PNMG (ct-1)L-5 ', 3 '-gag 1-PNMG (ct-9)L-5 ', 3 '-gag 1-PMNG (ct-1)L-5 ', 3 '-gag 1-PMNG (ct-9)L-5 ', 3 '-PNMG (ct-1)L-gag 5-5 ', 3 '-PNMG (ct-9)L-gag 5-5 ', 3 '-PMNG (ct-1)L-gag 5-5 ' or 3 '-PMNG (ct-9)L-gag 5-5 '.
In another embodiment, foreign rna expressing tumor specific antigen or tumor associated antigen are to induce the immune response at the protectiveness of tumour (for example, malignant tumour).Such tumour specific antigen or tumor associated antigen include, but not limited to KS 1/4 pan-carcinoma antigen; Ovarian cancer antigen (CA125); The prostanoic acid acid phosphate; The antigen of prostate specific; Melanoma associated antigen p97; Melanoma antigen gp75; The membrane antigen of high molecular melanoma antigen and prostate specific.
In other the embodiment, two kinds of VSV carrier sudden changes are G at some (ct)Sudden change and M (ncp)Sudden change.In certain embodiments, the G albumen by the G genes encoding of brachymemma has the kytoplasm stern construction territory (G that is made up of an amino acid (ct-1)) or the kytoplasm stern construction territory (G that forms by 9 amino acid (ct-9)).In other embodiments, M (ncp)Sudden change is the sudden change (M51A) of methionine(Met) to the methionine(Met) at the sudden change (M33A) of L-Ala and 51 places, position to L-Ala at 33 places, the proteic position of M.In a specific embodiment, the genome of sudden change is 3 '-NPM (ncp)G (ct-1)L-5 ' or 3 '-NPM (ncp)G (ct-9)L-5 '.In another embodiment, carrier also comprises the 3rd class and suddenlys change in its genome, and wherein this sudden change is ts sudden change, point mutation, gene reorganization sudden change, the change of G-belemnoid, ambisense RNA sudden change, G gene insertion sudden change and genetically deficient sudden change.In certain embodiments, the VSV carrier comprises and is selected from following HIV gene: gag, env, pol, vif, nef, tat, vpr, rev or vpu.In-individual specific embodiment, the HIV gene is gag, wherein Tu Bian genome be 3 '-gag 1-NPM (ncp)G (ct-1)L-5 ', 3 '-gag 1-NPM (ncp)G (ct-9)L-5 ', 3 '-NPM (ncp)G (ct-1)L-gag 5-5 ' or 3 '-NPM (ncp)G (ct-9)L-gag 5-5 '.
In other embodiments, two kinds of VSV carrier sudden changes are N (ts)Transgenation and L (ts)Transgenation.In a specific embodiment, carrier comprises 3 '-N (ts)PMGL (ts)-5 ' the genome of sudden change.In other embodiments, carrier also comprises the sudden change of the 3rd class in its genome, wherein this sudden change is G transgenation, G gene insertion sudden change and the genetically deficient sudden change of point mutation, gene reorganization sudden change, the change of G-belemnoid, non-cytopathogenic M transgenation, ambisense RNA sudden change, brachymemma.In certain embodiments, the VSV carrier comprises and is selected from following HIV gene: gag, env, pol, vif, nef, tat, vpr, rev or vpu.In a specific embodiment, the HIV gene is gag, and wherein Tu Bian genome is 3 ' gag 1-N (ts)PMGL (ts)-5 ' or 3 '-N (ts)PMGL (ts)-gag 5-5 '.
As described in following A.1 part, the insertion of 3 of foreign nucleus acid sequence (for example, HIV gag) any in the VSV genome in N, P, M, G or the L gene ' end can cause " gene reorganization sudden change " effectively.Thereby in certain embodiments, two kinds of VSV carrier sudden changes are G (stem)Sudden change and gene reorganization sudden change.In one embodiment, the vector gene group of sudden change be 3 '-gag1-NPMG (stem)L-5 '.In other embodiments, the VSV carrier also comprises the sudden change of the 3rd class in its genome, wherein this sudden change is G transgenation, G gene insertion sudden change and the genetically deficient sudden change of point mutation, ts sudden change, gene reorganization sudden change, non-cytopathogenic M transgenation, ambisense RNA sudden change, brachymemma.
In another embodiment, the present invention relates to immunogenic composition, it comprises the genetic modification VSV carrier of immunogenicity dosage, described carrier comprises at least 2 kinds of dissimilar sudden changes in its genome, with at least one foreign rna sequence as independent transcription unit, the non-essential VSV genome area that duplicates is inserted or replaced to described foreign rna sequence, and wherein collaborative to weaken VSV pathogenic for these two kinds sudden changes.In another embodiment, mutation type is selected from: ts sudden change, point mutation, gene reorganization sudden change, the change of G-belemnoid, non-cytopathogenic M transgenation, ambisense RNA sudden change, the G transgenation of brachymemma, G gene insert sudden change and genetically deficient sudden change.
In certain embodiments, two kinds of G transgenation and N gene reorganization sudden changes that sudden change is brachymemma.In specific embodiment, has the kytoplasm stern construction territory (G that forms by an amino acid by the G albumen of the G genes encoding of brachymemma (ct-1)) or the kytoplasm stern construction territory (G that forms by 9 amino acid (ct-9)).In other embodiments, with wild-type VSV genome 3 '-NPMGL-5 ' compares, the N gene reorganizes into 3 '-PNMGL-5 ' or 3 '-PMNGL-5 '.In certain embodiments, the VSV carrier of immunogenic composition comprise 3 '-PNMG (ct-1)L-5 ', 3 '-PNMG (ct-9)L-5 ', 3 '-PMNG (ct-1)L-5 ' or 3 '-PMNG (ct-9)The genome of the sudden change of L-5 '.In a specific embodiment, the vector gene group of the sudden change of immunogenic composition is 3 ' PMNG (ct-1)L-5 '.In another embodiment, the vector gene group of sudden change be 3 '-PNMG (ct-1)L-5 '.In other embodiments, the VSV carrier of immunogenic composition also comprises the sudden change of the 3rd class in its genome, wherein this sudden change is ts sudden change, ambisense RNA sudden change, point mutation, the change of G-belemnoid, G gene insertion sudden change, genetically deficient sudden change or non-cytopathogenic M transgenation.
In other the embodiment, the foreign rna in the genetic modification VSV carrier of insertion immunogenic composition is selected from: the HIV gene at some, the HTLV gene, the SIV gene, the RSV gene, the PIV gene, the HSV gene, the CMV gene, the Epstein-Barr virus gene, the varicella zoster virus gene, the mumps virus gene, the Measles virus gene, influenza virus gene, virogene of poliomyelitis, the rhinovirus gene, hepatitis A virus genes, hepatitis B virogene, hepatitis c virus gene, the Norwalk virus gene, the togavirus gene, the Alphavirus gene, rubella virus genome, the rabies virus gene, the Marburg virus gene, the Ou Baola virogene, the papillomatosis virus gene, the polyomavirus gene, between matter pneumonitis virus gene, the coronavirus gene, cholera vibrio gene, the streptococcus pneumoniae gene, the streptococcus pyogenes gene, the streptococcus agalactiae gene, meningitis naphthalene Se Shi coccus gene, gonorrhoea naphthalene Se Shi coccus gene, the corynebacterium diphtheriae gene, the clostridium tetani gene, the Bordetella pertussis gene, the helicobacter pylori gene, the Haemophilus spp gene, the chlamydiaceae gene, bacillus coli gene, cytokine gene, T-assists epi-position, the CTL epi-position, adjuvant gene and cofactor gene.In a specific embodiment, the foreign rna coding is selected from following HIV albumen: gag, env, pol, vif, nef, tat, vpr, rev and vpu.In a specific embodiment, the HIV gene is gag, wherein this gag gene in the genomic position 1 of VSV or position 5 insert genomes.In another embodiment, the VSV carrier of immunogenic composition comprise 3 '-gag 1-PNMG (ct-1)L-5 ', 3 '-gag 1-PNMG (ct-9)L-5 ', 3 '-gag 1-PMNG (ct-1)L-5 ', 3 '-gag 1-PMNG (ct-9)L-5 ', 3 '-PNMG (ct-1)L-gag 5-5 ', 3 '-PNMG (ct-9)L-gag 5-5 ', 3 '-PMNG (ct-1)L-gag 5-5 ' or 3 '-PMNG (ct-9)L-gag 5-5 ' the genome of sudden change.
In other the embodiment, the VSV carrier of immunogenic composition comprises G at some (ct)Sudden change and M (ncp)Sudden change.In another embodiment, the G albumen by the G genes encoding of brachymemma has the kytoplasm stern construction territory (G that is made up of an amino acid (ct-1)) or the kytoplasm stern construction territory (G that forms by 9 amino acid (ct-9)).In another embodiment, M (ncp)Sudden change is the sudden change (M51A) of methionine(Met) to the methionine(Met) at the sudden change (M33A) of L-Ala and 51 places, position to L-Ala at 33 places, the proteic position of M.In a specific embodiment, immunogenic composition comprises 3 '-NPM (ncp)G (ct-1)L-5 ' or 3 '-NPM (ncp)G (ct-9)The VSV genome of the sudden change of L-5 '.In other embodiments, the VSV carrier of immunogenic composition also comprises the 3rd class and suddenlys change in its genome, wherein this sudden change is ts sudden change, point mutation, gene reorganization sudden change, the change of G-belemnoid, ambisense RNA sudden change, G gene insertion sudden change and genetically deficient sudden change.In other embodiments, the foreign rna in the genetic modification VSV carrier of insertion immunogenic composition is selected from: the HIV gene, the HTLV gene, the SIV gene, the RSV gene, the PIV gene, the HSV gene, the CMV gene, the Epstein-Barr virus gene, the varicella zoster virus gene, the mumps virus gene, the Measles virus gene, influenza virus gene, virogene of poliomyelitis, the rhinovirus gene, hepatitis A virus genes, hepatitis B virogene, hepatitis c virus gene, the Norwalk virus gene, the togavirus gene, the Alphavirus gene, rubella virus genome, the rabies virus gene, the Marburg virus gene, the Ou Baola virogene, the papillomatosis virus gene, the polyomavirus gene, between matter pneumonitis virus gene, the coronavirus gene, cholera vibrio gene, the streptococcus pneumoniae gene, the streptococcus pyogenes gene, the helicobacter pylori gene, the streptococcus agalactiae gene, meningitis naphthalene Se Shi coccus gene, gonorrhoea naphthalene Se Shi coccus gene, the corynebacterium diphtheriae gene, the clostridium tetani gene, the Bordetella pertussis gene, the Haemophilus spp gene, the chlamydiaceae gene, bacillus coli gene, the gene of the Codocyte factor, the gene of the auxiliary epi-position of coding T-, the gene of coding CTL epi-position, the gene of the gene of coding adjuvant and coding cofactor.In certain embodiments, the HIV gene is selected from: gag, env, pol, vif, nef, tat, vpr, rev or vpu.In a specific embodiment, the HIV gene is gag, wherein Tu Bian genome be 3 '-gag 1-NPM (ncp)G (ct-1)L-5 ', 3 '-gag 1-NPM (ncp)G (ct-9)L-5 ', 3 '-NPM (ncp)G (ct-1)L-gag 5-5 ' or 3 '-NPM (ncp)G (ct-9)L-gag 5-5 '.
In other the embodiment, immunogenic composition comprises N at some (ts)Transgenation and L (ts)Transgenation.In a specific embodiment, immunogenic composition comprises 3 '-N (ts)PMGL (ts)-5 ' the VSV genome of sudden change.In other embodiments, immunogenic composition also comprises the sudden change of the 3rd class in its genome, wherein this sudden change is G transgenation, G gene insertion sudden change and the genetically deficient sudden change of point mutation, gene reorganization sudden change, the change of G-belemnoid, non-cytopathogenic M transgenation, ambisense RNA sudden change, brachymemma.In other embodiments, the foreign rna in the genetic modification VSV carrier of insertion immunogenic composition is selected from: the HIV gene, the HTLV gene, the SIV gene, the RSV gene, the PIV gene, the HSV gene, the CMV gene, the Epstein-Barr virus gene, the varicella zoster virus gene, the mumps virus gene, the Measles virus gene, influenza virus gene, virogene of poliomyelitis, the rhinovirus gene, hepatitis A virus genes, hepatitis B virogene, hepatitis c virus gene, the Norwalk virus gene, the togavirus gene, the Alphavirus gene, rubella virus genome, the rabies virus gene, the Marburg virus gene, the Ou Baola virogene, the papillomatosis virus gene, the polyomavirus gene, between matter pneumonitis virus gene, the coronavirus gene, cholera vibrio gene, the streptococcus pneumoniae gene, the streptococcus pyogenes gene, the helicobacter pylori gene, the streptococcus agalactiae gene, meningitis naphthalene Se Shi coccus gene, gonorrhoea naphthalene Se Shi coccus gene, the corynebacterium diphtheriae gene, the clostridium tetani gene, the Bordetella pertussis gene, the Haemophilus spp gene, the chlamydiaceae gene, bacillus coli gene, the gene of the Codocyte factor, the gene of the auxiliary epi-position of coding T-, the gene of coding CTL epi-position, the gene of the gene of coding adjuvant and coding cofactor.In certain embodiments, the HIV gene is selected from: gag, env, pol, vif, nef, tat, vpr, rev or vpu.In a specific embodiment, the HIV gene is gag, wherein Tu Bian genome be 3 '-gag 1-N (ts)PMGL (ts)-5 ' or 3 '-N (ts)PMGL (ts)-gag 5-5 '.
In other the embodiment, immunogenic composition comprises G at some (stem)Sudden change and gene reorganization sudden change.In a specific embodiment, immunogenic composition comprises 3 '-gag 1-NPMG (stem)The genome of the sudden change of L-5 '.In other embodiments, immunogenic composition also comprises the sudden change of the 3rd class in its genome, wherein this sudden change is G transgenation, G gene insertion sudden change and the genetically deficient sudden change of point mutation, ts sudden change, gene reorganization sudden change, non-cytopathogenic M transgenation, ambisense RNA sudden change, brachymemma.
In another embodiment, by being selected from following conventional route arbitrarily, use immunogenic composition of the present invention: intravenous, intracutaneous, subcutaneous, intramuscular, endoperitoneal, per os, rectum, that nose is interior, mouth, vagina and exsomatize (ex vivo).
In another embodiment, the present invention relates to method at HIV infection immunity mammalian subject, comprise the genetic modification VSV carrier of using immunogenicity dosage to the experimenter, described carrier comprises at least 2 kinds of dissimilar sudden changes in its genome, with at least one HIV RNA sequence as independent transcription unit, the non-essential VSV genome area that duplicates is inserted or replaced to described HIV RNA sequence, wherein collaborative to weaken VSV pathogenic for these two kinds sudden changes, and HIV RNA coding is selected from following antigen: gag, env, pol, vif, nef, tat, vpr, rev and vpu.In certain embodiments, the VSV carrier be 3 '-gag 1-PNMG (ct-1)L-5 ', 3 '-gag 1-PNMG (ct-9)L-5 ', 3 '-gag 1-PMNG (ct-1)L-5 ', 3 '-gag 1-PMNG (ct-9)L-5 ', 3 '-PNMG (ct-1)L-gag 5-5 ', 3 '-PNMG (ct-9)L-gag 5-5 ', 3 '-PMNG (ct-1)L-gag 5-5 ', 3 '-PMNG (ct-9)L-gag 5-5 ', 3 '-gag 1-NPM (ncp)G (ct-1)L-5 ', 3 '-gag 1-NPM (ncp)G (ct-9)L-5 ', 3 '-NPM (ncp)G (ct-1)L-gag 5-5 ', 3 '-NPM (ncp)G (ct-9)L-gag 5-5 ', 3 '-gag 1-N (ts)PMGL (ts)-5 ' or 3 '-N ( Ts)PMGL (ts)-gag 5-5 '.
At some in other the embodiment, the present invention relates to the method for directed toward bacteria infection immunity mammalian hosts, comprise the genetic modification VSV carrier of using immunogenicity dosage, described carrier comprises: (a) the dissimilar sudden change of at least 2 kinds in its genome, described sudden change is selected from: the ts sudden change, point mutation, gene reorganization sudden change, the G-belemnoid becomes, non-cytopathogenic M transgenation, the ambisense RNA sudden change, the G transgenation of brachymemma, the G gene inserts sudden change and genetically deficient sudden change, wherein collaborative to weaken VSV pathogenic for these two kinds sudden changes, (b) at least one foreign rna sequence, the non-essential VSV genome area that duplicates is inserted or replaced to described foreign rna sequence, and wherein this RNA coding is selected from following bacterioprotein: vibrio cholerae albumen, the pneumonia streptococcus mycoprotein, streptococcus pyogenes albumen, streptococcus agalactiae albumen, helicobacter pylori protein, meningitis naphthalene Se Shi coccus albumen, gonorrhoea naphthalene Se Shi coccus albumen, corynebacterium diphtheriae albumen, clostridium tetani albumen, Bordetella pertussis albumen, Haemophilus spp albumen, chlamydiaceae albumen and e. coli protein.
In a specific embodiment, two kinds of sudden changes are G (ct)Sudden change and N gene reorganization sudden change.In certain embodiments, the G albumen by the G genes encoding of brachymemma has the kytoplasm stern construction territory (G that is made up of an amino acid (ct-1)) or the kytoplasm stern construction territory G that forms by 9 amino acid (ct-9)At some in other the embodiment, with wild-type VSV genome 3 '-NPMGL-5 ' compares, the N gene reorganizes into 3 '-PNMGL-5 ' or 3 '-PMNGL-5 '.In other embodiments, the VSV genome of sudden change is 3 ' PNMG (ct-1)L-5 ', 3 '-PNMG (ct-9)L-5 ', 3 '-PMNG (ct-1) L-5 ' or 3 '-PMNG (ct-9)L-5 '.In a specific embodiment, the genome of sudden change is 3 ' PMNG (ct-1)L-5 ' or 3 '-PNMG (ct-1)L-5 '.
In other embodiments, VSV also comprises the 3rd class sudden change in its genome, wherein this sudden change is that ts sudden change, point mutation, ambisense RNA sudden change, genetically deficient sudden change, G-belemnoid become, the G gene inserts sudden change, gene inserts sudden change or non-cytopathogenic M transgenation.
In another embodiment, the present invention relates to method at virus infection immunity mammalian hosts, comprise the genetic modification VSV carrier of using immunogenicity dosage, described carrier comprises: (a) the dissimilar sudden change of at least 2 kinds in its genome, described sudden change is selected from: the ts sudden change, point mutation, gene reorganization sudden change, the G-belemnoid becomes, non-cytopathogenic M transgenation, the ambisense RNA sudden change, the G transgenation of brachymemma, the G gene inserts sudden change and genetically deficient sudden change, wherein collaborative to weaken VSV pathogenic for these two kinds sudden changes, (b) at least one foreign rna sequence, the non-essential VSV genome area that duplicates is inserted or replaced to described foreign rna sequence, and wherein this RNA coding is selected from following viral protein: HIV albumen, HTLV albumen, SIV albumen, rsv protein, PIV albumen, HSV albumen, CMV albumen, Epstein-Barr virus albumen, varicella zoster virus albumen, mumps virus albumen, Measles virus albumen, influenza virus protein, poliovirus albumen, rhinovirus albumen, hepatitis A virus albumen, hepatitis b virus protein, hepatitis C virus protein, Norwalk virus albumen, togavirus albumen, Alphavirus albumen, rubella virus protein, rabies virus albumen, the Marburg toxalbumin, the Ou Baola viral protein, papillomavirus protein, polyomavirus albumen, between matter pneumonitis virus albumen and coronavirus albumen.In a specific embodiment, RNA is selected from following HIV gene: gag, env, pol, vif, nef, tat, vpr, rev or vpu.
In certain embodiments, two kinds of sudden changes are G (ct)Sudden change and N gene reorganization sudden change.In a specific embodiment, the VSV genome of sudden change is 3 '-PNMG (ct-1)L-5 ', 3 '-PNMG (ct-9)L-5 ', 3 '-PMNG (ct-1)L-5 ' or 3 '-PMNG (ct-9)L-5 '.In another embodiment, the HIV gene is gag, wherein this gag gene in the position 1 or position 5 insert the VSV genomes, wherein Tu Bian genome be 3 '-gag 1-PNMG (ct-1)L-5 ', 3 '-gag 1-PNMG (ct-9)L-5 ', 3 '-gag 1-PMNG (ct-1)L-5 ', 3 '-gag 1-PMNG (ct-9)L-5 ', 3 '-PNMG (ct-1)L-gag 5-5 ', 3 '-PNMG (ct-9)L-gag 5-5 ', 3 '-PMNG (ct-1)L-gag 5-5 ' or 3 '-PMNG (ct-9)L-gag 5-5 '.In another embodiment, VSV also comprises the 3rd class sudden change in its genome, wherein this sudden change is that ts sudden change, point mutation, ambisense RNA sudden change, genetically deficient sudden change, G-belemnoid become, the G gene inserts sudden change, gene inserts sudden change or non-cytopathogenic M transgenation.
In other embodiment at the method for virus infection immunity mammalian hosts, two kinds of VSV sudden changes are G (ct)Sudden change and M (ncp)Sudden change.In-individual specific embodiment, the VSV genome of sudden change is 3 '-NPM (ncp)G (ct-1)L-5 ' or 3 '-NPM (ncp)G (ct-9)L-5 '.In another embodiment, the VSV genome of sudden change be 3 '-gag 1-NPM (ncp)G (ct-1)L-5 ', 3 '-gag 1-NPM (ncp)G (ct-9)L-5 ', 3 '-NPM (ncp)G (ct-1)L-gag 5-5 ' or 3 '-NPM (ncp)G (ct-9)L-gag 5-5 '.In another embodiment, the VSV genome also comprises the 3rd class and suddenlys change in its genome, and wherein this sudden change is ts sudden change, point mutation, gene reorganization sudden change, the change of G-belemnoid, ambisense RNA sudden change, G gene insertion sudden change and genetically deficient sudden change.
In other embodiment at the method for virus infection immunity mammalian hosts, two kinds of VSV sudden changes are N (ts)Transgenation and L (ts)Transgenation.In a specific embodiment, the VSV genome of sudden change is 3 '-N (ts)PMGL (ts)-5 ', 3 '-gag 1-N (ts)PMGL (ts)-5 ' or 3 '-N (ts)PMGL (ts)-gag 5-5 '.In another embodiment, the VSV genome also comprises the sudden change of the 3rd class in its genome, wherein this sudden change is G transgenation, G gene insertion sudden change and the genetically deficient sudden change of point mutation, gene reorganization sudden change, the change of G-belemnoid, non-cytopathogenic M transgenation, ambisense RNA sudden change, brachymemma.
In other embodiment at the method for virus infection immunity mammalian hosts, two kinds of VSV sudden changes are G (stem)Sudden change and gene reorganization sudden change.In one embodiment, the genome of sudden change be 3 '-gag 1-NPMG (stem)L-5 '.In another embodiment, the VSV genome also comprises the sudden change of the 3rd class in its genome, wherein this sudden change is G transgenation, G gene insertion sudden change and the genetically deficient sudden change of point mutation, ts sudden change, gene reorganization sudden change, non-cytopathogenic M transgenation, ambisense RNA sudden change, brachymemma.
From following detailed, its preferred embodiment and claims, can understand other features and advantages of the present invention.
The accompanying drawing summary
Fig. 1 shown wild-type VSV (3 '-NPMGL-5 '), N reorganization VSV mutant (3 '-PNMGL-5 ' [N2], 3 '-PMNGL-5 ' [N3] and 3 '-PMGNL-5 ' [N4]), G albumen kytoplasm tail (ct) brachymemma VSV mutant (3 '-NPMG (ct-9)L-5 ' [CT9] and 3 '-NPMG (ct-1)L-gag 5-5 ' [CT1-GAG 5]) and the VSV N reorganization of combination /G albumen ct truncated mutant (3 '-PNMG (ct-1)L-5 ' [N2CT1], 3 '-PNMG (ct-9)L-5 ' [N2CT9], 3 '-PMNG (ct-1)L-5 ' [N3CT1] and 3 '-PMNG (ct-9)L-5 ' [N3CT9]) growth kinetics (pfu/mL is to the time).At, seal ground (Indiana) strain of VSV is being represented in the abbreviation " in " that shows in little figure legend.
The VSV mutant (3 '-PNMGL-5 ', 3 '-PMNGL-5 ' and 3 '-PMGNL-5 ') that Fig. 2 has contrasted N reorganization with respect to wild-type VSV (3 '-NPMGL-5 ') and G albumen ct-1 VSV mutant (3 '-NPMG (ct-9)L-gag 5-5 ') growth kinetics.
Fig. 3 contrasted combination VSV N reorganization /G albumen ct-1 mutant (3 '-PNMG (ct-1)L-5 ' and 3 '-PMNG (ct-1)L-5 ') with respect to wild-type VSV (3 '-NPMGL-5 ') and G albumen ct-1 VSV mutant (3 '-NPMG (ct-1)L-gag 5-5 ') the speed of growth.
Detailed Description Of The Invention
The invention of Miao Shuing has solved the needs of this area to stomatitis herpesvirus (VSV) carrier hereinafter, described carrier has significantly weaken pathogenic in Mammals, especially the neuropathogenicity that weakens is as disclosing in animal nerve virulence model.As mentioned above, VSV has many features that it becomes the attractive carrier of immunogenic composition and/or gene therapy that make.For example, it is rarer that VSV infects the mankind, and be asymptomatic or be characterised in that gentle influenza-like symptom, and it can disappear at 3-8 days, and did not have complication, and same, and VSV is not considered human pathogen.The further feature that makes it become the VSV of attractive carrier comprises: the ability of (a) duplicating strongly in cell culture; (b) can not be integrated in the host cell DNA or the experience genetic recombination; (c) there is multiple serotype, thereby makes basis-booster immunization strategy become possibility; (d) the target alien gene can be inserted the VSV genome, and by virus transcription enzyme great expression; (e) be used for from exploitation (United States Patent (USP) 6,033,886 of the system of the eggcase of virus genomic cDNA copy rescue infective virus; United States Patent (USP) 6,168,943) and (f) very rare in people colony to the pre-existing immunity of VSV.
The VSV carrier of describing in this area of the attenuation of type early is called temperature sensitive (ts) mutant, and wherein the ts mutant can not produce virosome in restrictive temperature.For example, various VSV ts mutant known in the art (for example, is seen Holloway etc., 1970; Pringle etc., 1971; Evans etc., 1979; Pringle etc., 1981; Morita etc., 1987; Gopalakrishna and Lenard, 1985).In addition, the VSV mutant of the attenuation of other type has also been described in this area, and it comprises kytoplasm tail (ct) sudden change (Schnell etc., 1998), gene reorganization (or gene order rearrangement) sudden change (Wertz etc., 1998 of the brachymemma of VSV G albumen; Ball etc., 1999; Flanagan etc., 2001; United States Patent (USP) 6,596,529), the G-belemnoid becomes (Jeetendra etc., 2003; Jeetendra etc., 2002; Robinson and Whitt, 2000), non-cytopathogenic M protein mutation (Jayakar etc., 2000; Jayakar and Whitt, 2002) and ambisense RNA sudden change (Finke and Conzelmann, 1997; Finke and Conzelmann 1999).But as mentioned above, when testing in animal model, the VSV carrier of current available attenuation can keep remaining virulence, and same, can not be the carrier material standed for that is used for further people's clinical trial.
As detailed in this article, the present invention relates to unexpected and surprising observation, be the combination of two or more known attenuation mutation types (gene reorganization sudden change, the insertion of G albumen and truncated mutant, ts sudden change and other point mutation, non-cytopathogenic M transgenation, the change of G-belemnoid, ambisense RNA sudden change, genetically deficient sudden change etc.), the pathogenic level that weakens of the realization that obtains is had synergistic effect (rather than additive effect).For example, confirm in this article that when with the N gene mutation body of reorganization when combined, VSV G albumen truncated mutant is known from experience collaborative the weakening of performance VSV growth (embodiment 2) and neurovirulence (embodiment 3).In addition, certain embodiments of the present invention relate to the combination of the sudden change of other type, and it also has synergistic effect to the VSV attenuation.Such type comprises, but be not limited to: G transgenation (for example, ct mutant), ambisense RNA sudden change and the genetically deficient sudden change of ts sudden change, point mutation, gene reorganization sudden change (comprising N, P, M, G and the reorganization of L gene), the change of G-belemnoid, the insertion of G gene, non-cytopathogenic M transgenation, brachymemma.
Thereby, in certain embodiments, the present invention relates to genetic modification VSV carrier, described carrier comprises at least 2 kinds of dissimilar sudden changes in its genome, with at least one foreign rna sequence as independent transcription unit, the non-essential VSV genome area that duplicates is inserted or replaced to described foreign rna sequence, and wherein collaborative to weaken VSV pathogenic for these two kinds sudden changes.At some in other the embodiment, the present invention relates to immunogenic composition, it comprises genetic modification VSV carrier, described carrier comprises at least 2 kinds of dissimilar sudden changes in its genome, with at least one foreign rna sequence as independent transcription unit, the non-essential VSV genome area that duplicates is inserted or replaced to described foreign rna sequence, and wherein collaborative to weaken VSV pathogenic for these two kinds sudden changes.
A. stomatitis herpesvirus mutation type
As mentioned above, genetic modification VSV carrier of the present invention comprises at least two kinds of dissimilar sudden changes in its genome.As defined hereinafter, term " mutation type " or " type of sudden change " are used interchangeably, and when using separately, refer to the sudden change of the VSV of weakening known in the art.For example, " mutation type " of the present invention includes, but not limited to VSV temperature sensitive N transgenation (" N hereinafter, (ts)"), temperature sensitive L transgenation (" L hereinafter, (ts)"), point mutation, G-belemnoid become (" G hereinafter, (stem)"), non-cytopathogenic M transgenation (" M hereinafter, (ncp)"), gene reorganization or reset G the transgenation (" G hereinafter, of sudden change, brachymemma (ct)"), ambisense RNA sudden change, G gene insert sudden change, genetically deficient sudden change etc.As defined hereinafter, " sudden change " comprises sudden change known in the art, modifies as insertion, disappearance, replacement, gene rearrangement or reorganization.
As defined hereinafter, term " collaborative " attenuation refers to the VSV attenuation level greater than additivity.For example, comprise according to the Synergistic attenuation of VSV of the present invention, the sudden change of at least two types of combinations in identical VSV genome, thus cause much larger than the pathogenic minimizing of VSV with the every kind of independent observed additivity attenuation of VSV mutation type level.Thereby, in certain embodiments, the Synergistic attenuation of VSV is defined as at least greater than the LD with the every kind of observed additivity attenuation of independent mutation type level (that is the summation of two kinds of mutation types) 50, wherein in animalcule neurovirulence model, measure attenuation level (that is LD, 50).
As limiting examples, if equation (1) described VSV's " attenuation adds up ":
(1)Δa LD50+Δb LD50＝X LD50；
Δ a wherein LD50Be the LD that has the VSV of first kind of mutation type at its genome 50, Δ b LD50Be the LD that has the VSV of second kind of mutation type at its genome 50, and X LD50Be Δ a LD50With Δ b LD50Summation; Equation (2) has been described VSV of the present invention " Synergistic attenuation " so, and it has at least greater than the LD with the every kind of observed additivity attenuation of independent mutation type level 50:
(2)Δa，b LD50＞(Δa LD50+Δb LD50)；
Δ a wherein, b LD50Be the LD of VSV that in its genome, has the combination of two kinds of mutation types 50, Δ a LD50Be the LD that has the VSV of first kind of mutation type at its genome 50, and Δ b LD50Be the LD that has the VSV of second kind of mutation type at its genome 50Thereby, in certain embodiments, with respect to the LD of two kinds of VSV constructs (every kind of VSV construct has single mutation type in its genome) 50The synergy of having described the VSV attenuation (promptly, two kinds of mutation types in identical VSV genome), the Synergistic attenuation that wherein will have the VSV of two kinds of mutation types in its genome is defined as at least the additivity LD greater than two kinds of VSV constructs that have single mutation type in their genome 50LD 50(for example, see Table VSV LD in 7 50Value).
At some in other the embodiment, with respect to the LD of wild-type VSV 50, the synergy of VSV attenuation has been described.Thereby, in one embodiment, the Synergistic attenuation of VSV is defined as at least LD greater than wild-type VSV 50LD 50, wherein in animal nerve virulence model, measure LD 50In one embodiment, the Synergistic attenuation of VSV being defined as is the LD of wild-type VSV at least 5010 times LD 50, wherein in animal nerve virulence model, measure LD 50In another embodiment, the Synergistic attenuation of VSV being defined as is the LD of wild-type VSV at least 50100 times LD 50, wherein in animal nerve virulence model, measure LD 50In another embodiment, the Synergistic attenuation of VSV being defined as is the LD of wild-type VSV at least 501,000 times LD 50, wherein in animal nerve virulence model, measure LD 50In other embodiments, the Synergistic attenuation of VSV being defined as is the LD of wild-type VSV at least 5010,000 times LD 50, wherein in animal nerve virulence model, measure LD 50At some in other the embodiment, it is the LD of wild-type VSV at least that the Synergistic attenuation of VSV is defined as 50100,000 times LD 50, wherein in animal nerve virulence model, measure LD 50Those skilled in the art uses known testing method and animal model (for example, seeing embodiment 1), can easily determine 50% lethal dose (LD of specific VSV carrier 50) mensuration.
Thereby, in certain embodiments, the present invention relates to genetic modification VSV, it comprises at least two kinds of following dissimilar sudden changes.
1. gene reorganization sudden change
In certain embodiments, genetic modification VSV of the present invention comprises gene reorganization sudden change in its genome.As herein defined, term " gene reorganization ", " gene of reorganization ", " reorganization ", " reorganization ", " gene rearrangement " and " gene displacement (genetranslocation) " are used interchangeably, refer to the variation (sudden change) of the genomic order of wild-type VSV.As herein defined, wild-type VSV genome has following gene order: 3 '-NPMGL-5 '.
Known in the art, the VSV gene is expressed with respect to the determining positions of 3 ' promotor and the level of viral attenuation (United States Patent (USP) 6,596,529 and Wertz etc., 1998, incorporated by reference particularly in this article separately).Coding VSV G known in the art, M, N, P and the proteic nucleotide sequence of L (Rose and Gallione, 1981; Gallione etc., 1981).For example, United States Patent (USP) 6,596,529 have described gene reorganization sudden change, wherein the proteic gene of N is shifted one by one from first position of its wild-type promotor-nearside, and (reorganization) more the position in distally is (for example to the genome, 3 '-PNMGL-5 ', 3 '-PMNGL-5 ', 3 '-PMGNL-5 ', be called N2, N3 and N4 respectively).Thereby in certain embodiments, genetic modification VSV comprises gene reorganization sudden change in its genome.In one type sudden change, in a specific embodiment, genetic modification VSV comprises gene reorganization sudden change, and described sudden change comprises the displacement (for example, 3 '-PNMGL-5 ' or 3 '-PMNGL-5 ') of N gene.
The insertion that should be pointed out that 3 of foreign nucleus acid sequence (for example, HIV gag) any in the VSV genome in N, P, M, G or the L gene ' end in this article causes as defined above " gene reorganization sudden change " effectively.For example, when HIV gag gene being inserted the genomic position 1 of VSV (for example, 3 '-gag 1-NPMGL-5 '), N, P, M, G and L gene each move on the genome the more position in distally since their wild-type position.Thereby, in certain embodiments of the invention, gene reorganization sudden change comprise foreign nucleus acid sequence 3 ' end of any in N, P, M, G or the L gene in the VSV genome insertion (for example, 3 '-gag 1-NPMGL-5 ', 3 '-N-gag 2-PMGL-5 ', 3 '-NP-gag 3-MGL-5 ' etc.).
2.G albumen inserts and truncated mutant
In other the embodiment, genetic modification VSV of the present invention comprises the G gene of sudden change at some, and wherein by brachymemma, this cytoplasmic structure territory is also referred to as G proteic " cytoplasmic tail " to Bian Ma G albumen in its cytoplasmic structure territory (C-terminal).Known in the art, the G transgenation of the C-terminal in brachymemma cytoplasmic structure territory influences VSV budding and attenuated virus production (Schnell etc., 1998; Roberts etc., 1999).The proteic cytoplasmic structure of wild-type VSV G territory comprises 29 amino acid (RVGIHLCIKLKHTKKRQIYTDIEMNRLGK-COOH; SEQ ID NO:1).
In certain embodiments, the VSV G genes encoding G albumen of brachymemma of the present invention wherein lacks last 28 C-terminal amino-acid residues (arginine that only keeps 29 amino acid wild-type cytoplasmic structure territories of SEQID NO:1) in cytoplasmic structure territory.At some in other the embodiment, the VSV G genes encoding G albumen of brachymemma of the present invention wherein lacks last 20 C-terminal amino-acid residues (with respect to 29 amino acid wild-type cytoplasmic structure territories of SEQ ID NO:1) in cytoplasmic structure territory.
At some in other the embodiment, the VSV G genes encoding G albumen of brachymemma of the present invention, its cytoplasmic structure territory (cytoplasmic tail) at it comprises single amino acids, and wherein this single amino acids is the amino acid of natural generation arbitrarily.In other embodiments, the VSV G genes encoding G albumen of brachymemma of the present invention, its cytoplasmic structure territory (cytoplasmic tail) at it comprises 9 amino acid, wherein these 9 amino acid that amino acid is natural generation arbitrarily.At some in other the embodiment, the VSV genes encoding G albumen of sudden change of the present invention, it contains the insertion of representing external epi-position.Such mutant is (for example, seeing Schlehuber and Rose, 2003) known in the art.
As herein defined, the proteic G gene mutation body of G of will encoding is called " G (ct-1)", described G albumen is compared last 28 C-terminal amino-acid residues, the wherein G in disappearance cytoplasmic structure territory with the wild-type sequence of SEQ ID NO:1 (ct-1)The cytoplasmic structure territory have the aminoacid sequence of (R-COOH).As herein defined, the proteic G gene mutation body of G of will encoding is called " G (ct-9)", described G albumen is compared last 20 C-terminal amino-acid residues, the wherein G in disappearance cytoplasmic structure territory with the wild-type sequence of SEQ ID NO:1 (ct-9)The cytoplasmic structure territory have (RVGIHLCIK-COOH; SEQ ID NO:2) aminoacid sequence.Thereby in certain embodiments of the invention, genetic modification VSV of the present invention comprises the G gene of sudden change, and wherein Bian Ma G albumen is G (ct-1)Or G (ct-9)
3. temperature sensitive and other point mutation
As defined hereinafter, VSV " temperature sensitive " (" ts ") sudden change is the VSV genome mutation of restriction VSV in non-permissive temperature growth.For example, VSV ts mutant of the present invention is at permissive temperature (for example, 31 ℃) normal growth, and reaches height and tire, but their growth or breeding can be restricted in non-permissive temperature (for example, 37 ℃ or 39 ℃).By chemistry to produce the ts mutant with site-directed mutagenesis be well-known in the artly (for example, to see Pringle, 1970; Li etc., 1988); And characterized and described many ts mutant and (for example, seen Flamand and Pringle, 1971; Flamand and Bishop, 1973; Printz and Wagner, 1971; Gopalakrishna and Lenard, 1985; Pringle etc., 1981; Morita etc., 1987; Li etc., 1988; Rabinowitz etc., 1977; Lundh etc., 1988; Dal Canto etc., 1976; Rabinowitz etc., 1976).In certain embodiments, genetic modification VSV of the present invention comprises the ts sudden change in its genome, and wherein this ts sudden change is one or more sudden changes of coding G, M, N, P or the proteic nucleotide sequence of L.
As herein defined, the ts of any VSV G, M, N, P or L gene sudden change is " mutation type " that separates of the present invention.For example, in certain embodiments of the invention, the genetic modification VSV that comprises at least 2 kinds of dissimilar sudden changes (wherein these two kinds sudden changes collaborative weaken VSV pathogenic) in its genome comprises one or more ts N transgenation (" N hereinafter, as first kind sudden change (ts)") and one or more ts L transgenation (" L hereinafter, as the sudden change of second class (ts)").As limiting examples, comprise such as 3 '-N (ts)PMGL (ts)-5 ' genomic genetic modification VSV comprise two classes sudden changes (that is (1) N, (ts)Transgenation and (2) L (ts)Transgenation), comprise such as 3 '-gag 1-N (ts)PMGL (ts)-5 ' genomic genetic modification VSV comprise three classes sudden changes (that is (1) N, (ts)Transgenation, (2) L (ts)Gag is passed through in transgenation and (3) 1Insert gene reorganization sudden change).
At some in other the embodiment, genetic modification VSV of the present invention comprises point mutation in its genome, wherein said point mutation is one or more sudden changes of coding G, M, N, P or the proteic nucleotide sequence of L, wherein said sudden change can be given the attenuation phenotype, the fusion of for example acclimatization to cold, reduction or cytopathogenic effect efficient are (for example, see Fredericksen and Whitt, 1998; Ahmed and Lyles, 1997).For example, Fredericksen and Whitt (1998) have described 3 kinds of attenuation point mutation (for example, D137-L, E139-L or DE-SS) of G gene, and its fusion-activity has the pH threshold value of variation.Ahmed and Lyles (1997) have described the attenuation point mutation (N163D) of M gene, and it is the height defective suppressing aspect the host gene expression, and rapider than the turnover of wild-type M albumen.Thereby in certain embodiments, genetic modification VSV of the present invention comprises one or more point mutation in its genome.
4. non-cytopathogenic M transgenation
In other the embodiment, genetic modification VSV of the present invention comprises the non-cytopathogenic sudden change in the M gene at some.VSV (At, seal ground serotype) 229 amino acid whose M of M genes encoding (matrix) albumen, wherein NH 2-terminal preceding 30 amino acid comprise PPPY (PY) motif (Harty etc., 1999) of proline rich.The proteic PY motif of VSV M is positioned at the amino acid position 24-27 of At (Genbank registration number X04452) and New Jersey (NewJersey) (Genbank registration number M14553), VSV seal ground serotype.Jayakar etc. (2000) confirm that the sudden change in the PY motif (for example, APPY, AAPY, PPAY, APPA, AAPA and PPPA) reduces viral yield by the late stage of blocking virus budding.Thereby in certain embodiments, genetic modification VSV of the present invention comprises the non-cytopathogenic sudden change in the M gene, and wherein this sudden change is in the proteic PPPY motif of M of coding.
Be reported that M mRNA 2 kinds of other the albumen of can also encoding recently, be called M2 and M3 (Jayakar and Whitt, 2002).M2 and M3 albumen are the downstream methionine(Met) synthetic from the identical frame of 229 the amino acid whose M albumen (being called M1) of encoding, and lack M1 proteic preceding 32 (M2 albumen) or 50 (M3 albumen) amino acid.Observe, the cell that has infected the reorganization VSV of expression M albumen (but not expressing M2 and M3) shows the outbreak (in some cell type) of the cytopathic effect of delay, yet produces normal viral yield.Thereby in certain embodiments, genetic modification VSV of the present invention comprises the non-cytopathogenic sudden change in the M gene, and wherein said M transgenation produces does not express M2 or the proteic virus of M3 (for example, seeing Jayakar and Whitt, 2002).
Also expect amino acid mutation (for example, disappearance, replacement, insertion etc.) in M albumen PSAP (PS) motif that Irie etc. (2004) describes herein.
5.G-belemnoid becomes
In certain embodiments, genetic modification VSV of the present invention comprises the sudden change in the G gene, and wherein Bian Ma G albumen has the sudden change in the film-nearside stem district (being called G-stem albumen) of G albumen ectodomain.G-stem district comprises the proteic amino-acid residue 421 to 462 of G.Nearest research is verified, weakens VSV (Robinson and Whitt, 2000 by insertion in the proteic G-stem of G and/or disappearance (for example, brachymemma) sudden change; Jeetendra etc., 2002; Jeetendra etc., 2003).Thereby in certain embodiments, genetic modification VSV comprises the G-stem and inserts, lacks, replaces or its combination.In a specific embodiment, comprise genetic modification VSV carrier of the present invention (with its immunogenic composition) that the G-belemnoid becomes and comprise 3 '-gag 1-NPMG (stem)The genome of L-5 '.
6. ambisense RNA sudden change
In certain embodiments, genetic modification VSV of the present invention comprises the ambisense RNA sudden change, and wherein 5 ' anti-genomic promoter (AGP) is replaced by the copy of 3 ' genomic promoter (GP).VSV and other 5 ' AGP unsegmented, minus-stranded rna virus play to duplicate by force promotor, and 3 ' GP play a part to transcribe promotor and a little less than duplicate promotor.In the normal processes that VSV infects, exist the genome copy to surpass the doubly super advantage of 3-to 4-of anti-genome copy; For rabies virus, i.e. another member of Rhabdoviridae, this ratio even higher (Finke and Conzelmann, 1999).The work of rabies virus in the past confirms, replaces the genome and the antigenomic RNA copy of levels such as 5 ' AGP (being called the ambisense RNA sudden change) causes in the cell that infects with the GP of a copy.In addition, express alien gene from being positioned at genomic 5 ' terminal GP copy.When continuous passage in cultured cells, the rabies virus that contains the ambisense RNA sudden change is copied to the 1/10-to 1/15 that tires (Finke and Conzelmann, 1997) of the wild-type rabies virus of reorganization consistently.Such sudden change is used for attenuated virus and duplicates and express alien gene in the VSV carrier.Thereby in certain embodiments, genetic modification VSV comprises the ambisense RNA sudden change.
7. genetically deficient
In other the embodiment, genetic modification VSV of the present invention comprises the virus that has lacked VSV gene (for example G or M) from genome at some.For example, Roberts etc. (1999) have described a kind of VSV carrier, have wherein lacked the proteic whole gene of coding G (Δ G), and replace with influenza hemagglutinin (HA) albumen, and wherein VSV carrier (Δ G-HA) has confirmed the pathogeny that weakens.
B. Chong Zu stomatitis herpesvirus carrier
In certain embodiments, the invention provides genetic modification (reorganization) VSV carrier, it comprises at least 2 kinds of dissimilar sudden changes in its genome, with at least one foreign rna sequence, the non-essential VSV genome area that duplicates is inserted or replaced to described foreign rna sequence as independent transcription unit.
In the art, the method for producing the RNA viruses of reorganization is called " rescue " or " reverse genetics " method.The rescue method of exemplary VSV is described in United States Patent (USP) 6,033, and 886, United States Patent (USP) 6,596,529 and WO 2004/113517, incorporated by reference in this article separately.The enzymatic activity of the multimeric protein mixture by acting on ribonucleoprotein core (nucleocapsid) is realized transcribing and duplicating of negative justice, strand, unsegmented rna virus cdna group.Uncorrected gene expression group RNA can not be used as template.On the contrary, have only when these genome sequences are advanced nucleocapsid structure by the complete encapsidate of N albumen, they just can be identified.Only under this background, genomic and anti-genomic terminal promoter sequence just can be identified, and transcribes or duplicate approach with startup.
Clone's the genomic DNA equivalent of VSV (is for example placed suitable rna polymerase promoter that depends on DNA, T7 rna polymerase promoter) and (for example cut ribozyme sequence certainly, δ hepatitis ribozyme) between, it is inserted in the suitable transcription vector (for example, fertile bacterial plasmid).This transcription vector can provide the dna profiling that can easily operate, RNA polymerase (for example, the T7 RNA polymerase) can verily transcribe the single stranded RNA copy of the anti-genome of VSV (or genome) from this template, described copy has accurate or is close to accurate 5 ' and 3 ' end.The orientation of VSV genomic dna copy and flank promotor and ribozyme sequence is determining it is that anti-genome or geneome RNA equivalent are transcribed.New VSV offspring's rescue also needs anti-genome of naked strand VSV or geneome RNA transcript encapsidate are advanced the specific trans-acting support of the required VSV-of functional nucleocapsid template albumen: virus nucleocapsid (N) albumen, polysaccharase-relevant phosphorprotein (P) and polysaccharase (L) albumen.These albumen comprise the RNA polymerase that depends on RNA of challenge virus, and it must engage this nucleocapsid template, transcribe and duplicate with realization.
Thereby, according to rescue method known in the art, produced the VSV of genetic modification of the present invention and attenuation, it comprises at least 2 kinds of dissimilar sudden changes (for example, seeing the A part) in its genome.For example, under the condition of the production of the VSV of coexpression that is enough to allow these carriers and reorganization, in host cell, use (1) transcription vector, it comprises isolated nucleic acid molecule, described nucleic acid molecule comprises genome or the anti-genomic polynucleotide sequence of the VSV that encodes, (2) at least a expression vector, it comprises at least a separated coding encapsidate, transcribes and duplicate essential trans-acting N, P and the proteic nucleic acid molecule of L, producer gene is modified the VSV carrier, and described carrier comprises at least 2 kinds of dissimilar sudden changes in its genome.According to the present invention, can use any suitable VSV strain or serotype, include, but not limited to At, VSV seal ground, VSV New Jersey, VSV Chandipura, VSV Isfahan, VSV San Juan, VSV Glasgow etc.
Except the polynucleotide sequence of the VSV of coding attenuation form, polynucleotide sequence also can encode one or more allogenic (or external) polynucleotide sequences or open reading-frame (ORF) (ORF) (for example, seeing the C part).Allogenic polynucleotide sequence can change with the need, and comprise, but be not limited to, cofactor, cytokine (for example interleukin-), T-assist the albumen of epi-position, CTL epi-position, limit markers, adjuvant or different microorganisms pathogenic agent (for example virus, bacterium, parasite or fungi), especially can cause the immunoreactive albumen of hope.In certain embodiments, allogenic ORF contains HIV gene (for example, gag, env, pol, vif, nef, tat, vpr, rev or vpu).In a specific embodiment, the HIV gene is gag, wherein this gag gene insert the genomic position 1 of VSV (3 '-gag 1-NPMGL-5 ') or position 5 (3 '-NPMG-gag 5-L-5 ').Allogenic polynucleotide also are used for being provided at the reagent that gene therapy is used.In another embodiment, allogenic polynucleotide sequence is the Codocyte factor also, and for example interleukin 12 selects described cytokine to improve prevention or the treatment feature of the VSV of reorganization.
In certain embodiments, by ordinary method, for example chemomorphosis, the VSV of suddenly change genetic modification of the present invention and attenuation.For example, in the process that virus is grown in cell culture, add chemical mutagen, subsequently: (a) select the virus gone down to posterity at the suboptimal temperature degree, to select the sudden change of temperature sensitive and/or acclimatization to cold, (b) differentiate and in cell culture, to produce the mutated viruses of little plaque and (c) in heterologous host, go down to posterity, to select host-range mutant.In other embodiments, the attenuation sudden change comprises, and uses site-directed mutagenesis (for example, seeing the A part), produces predetermined sudden change, and rescue contains the virus of these sudden changes then.As previously mentioned, genetic modification VSV of the present invention comprises at least two types sudden change in its genome.In certain embodiments, the sudden change of one or more types also comprises a plurality of sudden changes, and the G-belemnoid that for example has dual sudden change (for example, disappearance, insertion, replacement etc.), triple mutant etc. becomes type.Then, at their the VSV carrier (for example, seeing embodiment 1 and embodiment 3) that weakens in animal model these attenuations of screening of virulence.
General (although not necessarily exclusive) rescue environment comprises suitable mammalian cell environment, wherein has the T7 polysaccharase, transcribes anti-genomic (or genomic) single stranded RNA to drive from the transcription vector that contains viral genome cDNA.When corotation is recorded or in the short period of time after this, the anti-genome of this virus (or genome) rna transcription thing is advanced functional template by the nucleocapsid protein encapsidate, and the polysaccharase component that is required joint, described component generates simultaneously from the virus-proteic expression plasmid of specific trans-acting of the coding needs of cotransfection.These incidents and process cause the prerequisite of virus mRNA to be transcribed, and new genomicly duplicate and increase, thereby and cause new VSV offspring's generation, i.e. rescue.
Transcription vector and expression vector generally are to be designed for the plasmid vector of expressing in host cell.The expression vector that comprises at least one separated coding encapsidate, transcribes and duplicate the essential proteic nucleic acid molecule of trans-acting is from identical expression vector or at least 2 these albumen of different vector expressions.From basic rescue method, can know these carriers usually, and they do not need promptly to can be used in the modification method of the present invention through changing.
Be used to save virus for example other technical description of VSV at United States Patent (USP) 6,673,572 and the interim patent 60/477,389 of the U.S. in, it is incorporated by reference hereby.
The host cell that uses in the rescue of VSV is those host cells that allow from the essential necessary component of the VSV of vector expression production reorganization.Can be from prokaryotic cell prokaryocyte or eukaryotic cell, and preferred vertebrate cells, such host cell selected.Usually, host cell is derived from people's cell, for example HEKC (for example, 293).Vero cell, and the cell of many other types are also as host cell.It is the limiting examples of proper host cell below: (1) people diploid primary cell line (for example WI-38 and MRC5 cell); (2) monkey diploid cell line (for example FRhL-fetus macaque pneumonocyte); (3) standard-former generation continuous cell line (Quasi-Primary Continues Cell Line) (for example AGMK-African green monkey kidney cell); (4) people's 293 cells and (5) other potential clone, for example, CHO, MDCK (Madin-Darby dog kidney), former generation chick embryo fibroblast.In certain embodiments, add transfection and promote reagent, take in the DNA that increases cell.A lot of such reagent known in the art (for example, calcium phosphate).Lipofectace (Life Technologies, Gaithersburg, MD) and Effectene (Qiagen, Valencia CA) are common example.Lipofectace and Effectene are cation lipids.They all wrap by DNA, and strengthen the DNA absorption of cell.Lipofectace forms the liposome around DNA, and the Effectene bag is not still formed liposome by DNA.
Then, at first by external mode, the required phenotype of the VSV of the attenuation of test rescue (temperature sensitivity, acclimatization to cold, plaque morphology and transcribe and duplicate and weaken).Also use small-sized replicon (minireplicon) system testing sudden change, trans-acting encapsidate that wherein needs and polymerase activity are provided by wild-type or vaccine helper virus, or the plasmid of the N, the P that are carried gene-specific attenuated mutant by expression and different L gene provides.Also in animal nerve virulence model, the Synergistic attenuation of the VSV of body build-in test attenuation.For example, for detecting neurovirulence, mouse and/or ferret model have been established.In brief, inject the LD that strides expection for the group encephalic ground (IC) of 10 mouse 50Each concentration in the virus concentration of the certain limit of dosage (making the lethal dosage of 50% animal).For example, with 10 2, 10 3, 10 4With 10 5The virus of pfu is carried out IC inoculation, the wherein LD of Bing Du expection 50In scope 10 3-10 4In the pfu.By the virus stock solution used of serial dilution purifying in PBS, the preparation virus formulation.Then, by the top of skull, give the dosage of injected in mice necessity, in 50-100 μ l PBS.The losing weight of monitor animal, sickness rate and death once a day.Then, dead from mouse accumulation in the concentration range of testing, the LD of calculating virus vector 50
C. allogenic nucleotide sequence and antigen
In certain embodiments, the invention provides the VSV of Synergistic attenuation, it also comprises the foreign rna sequence as independent transcription unit, the non-essential genomic locus that duplicates is inserted or replaced to described foreign rna sequence, wherein said foreign rna sequence (it is negative justice) guidance can be in the host cell that VSV infects the production of expressed proteins.Insert VSV cDNA, the genome of original this reorganization of generation by foreign DNA with proteins encoded.In certain embodiments, separate the antigenic dna sequence dna arbitrarily of coding immunogenicity, and it is integrated in the VSV carrier that uses in immunogenic composition of the present invention, when individually or with the combined ground of other antigen of expressing by identical or different VSV, in the VSV of the Synergistic attenuation of reorganization of the present invention, be expressed as and merge or during non-fusion rotein, described immunogenicity antigen produces at disease or prevention of disorder or therapeutic immunization.
In certain embodiments, the VSV of the reorganization of Synergistic attenuation is to the immune response of antigenic induced expression at pathogenic micro-organism.For example, antigen can show immunogenicity of antigens or the antigenicity of finding on bacterium, parasite, virus or fungi as the pathogenic agent of disease or obstacle.In one embodiment, use shows the antigen of human pathogen or the antigenicity or the immunogenic antigen of other target antigen.
For by detect with antibody combine to determine immunogenicity or antigenicity, various immunoassay known in the art have been used, include but not limited to, use the emulative and noncompetitive mensuration system of following technology, radioimmunoassay for example, ELISA (enzyme-linked immunosorbent assay), " sandwich " immunoassay, immunoradiometric assay, the GDP reaction, immunodiffusion(ID) is measured, the original position immunoassay (are used for example Radioactive colloidal gold, enzyme or labelled with radioisotope), Western blot, immunoprecipitation, CA (for example, gel CA, hemagglutination is measured), complement is in conjunction with mensuration, immunofluorescence assay, A protein determination and immunoelectrophoresis are measured, neutralization mensuration etc.In one embodiment, by detecting the mark on the first antibody, measure antibodies.In another embodiment, combine the detection first antibody by what measure second antibody or reagent and first antibody.In another embodiment, second antibody is labeled.Bonded mode in many detection immunoassay known in the art.In detecting an immunogenic embodiment, by standard method, for example, external or cells in vivo toxicity test, tetramer mensuration, enzyme linked immunological spot (elispot) mensuration or the interior delayed type hypersensitivity of body are measured, and measure the cell-mediated reaction of T.
The parasite of the epi-position (antigenic determinant) that expression can be expressed by the VSV of Synergistic attenuation and bacterium (wherein foreign rna instructs the production of antigen or its derivative that contains its epi-position of parasite or bacterium) include but not limited to those that table 1 is listed.
Table 1
The parasite and the bacterium of the epi-position that expression can be expressed by VSV
Parasite
Malaria Eimeria species (plasmodium spp.)
Eimeria (Eimeria spp.)
Nematode
Schistosomicide (schisto)
Leshmania
Bacterium
Vibrio cholerae
Streptococcus pneumoniae
Streptococcus agalactiae
Meningitis naphthalene Se Shi coccus
Gonorrhoea naphthalene Se Shi coccus
Corynebacterium diphtheriae
Clostridium tetani
Bordetella pertussis
Haemophilus spp species (for example, influenza)
The chlamydiaceae species
Enterotoxigenic E.Coli
Helicobacter pylori
Mycobacterium
In another embodiment, antigen comprises the antigenic epi-position of nematode, to be protected from the obstacle that is caused by such worm.In another embodiment, separate any dna sequence dna of coding malaria worm epi-position, to be used for inserting according to VSV of the present invention (-) DNA, when the VSV by reorganization expressed, this dna sequence dna was immunogenic in vertebrate host.Malaria worm species as the DNA source include but not limited to people's malaria parasite subtertian malaria worms (P.falciparum), quartan malaria worm (P.malariae), ovale malaria worm (P.ovale), vivax (P.vivax) and animal malaria parasite Bei Shi malaria worms (P.berghei), Yue Shi malaria worm (P.yoelii), Nuo Shi monkey malaria worm (P.knowlesi) and dog malaria worm (P.cynomolgi).In another embodiment, antigen comprises the β-subunit peptides of Toxins,exo-, cholera.
The virus of the epi-position that expression can be expressed by the VSV of Synergistic attenuation (the wherein antigen of foreign rna guidance virus or the production of its derivative that contains its epi-position) includes but not limited to those that table 2 is listed, table 2 has been listed such virus by section, to be used for convenient and nonrestrictive purpose.
Table 2
The virus of the epi-position that expression can be expressed by VSV
I. Picornaviridae (Picornaviridae)
Enterovirus
Poliovirus
Coxsackie virus
Europe can virus
Rhinovirus
Hepatitis A virus
II. embedding Caliciviridae (Caliciviridae)
The Norwalk papova
III. Togaviridae (Togaviridae) and flaviviridae (Flaviviridae)
Togavirus (for example, dengue virus)
Alphavirus
Flavivirus (for example, hepatitis C virus)
Rubella virus
IV. coronaviridae (Coronaviridae)
Coronavirus
V. Rhabdoviridae (Rhabdoviridae)
Rabies virus
VI. filovirus section (Filoviridae)
Marburg virus
Ou Baola virus
VII. Paramyxoviridae (Paramyxoviridae)
Parainfluenza virus
Mumps virus
Measles virus
Respiratory syncytial virus
Between the matter pneumonitis virus
VIII. orthomyxoviridae family (Orthomyxoviridae)
Orthomyxovirus (for example, influenza virus)
IX. Bunyaviridae (Bunyaviridae)
Cloth Buddhist nun virus
X. Arenaviridae (Arenaviridae)
Arenavirus
XI. Reoviridae (Reoviridae)
Reovirus
Rotavirus
Orbivirus
XII. retrovirus section (Retroviridae)
Human T-cell leukemia virus I type
Human T-cell leukemia virus II type
Human immunodeficiency virus's (for example, I type and II type)
Simian immunodeficiency virus
Slow virus
XIII.Papoviridae
Polyomavirus
Papilloma virus
XIV. Parvoviridae (Parvoviridae)
Parvovirus
XV. herpetoviridae (Herpesviridae)
Hsv
Epstein-Barr virus
Cytomegalovirus
Varicella zoster virus
Herpes virus hominis-6
Herpes virus hominis-7
Cercopithecid herpesvirus-1 (B virus)
XVI. Poxviridae (Poxviridae)
Poxvirus
XVIII. Hepadnaviridae (Hepadnaviridae)
Hepatitis B virus
XIX. Adenoviridae (Adenoviridae)
In specific embodiment, show the antigenicity or the immunogenicity of influenza virus haemagglutinin, human respiratory syncytial precursor virus G glycoprotein (G), Measles virus hemagglutinin or herpes simplex virus type 2 glycoprotein gd by the antigen of the external sequence encoding of expressing after at VSV the host being infected with attenuation.
Other antigen of expressing by the VSV of attenuation include, but not limited to show following antigenic antigenicity or immunogenic those: poliovirus I VP1; The envelope glycoprotein of HIV I; Hepatitis B surface antigen(HBsAg); Diphtheria toxin; Suis 24M epi-position, SpeA, SpeB, SpeC or C5a peptidease; With the gonococcus pilin.
In other embodiments, the antigen of being expressed by the VSV of attenuation shows the antigenicity or the immunogenicity of following material: pseudorabies virus g50 (gpD); pseudorabies virus II (gpB); pseudorabies virus gIII (gpC); the pseudorabies virus glycoprotein h; the pseudorabies virus glycoprotein E; transmissible gastroenteritis glycoprotein 195; the transmissible gastroenteritis stromatin; porcine rotavirus glycoprotein 38; the pig parvoviral capsid protein; the little snake bacterium of swine dysentery (Serpulinahyodysenteriae) protection antigen; bovine viral diarrhoea glycoprotein 55; ewcastle disease virus hemagglutinin-neuraminidase; porcine influenza hemagglutinin or porcine influenza neuraminidase.
In certain embodiments, the antigen of being expressed by the VSV of attenuation shows antigenic antigenicity or the immunogenicity that is derived from dog or cat disease substance, and described pathogenic agent includes, but not limited to feline leukaemia virus, canine distemper virus, hepatitis infectiosa canis virus, canine parvovirus etc.
At some in other the embodiment, the antigen of being expressed by the VSV of attenuation shows and is derived from following antigenic antigenicity or immunogenicity: the little snake bacterium of swine dysentery, foot and mouth disease virus, Pestivirus suis, swine influenza virus, African swine fever virus, mycoplasma hyopneumoniae (Mycoplasmahyopneumoniae), infectious bovine rhinotracheitis virus are (for example, infectivity trachitis bovine rhinovirus glycoprotein E or glycoprotein G) or infectious laryngotracheitis virus (for example, infectious laryngotracheitis virus glycoprotein G or glycoprotein I).
In another embodiment, antigen shows the antigenicity or the immunogenicity of the glycoprotein of following virus: La Crosse virus, newborn calf diarrhea virus, peste loca virus, Punta Toro virus, murine leukemia virus or mouse mammary tumour virus.
In other embodiments, antigen shows antigenic antigenicity of human pathogen or immunogenicity, and described human pathogen includes but not limited to herpes virus hominis, hsv-1, hsv-2, human cytomegalic inclusion disease virus, Epstein-Barr virus, varicella zoster virus, herpes virus hominis-6, herpes virus hominis-7, human influenza virus, human immunodeficiency virus's (1 type and/or 2 types), rabies virus, Measles virus, hepatitis B virus, hepatitis C virus, subtertian malaria worm and Bordetella pertussis.
By various standards; for example antigen in and participation in the infectivity of pathogenic agent; type or group-specific; patient's the antiserum(antisera) or the identification of immunocyte; and/or to specific antiserum(antisera) of antigen or immunocyte the demonstration of provide protection; can identify the antigen or derivatives thereof of potentially useful, with the antigen of expressing as VSV by attenuation.
In another embodiment; the foreign rna of the VSV of attenuation instructs the antigenic production that comprises epi-position; when the VSV with attenuation imports required host, this epi-position induction of immunity reaction, described immune response is protected from situation or the obstacle that is caused by the entity that contains this epi-position.For example, antigen can be tumour specific antigen or tumor associated antigen, to be used to induce the immune response at the protectiveness of tumour (for example, malignant tumour).Such tumour specific antigen or tumor associated antigen include, but not limited to KS 1/4 pan-carcinoma antigen; Ovarian cancer antigen (CA125); The prostanoic acid acid phosphate; The antigen of prostate specific; Melanoma-related antigen p97; Melanoma antigen gp75; The membrane antigen of high molecular melanoma antigen and prostate specific.
Insert the foreign DNA of coding for antigens in nonessential site of the VSV DNA of attenuation, also randomly comprise the foreign DNA sequence of the Codocyte factor, express among the host that described cytokine can infect at the VSV by attenuation, and immune response stimulating.For example, such cytokine includes but not limited to interleukin 1 α, 1 β, 2,4,5,6,7,8,10,12,13,14,15,16,17 and 18, interferon-' alpha ', interferon-beta, interferon-, granulocyte colony-stimulating factor, rHuGM-CSF and tumor necrosis factor alpha and β.
D. immunogenic and pharmaceutical composition
In certain embodiments, the present invention relates to immunogenic composition, it comprises the genetic modification VSV carrier of immunogenicity dosage, described carrier comprises at least 2 kinds of dissimilar sudden changes in its genome, with at least one insertion or replace non-foreign rna sequence of duplicating essential VSV genome area, wherein collaborative to weaken VSV pathogenic for these two kinds sudden changes.
The VSV carrier of preparing Synergistic attenuation of the present invention is to be used to be administered to mammalian subject (for example, people).Such composition generally comprises VSV carrier and pharmaceutically acceptable carrier.As using hereinafter, statement " pharmaceutically acceptable carrier " be intended to comprise compatible with medicament administration arbitrarily with all solvent, dispersion medium, dressing, antibacterium and anti-mycotic agent, isotonic agent and absorption delay agent etc.The such medium of pharmaceutically active substance and the application of reagent are well-known in the art.Except conventional arbitrarily medium or reagent are incompatible with the VSV carrier, the such medium of use in immunogenic composition of the present invention.Also the active ingredient polymer blends that replenishes can be advanced in the composition.
Thereby, VSV immunogenic composition of the present invention is mixed with compatible with its expection route of administration.The example of route of administration comprises parenteral (for example, intravenous, intracutaneous, subcutaneous, intramuscular, endoperitoneal) and mucous membrane (for example, in per os, rectum, the nose, mouthful, vagina, breathe).The solution or the suspension that are used for parenteral, intracutaneous or subcutaneous application comprise following component: sterile diluent is water for injection, salt brine solution, fixed oil, polyoxyethylene glycol, glycerine, propylene glycol or other synthetic for example; Antibacterial agent is phenylcarbinol or methyl p-hydroxybenzoate for example; Antioxidant is xitix or sodium bisulfite for example; Sequestrant is ethylenediamine tetraacetic acid (EDTA) for example; Buffer reagent is for example sodium-chlor or dextrose of acetate, Citrate trianion or phosphoric acid salt and tension regulator for example.Regulate pH, for example hydrochloric acid or sodium hydroxide with acid or alkali.Parenteral administration can be packaged in ampoule, disposable syringe or the multi-agent bottle made from glass or plastics.
The pharmaceutical composition that is applicable to the injection purposes comprises aseptic aqueous solution (at this moment being water miscible) or dispersion and the sterilized powder that is used for temporarily preparing sterile injectable solution or dispersion.Use for intravenously, suitable carriers comprises physiological saline, system bacterium water, Cremophor EL TM(BASF, Parsippany, NJ) or phosphate-buffered saline (PBS).In all cases, composition must be aseptic, and should be to reach the fluid that has the degree that is easy to syringeability (Syringability).It must be stable under production and storage conditions, and must prevent that the contamination ground of microorganism (for example bacterium and fungi) from storing.Carrier is solvent or dispersion medium, and it contains, for example, and water, ethanol, polyvalent alcohol (for example, glycerine, propylene glycol and liquid macrogol etc.) and its suitable mixture.Keep suitable flowability, for example, by using for example Yelkin TTS of dressing, by under the situation of dispersion, keeping the size of particles that needs and passing through to use tensio-active agent.By various antibacterial agents and anti-mycotic agent, for example, metagin, chlorobutanol, phenol, xitix etc. are realized the prevention of microbial process.In many cases, preferably in composition, comprise isotonic agent, for example, sugar, polyvalent alcohol is mannitol for example, Sorbitol Powder, sodium-chlor.By comprise the reagent that can postpone absorption in composition, for example aluminum monostearate and gelatin are realized the absorption of the delay of Injectable composition.
By in the suitable solvent that the VSV carrier fusion of requirement (or dosage) is advanced to have a kind of of mentioned component or combination, prepare sterile injectable solution by filtration sterilization then as required.Usually, by the active ingredient polymer blends is advanced in the sterile carrier, described carrier contains other composition from mentioned component of basic dispersion medium and needs, prepares dispersion.Under the situation of the sterilized powder that is used to prepare sterile injectable solution, preferred manufacturing procedure is vacuum-drying and lyophilize, and it can produce activeconstituents and add powder from the composition of any other needs of the solution of sterile filtration before it.
Use for suction, since from containing the pressure-vessel of suitable propelling agent (for example, gas is carbonic acid gas for example, or atomizer) or the aerosol form of divider, send and pass compound.Systemic administration can be by mucous membrane or through the mode of skin.For mucous membrane or through the using of skin, in preparation, use the permeate agent that is applicable to the barrier that will permeate.Such permeate agent is normally known in the art, and comprises, for example, for mucosal administration, stain remover, cholate and fusidic acid derivatives.By using nasal spray or suppository, finish mucosal administration.Also compound can be become to be used for rectum and send the suppository (for example, having conventional suppository base for example theobroma oil and other glyceryl ester) passed or the form of enema,retention.
In certain embodiments, advantageously with per os or the parenteral composition of dosage unit form preparation, so that use consistence with dosage.As using hereinafter, dosage unit form refers to be suitable for the physically discrete unit of the unitary dose of doing experimenter to be treated; Each unit contains calculating with the active compound of the result of treatment that produces needs and the pharmaceutical carrier combination of needs of predetermined amount.The technical requirements of dosage unit form of the present invention is depended on and is directly depended on the specific characteristic of active compound and the particular treatment effect that will realize, and this area inherent prepares such restriction that is used for the treatment of individual active compound.
All patents and the publication quoted in this article, all incorporated by reference hereby.
D. embodiment
Except the situation that other has a detailed description, use the well-known and conventional standard technique of those skilled in the art, carry out the following examples.The purpose that the following examples are used to illustrate, and should not understand with any way that limits the scope of the invention.
Embodiment 1
Material and method
VSV G albumen kytoplasm urogomphus variant
The method that is used to produce G albumen kytoplasm urogomphus variant of the present invention is known in the art, and is described in detail by (1998) such as Schnell.Compare with 29 amino acid kytoplasm stern construction territories (SEQ ID NO:1) of wild-type VSV seal ground At strain, these G protein mutants keep single amino acids (G in G kytoplasm stern construction territory (ct-1)) or 9 amino acid (G (ct-9)).By translation stop codon being moved to real terminator codon upstream 60 Nucleotide or 84 Nucleotide (being respectively nine amino acid kytoplasm tail and 1 amino acid kytoplasm tail), and cause the proteic brachymemma of G, produce the brachymemma of kytoplasm tail.
The mutant of VSV N gene reorganization
Be the the 2nd, the 3rd or the 4th gene by the N gene is reapposed, produce N gene displacement mutant (N reorganization) from viral genome 3 ' end beginning.For example, with the real gene order 3 of wild-type VSV '-NPMGL-5 ', be mutated into 3 '-PNMGL-5 ' and 3 '-PMNGL-5 '.The displacement of the N gene of Du Te 3 ' rna transcription promotor causes the proportional decline of N gene expression dose (for example, see United States Patent (USP) 6,596,529, it is whole in this article incorporated by reference) further away from each other.The decline of N protein level slows down virus nucleocapsid formation in the infected cell, thereby finally reduces genome duplication speed and virion formation.The method that is used for the displacement of N gene is described below.
For the first step that produces the reorganization of N gene, cDNA removes the N gene fully from the total length viral genome, and the result is to make the P gene be close to the virus leader sequence, to substitute the N gene.In order to delete the N gene, use full-length gene group cDNA as template, prepared two kinds of PCR products.First kind of PCR product contains sequence and the BsmBI site, additional downstream that extends to virus leader sequence end from the natural B saAI site of T7 promotor upstream.Second kind of PCR product contains the sequence that extends to the P gene transcription start signal of closing on BsmBI site, additional upstream from the natural XbaI site of P gene.Can seamlessly connect the mode of (after digesting and being connected) two kinds of PCR products, arrange the BsmBI site, contain the one dna fragmentation of the virus leader sequence that is close to the P gene with generation.Then, this dna fragmentation is connected the into XbaI/BsaAI site of full-length gene group cDNA, thereby eliminate the N gene effectively from viral genome.
In producing next step of N gene reorganization, the N gene is inserted between the P and M gene of genome cDNA of disappearance N, or between M and the G gene, or between G and the L gene.For the N gene is inserted between P and the M gene, as template, 3 kinds of PCR products have been prepared with full-length gene group cDNA.First kind of PCR product contains the sequence that extends to the M gene transcription start signal with additional flank BsmBI site from the natural XbaI site of P gene.Second kind of PCR product contain from N gene transcription start signal extend to have additional flank BsmBI site close on 3 of N gene '-sequence of the conservative TATG sequence of AAAAAAA-polyadenylation signal.The third PCR product contains the sequence that extends to the P gene conservative TATGAAAAAAA polyadenylation signal with additional flank BsmBI site from the natural MluI site that the G gene begins.Then, digest all 3 kinds of fragments with BsmBI, and reconnect, to form one dna fragmentation, it has the N gene of the part of side joint P gene and M gene.Then, digest this dna fragmentation, and connect the virus genomic XbaI/MluI of δ-N site with XbaI and MluI, with form 3 '-PNMGL-5 ' cDNA.
For produce 3 '-PMNGL-5 ' genome cDNA, prepared 2 kinds of PCR products that separate.First kind of PCR product contains the sequence that extends to G gene transcription start signal with additional flank BsmBI site (5 '-AACAG-3 ') from the natural XbaI site of P gene.Second kind of PCR product contains complete N gene order, it is from having the transcription initiation signal in BsmBI site, additional flank upstream, to N genetic transcription termination/polyadenylation signal, the latter has the flanking sequence that extends to the natural MluI site the G gene from G transcription initiation signal.The sequence of G gene specific is added the N gene order, as the part of one of PCR primer.With two kinds of PCR products of BsmBI digestion, and connect,, and connect the XbaI/MluI site of the genome cDNA that advances to lack N then with XbaI and MluI digestion to form one dna fragmentation, with form 5 '-PMNGL-3 ' sequence in the gene.
For produce 5 '-PMGNL-5 ' genome cDNA, from complete genome cDNA template, prepared 3 kinds of PCR products.First kind of PCR product contains the sequence that extends to the L gene transcription start signal in the additional BsmBI of side joint site from the natural SwaI site of G gene.Second kind of PCR product contains the sequence of whole N gene, and to transcription termination signal, the latter connects additional BsmBI site at two end sides from the transcription initiation signal for it.The third PCR product contains from the sequence in the natural HpaI site of the initial L of the extending to gene of L genetic transcription in the additional BsmBI of side joint site.Digest all 3 kinds of PCR products with BsmBI, and connect to form one dna fragmentation, then with SwaI and HpaI digestion, and connect the SwaI/HpaI site of the genome cDNA that advances to lack N, thereby form 5 '-PMGNL-3 ' sequence in the gene.In the genome of all 3 kinds of rearrangements, the sequence integrity of each gene is all identical with unaltered virus with flank adjusting sequence, just the position difference of N gene.
The combination that G albumen kytoplasm urogomphus becomes and N reorganization suddenlys change
The genome (that is, two kinds of mutation types) of the dual sudden change of combination results of N gene reorganization and the brachymemma of G albumen kytoplasm tail, for example 3 '-PNMG (ct-9)L-5 ', 3 '-PNMG (ct-1)L-5 ' and 3 '-PMNG (ct-9)L-5 ', 3 '-PMNG (ct-1)L-5 '.By G with above-mentioned brachymemma (ct-1)Or G (ct-9)Natural G gene in the genome of gene swapping N reorganization makes up dual mutator gene group cDNA.By with MluI and HpaI digestion donor cDNA (5 '-NPMG (ct-1)L-3 ' and 5 '-NPMG (ct-9)L-3 '), then G gene purifying, brachymemma is connected the into genomic MluI/HpaI of the cDNA site of N reorganization, exchange.Save these double mutants from cDNA, by plaque size and growth kinetics as described below, the triple plaques of purifying, amplification and sign in cell culture.
Non-cytopathogenic M transgenation
VSV M genes encoding viroplast (M) albumen and 2 kinds of interior polypeptide (M2 and M3) of littler frame.M2 translates from the open reading-frame (ORF) (ORF) identical with M albumen with the M3 polypeptide, and lacks preceding 33 and 51 amino acid respectively.The reorganization VSV carrier that generation as described below comprises non-cytopathogenic M transgenation (promptly, do not express the proteic VSV carrier of M2 and M3) yet, and described carrier comprises one or more other sudden change in addition, thereby is created in the VSV carrier of cell culture and animal camber attenuation.
Use clone's strategy of PCR-based, wherein the Nucleotide replacement (AUG to GCT) with necessity is integrated into PCR primer (Jayakar and Whitt, 2002; Jayakay etc., 2000), generate non-cytopathogenic M transgenation (M (ncp)), it causes methionine(Met) 33 and 51 transformations to L-Ala (M33A, M51A).Then, with the M33 that contains that obtains, the PCR product cloning of 51A sudden change is advanced total length VSV cDNA genome, thereby allows not express the rescue of the virus of M2 and M3 polypeptide.
By exchange XbaI-MluI fragment (striding whole M gene and part P gene), will be present in the M33 among the reorganization VSV carrier cDNA of Jayakar and Whitt design, VSV carrier cDNA is transferred in 51A sudden change.CDNA fragment exchange can not cause removing M33, any other the amino acid code change outside the 51A sudden change.
The combination of change of G albumen kytoplasm urogomphus and non-cytopathogenic M transgenation
The genome of the dual sudden change of combination results of tail brachymemma of G albumen kytoplasm and non-cytopathogenic M transgenation (that is, two kinds of mutation types), for example 3 '-NPM NcpG Ct-1L-5 ' or 3 '-NPM NcpG Ct-9L-5 '.Exchange by the M gene cDNA that will contain the sudden change that produces non-cytopathogenic phenotype and into to contain G (ct-1)Or G (ct-9)The full-length gene group cDNA of sudden change makes up dual mutator gene group cDNA.In each case, the cDNA fragment of exchange extends to the Mlu I site of the uniqueness 5 ' non-translational region of G gene from the Xba I site of the uniqueness of P gene, and comprises whole non-cytopathogenic M gene order.
As previously mentioned, the proteic difference of M that replace non-cytopathogenic M albumen and it is only 2 aminoacid replacement (M33A and M51A), and the latter produces non-cytopathogenic phenotype.Then,, further modify the genome of these dual sudden changes, to allow to be used for the proteic expression of gag of immunogenicity research by HIV-1 gag gene is inserted between the G and L gene at 5 places, genomic position.As for other viral rVSV carrier, Xho I/Nhe I site in the uniqueness at 5 places, position of genome cDNA is entered in the gag gene clone.
VSV N gene temperature sensitive mutation and/or VSV L gene temperature sensitive mutation
By substituting N gene and/or L gene (Pringle with the same source coding sequence that is derived from known biologically-derived VSV temperature sensitive (ts) mutant, 1970), modification is from VSV (the rVSV) (rVSV-Gag of the proteic reorganization of coding HIV Gag of first the 3 ' cistron in the viral genome 1).The carrier that obtains, (i) rVSV-Gag 1TsN (that is, 3 '-gag 1-N (ts)PMGL-5 ') contains ts N gene, (ii) rVSV-Gag from VSV strain ts41 1TsL (that is, 3 '-gag 1-NPMGL (ts)-5 ') contain from the L gene of VSV strain ts11 and (iii) rVSV-Gag 1TsN+L (that is, 3 '-gag 1-N (ts)PMGL (ts)-5 ') contain from the ts N gene of VSV strain ts41 with from the L gene of VSV strain ts11.VSV strain ts41 and ts11 also are called tsG41 and tsG11 respectively in this area.
After the VSV (the Glasgow strain of At, seal ground serotype) to laboratory-adaptation carried out chemomorphosis, Pringle had separated biologically-derived ts gene-donor strain (Pringle, 1970).Pringle has also drawn the collection of illustrative plates of the ts sudden change of N or L gene.
From infected cell RNA, clone ts41 N and ts11 L gene.In brief, at permissive temperature (31-32 ℃), infect bhk cell with ts11 or ts41.Allow to infect, surpassing in 75% cell monolayer up to cytopathic effect is significantly, extracts constantly and the total RNA of purifying at this.Then, use gene-specific primer, reverse transcription RNA, synthetic to instruct cDNA, after this by pcr amplification cDNA.Then, the cDNA that increases is cloned into rVSV vector gene group cDNA, and verify by sequential analysis.
Determine the complete genome group sequence of ts11, ts41 and their ancestors' strain (Glasgow), facilitate the code change of ts phenotype with discriminating.From the encoding sequence of rVSV carrier background, Pringlets mutant and Glasgow ancestors virus, may predict which code change can facilitate rVSV-Gag by contrast 1TsN, rVSV-Gag 1TsL and rVSV-Gag 1The ts phenotype of tsN+L carrier.
Table 3 has contrasted N Argine Monohydrochloride sequence.Can obviously find out from data, substitute rVSV carrier N gene with the ts41 homologue and cause 4 aminoacid replacement.During these change any all may influence the N protein function under the carrier genetic background, and facilitates the ts phenotype.Should be noted that an only variation (Tyr to Cys at 74 places, position is with the residue of italic demonstration), ts41 and its ancestors' virus (Glasgow) are distinguished, show that this replacement may be crucial ts determinative.
Table 3
The proteic contrast of VSV N
The amino acid numbering Virus strain
XN2 ts41 Glasgow
14 Val Ile lle
74 Tyr Cys Tyr
128 Ser Arg Arg
353 Asn Ser Sar
Similarly, table 4 provides the contrast of L albumen.The L gene that substitutes in the rVSV carrier with the ts11 resemblance causes 13 amino acid code change.As top about as described in the N gene, in these code change any all may be facilitated the observed ts phenotype that is produced that substitutes by the L gene, but several (showing with italic) in these encoding mutants are more interesting, because they also distinguish ts11 and its Glasgow ancestors virus, thereby potentially these aminoacid replacement discriminatings are the crucial origin cause of formation of ts phenotype.
Table 4
The proteic contrast of VSV L
The amino acid numbering Virus strain
XN2 ts11 Glasgow
87 Pro Ser Ser
88 Thr Ala Thr
202 Ile Leu Leu
203 Arg Lys Lys
268 Tyr His Tyr
367 Thr Ala Ala
1112 Pro Ser Pro
1374 Ala Val Val
1519 Ile Leu Leu
1792 Leu Val Val
1793 Ile Val Ile
2042 Leu Ser Leu
2075 Arg Lys Lys
The G-belemnoid becomes and G-stem/gene reorganization sudden change
In certain embodiments, genetic modification VSV of the present invention comprises the sudden change in the G gene, and wherein Bian Ma G albumen has the sudden change in the film-nearside stem district (being called G-stem albumen) of G albumen ectodomain.By with the G gene (Schnell etc., 1996) in the G gene substitution VSV XN carrier genetic background of the coding G-stem of modifying, import the G-belemnoid and become.G-stem (Robison, 2000) is formed by 108 in 512 G Argine Monohydrochlorides, comprising: 1) proteic preceding 17 amino acid of G, and it comprises the signal sequence that polypeptide target is inserted to film; 2) be called 42 amino acid of the film-nearside ectodomain of stem; 3) 20 amino acid whose membrane spaning domains; With 4) tail in 29 amino acid whose C-terminal born of the same parents.This configuration of G-stem polypeptide contains the sophisticated G protein sequence that is enough to mediate virion, but lacks the necessary sequence of performance CAP effect.As a result, the cell that has infected G-stem carrier can be expressed the exotic antigen of viral protein and coding, but can generate noninfective progeny virus particle, the global function G albumen because G-stem carrier can not be encoded.
To contain the required proteic G-stem of the functional G carrier granule of target cell infection in order generating, must transly to provide total length G albumen.This can realize in virus rescue and the production of vaccine process of passing through one of following method subsequently: 1) can develop and express the proteic clone of G; 2) can adopt the proteic complementary virus vector of expression G, for example adenovirus, MVA or VEE; Or 3) cell that can be used to produce with the coding proteic plasmid DNA carrier of G or mRNA transfection.
Now, by the instantaneous complementation in being designed for the cell of expressing the proteic plasmid transfection of G, produce G-stem carrier.This can be avoided generating the needs of the proteic clone of expression G, and described clone is difficult to produce, because G albumen is deleterious, and also can avoid biological reagent (as helper virus) is imported in the production process.In some configurations of G-stem carrier, the cistron of the viral protein of will having encoded is reorganized to the downstream, inserts first genome position to allow alien gene.This understands attenuated virus, and the exotic antigen gene is placed the promotor nearside, thereby guarantees high-caliber expression.
As described in top A1 part, HIV gag gene (or any other gene) to the insertion of the genomic position 1 of VSV (3 '-gag 1-NPMGL-5 ') cause gene reorganization sudden change effectively, each moves to position farther on the genome since their wild-type position wherein said N, P, M, G and L gene.Thereby, G (stem)Sudden change and gag are to the insertion (gag of the genomic position 1 of VSV 1) the dual sudden change of combination results genome 3 '-gag 1-NPMG (stem)L-5 '.
The rescue of the stomatitis herpesvirus in 293 cells
According to the rules of following correction, use International Application No. WO 2004/113517 (specifically incorporated by reference in this article) described basic heat shock/plasmid-T7 system, successfully save VSV from 293 cells.
Material
Plasmid DNA: 1) total length viral genome cDNA, 2) pT7-N, 3) pT7-P, 4) pT7-L, 5) pT7-M, 6) pT7-G and 7) pCI-Neo-bcl-T7 (p0061).
Calcium phosphate transfection reagent: 1) 2X BES-buffer saline: 50mM BES (pH 6.95-6.98), 280mM NaCl, 1.5mM Na 2HPO 4, 2) and 2.5M CaCl 2With 3) Hepes-buffer saline washing soln (HBS): 20mM hepes (pH7.0-7.5), 140mM KCl, 1mM MgCl 2
Cell cultures solution: 1) added DMEM 10% qualify, heat-inactivated FBS (DMEM/FBS), 2) MEM (IMEM) of having added Iscoves 10% qualify, heat-inactivated FBS improvement (IMEM/FBS), 3) poly-L-Lysine: 0.01%, be dissolved in H 2Among the O, 4) PBS and 5) pig trypsinase/EDTA.
Method
293 cell cultures: 293 cells are difficult to cultivate, and many different their methods of processing are arranged.Present method is successfully as the part of the rescue system of the VSV vector construction body of VSV and modification.
The routine cultivation of going down to posterity:
1) removes substratum, and wash the individual layer (10cm flat board) that converges with 5ml temperature PBS; Side along ware is moved liquid gently, with the desorption of prevention cell (placed 293 cells of a specified duration or that becoming in the substratum of alkalescence (redness) in room temperature and understand desorption).
2) add 2ml trypsinase gently, and swing plate, to cover whole individual layer.Suction trypsinase, and allow flat board to place about 1 minute in room temperature.With the miter angle tilt flat plate, on the working-surface of stink cupboard tip-tap it so that the cell desorption.If cell does not have desorption, then cultivate 1 minute (guarantee cell at this stage desorption, thereby can avoid acutely moving liquid) in addition in room temperature.
3) add 5ml DMEM/FBS gently, and move down liquid lentamente, with cell dispersion.
4) the 1ml cell is added to the flat board that contains 9ml DMEM/FBS.
5) at 37 ℃, 5%CO 2Cultivate.
The cultivation of going down to posterity before the transfection:
1) is required the flat board of number with poly--L Methionin bag.The about 3-4ml 0.001% poly--L Methionin of each dull and stereotyped adding, and allow it to place at least 30 minutes in room temperature.Suction gathers-the L lysine solution.With 5ml substratum washing plate.
2) trypsin digestion and cell as mentioned above.Use the separation (split) that can produce the flat board that 50-75% converges next day than (1: 3 to 1: 6).
3) make the cell desorption after, add IMEM/FBS, and with the flat board of cell transfer to the bag quilt that contains 9mlIMEM/FBS.Before transfection, isolated cell also allows its overnight growth in IMEM/FBS seemingly important.
4) at 37 ℃, 5%CO 2Cultivate.
Transfection:
1) before transfection 1-3 hour,, and is being set at 3%CO to the cell 9ml IMEM/FBS that feeds 232 ℃ of incubators in culturing cell.
2) be prepared as follows calcium phosphate-DNA transfection mixture:
A) in the 5ml polypropylene tube, merge following DNA:(i) 8 μ g T7-N,
(ii) 4 μ g T7-P, (iii) 1.2 μ g T7 L, (iv) 1.0 μ g T7-M, (v) 1.0 μ g T7-G, (vi) 10 μ g viral genome cDNA clone and (vii) 10 μ g hCMV-T7 expression vectors.
B) water is with the final volume of volume-adjustment to 450 μ l.
C) add 50 μ l 2.5M CaCl 2
D) in vortex tube gently, add 500 μ l 2XBBS, allow pipe to place 15-20 minute then in room temperature.
3) take out cell from incubator, and in substratum, slowly add calcium phosphate-DNA mixture, and rotation gently, to distribute precipitation.Immediately cell is returned 32 ℃-3%CO 2Incubator.
4) after the beginning transfection 3 hours, culture dish is sealed in the plastics bag, and is immersed in the water-bath that is set at 43 ℃ 2 hours fully, with the inducing cell heat shock response.
5) after the heat shock, cell is returned 32 ℃-3%CO 2Incubator, and continue overnight incubation.
6) next day, with HBS washed cell 2 times, and to the cell 10ml IMEM/FBS that feeds.At 37 ℃, 5%CO 2Cultivate.
7) the beginning transfection after 48-72 hour, set up enough T150 bottles, wherein contain 20ml DMEM/FBS, be used for cells transfected is transferred to more large container.1 T150 bottle is used for each 10cm flat board of transfection.
8) by moving substratum on the liquid individual layer gently, shift 293 cells of transfection so that it is removed from cell surface.Avoid acutely moving liquid, and use just enough power, to remove cell.After removing cell, on move down liquid about 5 times, to reduce the size of cell lump, then with substratum and cell transfer to the T150 bottle that contains 20ml IMEM/FBS.
9) after 4-6 hour, (note: if cell does not adhere on the flat board, then this step can postpone up to 24 hours to replace substratum with the fresh DMEM that has added 10%FBS.Equally, successfully skipped this step).
10) the monitoring cell is 5-7 days, to detect the evidence of cytopathic effect.
11) show as when obvious as CPE, the 50ul medium supernatant is transferred in the hole of 6 hole flat boards, the Vero cell monolayer of substratum and establishment is contained in described hole.If save, then can see CPE (note: this step is important, because 293 cells are sometimes really from the surface desorption of T150 bottle, and does not in fact have to show VSV-and infect when infected at them) in next day.
12) after sample transfer arrives the Vero cell monolayer in a small amount, from T150 bottle harvested cell and substratum, and freezing-70.Usually, can be by moving the substratum on the liquid individual layer so that the cell desorption is gathered in the crops 293 cells.
The rescue of the stomatitis herpesvirus in the Vero cell
Solution
Usually, following solution can be used for the host cell transfection:
1) 280mM NaCl[16.4g NaCl (or 56ml 5M NaCl)], 50mMBES[10.7g BES (free acid form)] and 1.5mM sodium phosphate [0.21g Na 2HPO 4] 2XBBS (every L) solution (2XBES-buffer saline).With NaOH BBS solution is adjusted to pH 6.95-6.98.Then, with the solution filtration sterilization, and freezing.
2) the 2.5M CaCl of the every 100ml cumulative volume 36.8g of preparation 2Solution, and-20 ℃ of preservations.Use nitrocellulose, with the solution filtration sterilization.Should avoid using the rhodia strainer, because they can block.Perhaps, with transfection solution autoclaving.But a kind of method in back may be not satisfactory, because 2XBBS solution may change in the high-pressure sterilizing course part omitted.
Usually, following solution can be used as substratum:
1) added DMEM+FBS solution (the high glucose that contains glutamine of the DMEM of the FBS of 10% heat-inactivated and qualify and 10-20 μ g/ml (randomly being up to 50 μ g/ml) gentamicin; Gibco/BRL, [Grand Island, NY]).
2) the MEM+FBS solution of MEM (has been added FBS and 10-20 μ g/ml (randomly being up to 50 μ g/ml) the 20-25mM Hepes damping fluid of glutamine, non-essential amino acid, 10% heat-inactivated and qualify; Gibco/BRL) (Grand Island NY), and randomly comprises the 1X amphotericin).
3) Hepes-buffer saline washing soln, 20mM hepes, pH 7.0,150mMNaCl, 1mM MgCl 2HBS solution.
Method
Can select useful host cell usually from isolating Vero cell, described Vero cell places DMEM+FBS the day before yesterday in transfection, thereby they can be about 50% to converge [for RSV, 80-90%] (at 6 hole flat board or 12.5cm in next day 2In the bottle).Higher cell density has lower efficient.Next day, every kind of culture was fed 1-4 hour with 4.5mlDMEM+FBS before transfection.Then, with cell transfer to being set in 3%CO 2CO with 32 ℃ 2Incubator.The time that Vero cell growth is spent the night, needing only them is about 50% to converge when transfection.
The following CaCl that obtains 2/ calcium phosphate precipitation: before beginning, keep BBS and CaCl in room temperature 2In the 5ml polypropylene tube that contains 250 μ l cumulative volumes, with being total to 2-20 μ g plasmid DNA and 25 μ l CaCl 2, preparation DNA mixture.The DNA that is used for the total length rescue comprises Full-length cDNA Construction body, 400ng N albumen, 300ng P albumen, 100-200ng L albumen and 5-10 μ g pCI-Neo-Bcl-T7 plasmid (the SEQ ID NO:1 of 5 μ g VSV; Fig. 2).Rescue efficient in the Vero cell is lower, thus every kind of total length construct transfection 3-6 hole to be saved.
Prepare all DNA/CaCl 2Behind the solution, add 2XBBS.This common following realization: by successive low speed vortex, stirring pipe gently, and, dropwise add 250 μ l2XBBS along the sidewall of pipe.For all pipes, repeat this step, allow described pipe to place other 15-20 minute, to allow to form the DNA-calcium phosphate precipitation in room temperature.Behind the room temperature incubation, will precipitate dropwise and add in the cell culture medium, and, it be distributed equably by swing plate.Then, be set in 3%CO 2Incubator in, incubation substratum 3 hours.3%CO 2Level for BBS/CaCl 2Rotaring dyeing technology is important; 5%CO 2Work non-constant, if it can be worked.3%CO 2Control the pH of substratum, and allowing in substratum, to form effective calcium phosphate-DNA precipitation.
Then, randomly carry out heat shock operation, for example after the beginning transfection 3 hours.With cell transfer to the water-bath that is set in 44 ℃.Cell is sealed in the plastics preservation bag, thereby culture can be immersed in the water fully.44 ℃ carry out 3 hours after, cell transfer returned is set in 3%CO 232 ℃ of incubators, and continue overnight incubation.
Next day, take out transfection media, and with HBS washed cell 2 times.After the washing, add the fresh DMEM+FBS of 2ml.For washing step, PBS and the work of HankShi damping fluid are relatively poor, may be because the phosphoric acid salt in these damping fluids can cause more CaCl 2From transfection media, be precipitated out.
Then, randomly carry out common cultivation operation.After transfection 48-72 hour, by they are scraped in the substratum, and cell is added substratum change the T25 bottle that contains the 50% Vero cell monolayer that converges over to, gather in the crops cells transfected.Begin this and cultivated altogether back 6 hours, substitute substratum with 4ml MEM+FBS.Then, the incubation culture is 5 days.Exhaust if substratum begins to show in this incubation period, then take out the 2ml substratum, and replace with fresh MEM+FBS.Do not recommend to replace all substratum, be kept at generate in the rescue process may be in substratum the virus of any a small amount of.During this common cultivation stage, CPE may become obviously, but often really not so.If CPE is not obvious, then can continue rescue.
Began common cultivation back 5 days, harvested cell.At first, with 0.5ml 2.18M sucrose, 37.6mMKH 2PO 4, 71.0mM K 2HPO 4, the 49.0mM Sodium Glutamate adds substratum, and mixes by shaking bottle.Then, cell is scraped into substratum, on move down liquid to mix, aliquots containig enters in the freeze pipe of transportation usefulness then, and quick freezing in dry ice/ethanol bath then, and preservation in-80 ℃.
VSV carrier purifying
From the supernatant liquor of cells transfected, the VSV carrier of plaque-purifying rescue.After 3 continuous rounds of plaque purification, amplicon virus on bhk cell is with seeding stoste, again with its further amplification on bhk cell, to produce virus work stoste.In order to prepare a large amount of zooperal virus that is used for,, use work stoste to infect the bhk cell that converges of 10-20 T-150 bottle with the infection multiplicity (MOI) of 0.5-1.0 plaque forming unit (pfu)/cell.32 ℃ carry out 48 hours after, by 4,000xg is centrifugal, the cell conditioned medium liquid that clarification is infected.Then, by in 10% sucrose bed course, 25,000rpm, in SW 28 rotors centrifugal 1 hour, concentrating virus from supernatant liquor.The virus precipitation is suspended in the phosphate-buffered saline (PBS) again, and in ethanol/the dry ice bath, freezes suddenly.Then, on the Vero cell monolayer, the spissated virus stock solution used of titration is to determine the number of infective granule in the preparation.
Titration of virus
By the plaque measurement of standard, determine the number of infectious virus particle in the virus formulation.In brief, infect the Vero cell monolayer that spends the night that newly converges in the 6 hole flat boards with 10 times of serial dilutions of virus formulation.For this reason, from cell monolayer suction growth medium, and 100 μ l aliquots containigs of every kind of viral dilution liquid that will be in DMEM are transferred to the center of cell monolayer in triplicate.In order to prevent cell dehydration, again 400 μ l DMEM are added each cell monolayer, and flat board was kept 15 minutes in room temperature, shake down in interruption then, at 37 ℃, 5%CO 2Incubation 30 minutes.Then, take out the virus inoculation thing, and cover 3ml for each cell monolayer to be dissolved in 0.8% agarose among the DMEM.Then, at 37 ℃, 5%CO 2Dull and stereotyped 1-4 days of incubation forms to allow plaque.Then, take out the agarose plug, and in room temperature, with Viola crystallina (being dissolved in 2% Viola crystallina in 50% methyl alcohol) with cell dyeing 10 minutes.Then, remove unnecessary dyestuff, and water cleaning down cell monolayer.Then, manifest the viral plaque in the cell monolayer, they are not dye blue aperture.
By the quantitative viral RNA of PCR in real time
Use quantitative PCR in real time (RT/PCR) to measure, the VSV genome in detection and the quantitative animal tissues.Measure and use 2-step RT/PCR method, it can detect the negative-sense viral geneome RNA specifically, and uses the synthetic oligonucleotide of whole amplicon to form typical curve.In brief, will become to be dissolved in 20%W/V slurries among the SPG from the brain tissue homogenate of monkey, ferret and mouse.3, the centrifugal slurries of 000xg 15 minutes are with the deposit seeds material.Then, 14, the further centrifuged supernatant of 000xg, and extract total RNA from the supernatant liquor that obtains.Use the template of this RNA, then, product is used for PCR in real time measures as the reverse transcription of the primer that uses virus-specific.
Measure the 50% lethal dose (LD of VSV carrier in mouse 50)
With mouse LD 50Model is as the tolerance of the relative attenuation of VSV carrier.With wild-type VSV, 3 '-NPMG (ct-1)L-5 ', 3 '-PNMG (ct-1)L-5 ' and 3 '-PMNG (ct-1)Several log-of L-5 ' are (log-fold) diluent doubly, is injected into 4.5 all female Swiss Webster mouse in age (6-10 mouse/group) encephalic.Observe losing weight, paralyse and dead (LD of mouse then 50), continued for 3 weeks.By the method for Reed and Muench,, calculate LD from the cumulative percentage mortality ratio 50
The former Journal of Sex Research of mouse immune
With 1 * 10 7The embodiment 4 described VSV carriers of the sign of pfu (At, seal ground serotype), intramuscular ground immune mouse (n=15).Start (priming) back 7 days, at the effective stage peak value, from one group of mouse (" basis ", n=5) separating Morr. cell.With 1 * 10 7The VSV carrier (NJ-G-changes version) that pfu indicates is strengthened 2 groups of mouse (n=10).Strengthen back 5 days, at the effective stage peak value, from one group of mouse (" reinforcement ", n=5) separating Morr. cell.Strengthen back 30 days, in the memory stage process, from another group mouse (memory, n=5) separating Morr. cell.By tetramer dyeing, determine the specific CD8 T of Gag cell frequency.With the immunodominant peptide of gag stimulate spend the night after, determine the specific IFN-γ secretion of Gag by ELISPOT.
Embodiment 2
The sign of VSV mutant
Observed the essence difference (table 5) of two kinds of mutation type VSV carriers (N reorganizes/G albumen ct brachymemma) of embodiment 1 described combination with respect to the plaque size of single type sudden change VSV carrier.Generally, single type sudden change VSV carrier formed the plaque of count enable size in the plaque measurement at 24 hours, and some N reorganization /G albumen ct brachymemma carrier needs to form over 3-4 days the plaque that is equal to size.The relative different of the plaque size of VSV carrier also with observed relative different similar (Fig. 1 to Fig. 3) in the growth kinetics research process of cell cultures.
Table 5
The relative plaque size of VSV carrier
Figure A20058001907200631
Embodiment 3
The research of VSV neurovirulence
In a series of mouse, ferret and the research of monkey neurovirulence, estimated with single type VSV mutational vector and compared, comprise the Synergistic attenuation of VSV of the combination of two or more mutation types, its method is described among the embodiment 1.Mouse highly allows VSV to duplicate, and this character allows them to be used to distinguish the different levels of viral growth and attenuation.For different VSV carriers, in mouse, observed the pathogenic/attenuation (table 6 and table 7) of different gradients.For example, encephalic inoculation 3 '-NPMGL-5 ', 3 '-NPMG (ct-1)L-5 ', 3 '-PNMG (ct-1)L-5 ' or 3 '-PMNG (ct-1)The LD of the mouse of L-5 ' 50Relative attenuation gradient below (table 6) shown: 3 '-PMNG (ct-1)L-5 ' (LD 50=2x10 5)＞3 '-PNMG (ct-1)L-5 ' (LD 50=1x10 4)＞3 '-NPMG (ct-1)L-5 ' (LD 50=14.5)＞3 '-NPMGL-5 ' (LD 50=3.2).
Table 6
The number of mouse death or paralysis
IC dosage (pfu)
Carrier 1 10 100 1000 10000 100000
3′-NPMG (ct-1)L-5′ (LD 50＝14.5pfu) ND 3 death 5 death 6 death 6 death ND
3’-PNMG (ct-1)L-5’ (LD 50＝1×10 4pfu) ND 2 paralysis 1 paralysis of 3 death 1 paralysis of 1 death 1 paralysis of 1 death ND
3′-PMNG (ct-1)L-5’ (LD 50＞2×10 5pfu) ND ND 1 paralysis of 1 death 1 paralysis 3 paralysis 4 paralysis of 2 death
3′-NPMGL-5′(wt) (LD 50＝3.2pfu) 1 death 5 death 6 death 6 death ND ND
Give 6 mouse intracranial (IC) inoculation every kind of above-mentioned carrier.
Below table 7 show have 0 (wild-type VSV), 1,2,3 and the mouse of the intracranial injection VSV carrier of 4 kind of (insertion of gag gene) mutation type in LD 50Also show and similarly weaken gradient.And, intracranial injection VSV carrier 3 '-gag 1-PMNG (ct1)L-5 ', 3 '-gag 1-N (ts)PMGL (ts)-5 ', 3 '-gag 1-NPMGL (ts)-5 ', 3 '-gag 1-NPM (ncp)G (ct1)L-5 ', 3 '-gag 1-PMNG (ct9)L (ts)-5 ' and 3 '-gag 1-NPMG (stem)The mouse of L-5 ' does not show mortality ratio.
Table 7
The encephalic neurovirulence of VSV carrier in mouse
Carrier LD 50 (pfu) The number of mutation type
3’-NPMGL-5’(VSV wt) 3.2 0
3’-NPMGL-5’(rVSV) 10 0
3’-NPMG-gag 5-L-5’ 10 1
3’-PNMGL-5’ 12 1
3’-PMNGL-5’ 14 1
3’-NPMG (ct-1)L-5′ 14.5 1
3’-NPMG (ct-1)-gag 5-L-5′ 24 2
3′-gag 1-NPMGL-5′ 115 1
3’-PNMG (ct-1)L-5’ 1×10 4 2
3’-PMNG (ct-1)L-5’ 2×10 5 2
3′-gag 1-PMNG (ct-9)L-5 1×10 6 3
3′-gag 1-PNMG (ct-1)L-5′ 1×10 6 3
3′-NPM (ncp)G-gag 5-L-5′ ＞1×10 6 2
3′-gag 1-N (ts)PMGL-5′ 8×10 6 2
3′-PMNG (ct-1)-gag 5-L-5′ 1×10 7 3
3′-NPM (ncp)G (ct-1)-gag 5-L-5′ ＞1×10 7 3
3′-gag 1-PMNG (ct-1)L-5′ ＞1×10 7 3
3′-gag 1-N (ts)PMGL (ts)-5′ ＞1×10 7 3
3′-gag 1-NPMGL (ts)-5′ ＞1×10 7 2
3′-gag 1-NPM (ncp)G (ct-1)L-5′ ＞1×10 7 3
3′-cgag 1-PMNG (ct-9)L (ts)-5′ ＞1×10 7 4
3′-gag 1-NPMG (stem)L-5′ ＞1×10 7 2
Histopathology data from the macaque of the carrier of (intrathalamically) inoculation same train in the thalamus show very similarly attenuation gradient.2 treated animal data have obtained the further confirmation from the result of a series of ferret neurovirulences research, wherein respectively by plaque measurement and PCR in real time, periodically measure the infective virus that exists in the brain of animal of encephalic inoculation and the level of geneome RNA.These data confirm jointly, and the combination of two or more mutation types has abundant attenuation level greater than single mutation type VSV carrier.Mouse LD 50Tiring shows strongly, by two kinds of dissimilar sudden changes of combination in identical VSV carrier, can have intensive attenuation synergy.
Embodiment 4
The enhanced immunogenicity of the VSV carrier of attenuation
With the VSV carrier 3 of attenuation '-gag 1-NPM (ncp)G (ct1)L-5 ', 3 '-gag 1-PMNG (ct9)L-5 ' and 3 '-gag 1-N (ts)PMGL (ts)-5 ' immunogenicity and VSV prototype carrier 3 '-NPMG-gag 5-L-5 ' and 3 '-NPMGL-5 ' contrasts.As described in embodiment 1, with one of above-mentioned VSV carrier immune mouse.The reaction of the VSV carrier induction of immunity of attenuation, it is greater than by prototype VSV-gag 5Carrier (3 '-NPMG-gag 5-L-5 ') those reactions of inductive.It should be noted that most 3 '-gag 1-PMNG (ct9)L-5 ', when starting and strengthening the back and assessing in the memory stage process of reaction, it can induce higher than the prototype inductive significantly Gag specific T cell frequency (table 8) of statistics.
Table 8
The specific CD8 T of GAG cell frequency
The per-cent of Gag tetramer male CD8 T-cell
Carrier The basis Strengthen Memory
3’-NPMGL-5’ 0.24 0.15 0.21
3’-NPMG-gag 5-L-5’ 1.46 2.29 0.92
3’-gag 1-NPM (ncp)G (ct1)L-5′ 0.96 3.42 2.25
3’-gag 1-PMNG (ct9)L-5’ 2.29 * 8.68 * 3.13 *
3’-gag 1-N (ts)PMGL (ts)-5’ 0.66 3.59 0.21
*=be significantly higher than with 3 '-NPMG-gag 5The reaction that-L-5 ' sees
(Si Shi t check, p＜0.05).
3 '-gag 1-PMNG (ct9)L-5 ' also induces IFN-γ secretion, and its tendency is higher than by prototype 3-NPMG-gag 5-L-5 ' inductive (table 9).To 3 '-gag 1-NPM (ncp)G (ct1)L-5 ' and 3 '-gag 1-N (ts)PMGL (ts)-5 ' reaction, also the tendency be higher than by prototype 3 '-NPMG-gag 5-L-5 ' inductive those (tables 9).
Table 9
GAG IFN-γ ELISPOT
IFN-γ spot/1 * 10 6Splenocyte
Carrier The basis Strengthen Memory
3’-NPMGL-5’ 0 1 0
3’-NPMG-gag 5-L-5’ 680 887 196
3’-gag 1-NPM (ncp)G (ct1)L-5’ 372 984 668 *
3’-gag 1-PMNG (ct9)L-5’ 908 1,552 496
3′-gag 1-N (ts)PMGL (ts)-5’ 129 632 413
*=be significantly higher than with 3 '-NPMG-gag 5The reaction that-L-5 ' sees
(Si Shi t check, p＜0.05).
Embodiment 5
Give to send in macaque intramuscular and the nose and pass expression
The immunogenicity of the V5V carrier of the attenuation of HIV gag
Following research and design is used to measure with after the VSV carrier immunity of expressing the proteic attenuation of HIV gag, the immune response that causes in macaque.
With totally 24 male rhesus macaques of selecting without heredity, carry out the described research of table 10, wherein with 1 * 10 7(pfu) dosage, with one of following VSV carrier, the immune interiorly every treated animal of intramuscular or nose (that is group 1-6): 3 '-gag 1-N (ts)PMGL (ts)-5 ' (TsN+L), 3 '-gag 1-PMNG (ct9)L-5 ' (N4CT9) or 3 '-NPMG-gag 5-L-5 ' (Gag5).
With totally 24 male rhesus macaques of selecting without heredity, carry out the described research of table 11, wherein with 1 * 10 7(pfu) dosage, with one of following VSV carrier, the immune interiorly every treated animal of intramuscular or nose (that is group 1-8): 3 '-gag 1-NPM (ncp)G (ct1)L-5 ' (MncpCT1), 3 '-gag 1-NPMG (stem)L-5 ' (G stem), 3 '-gag 1-PMNG (ct1)L-5 ' (N4CT1) or 3 '-NPMG-gag 5-L-5 ' (Gag5).
Usually, discharging of the whole body of every animal of the inspection of the mensuration below the use with humoral immune reaction and VSV:
Cell immune response: the IFN-γ ELISPOT reaction of HIV gag peptide specific and the IFN-γ ELISPOT reaction of VSV N peptide specific.Humoral immune reaction: measure serum anti-HIV gag antibody titer by ELISA, by ELISA measure serum anti--VSV antibody titer and serum is anti--the VSV NAT.
Table 10
The research of primate immunogenicity
Group The # animal Basis (At, seal ground serotype) Strengthen (New Jersey's serotype) Dosage Approach
1 5 TsN+L TsN+L 1×10 7 IM
2 5 TsN+L TsN+L 1×10 7 IN
3 5 N4CT9 N4CT9 1×10 7 IM
4 5 N4CT9 N4CT9 1×10 7 IN
5 2 Gag 5 Gag 5 1×10 7 IM
6 2 Gag 5 Gag 5 1×10 7 IN
Approach IM is an intramuscular.
Approach IN is in the nose.
Table 11
The research of primate immunogenicity
Group The # animal Basis (At, seal ground serotype) Strengthen (New Jersey's serotype) Dosage Approach
1 5 Mncp CT1 Mncp CT1 1×10 7 IM
2 5 Mncp CT1 MncpCT1 1×10 7 JN
3 5 The G stem The G stem 1×10 7 IM
4 5 The G stem The G stem 1×10 7 IN
5 5 N4CT1 N4CT1 1×10 7 JM
6 5 N4CT1 N4CT1 1×10 7 IN
7 2 Gag 5 Gag 5 1×10 7 IM
8 2 Gag 5 Gag 5 1×10 7 IN
Approach IM is an intramuscular.
Approach IN is in the nose.
Reference
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Sequence table
<110>Wyeth
Clarke，David K
Hendry，Michael
Udem，Stephen
Parks，Christopher
＜120〉Synergistic attenuation of stomatitis herpesvirus, its carrier and its immunogenic composition
<130>AM101651PCT
<160>2
<170>PatentIn version 3.3
<210>1
<211>29
<212>PRT
＜213〉stomatitis herpesvirus
<400>1
Arg Val Gly Ile His Leu Cys Ile Lys Leu Lys His Thr Lys Lys Arg
1 5 10 15
Gln Ile Tyr Thr Asp Ile Glu Met Asn Arg Leu Gly Lys
20 25
<210>2
<211>9
<212>PRT
＜213〉stomatitis herpesvirus
<400>2
Arg Val Gly Ile His Leu Cys Ile Lys
1 5

Claims (144)

1. genetic modification stomatitis herpesvirus (VSV), it comprises at least 2 kinds of dissimilar sudden changes in its genome, described sudden change is selected from temperature sensitive (ts) sudden change, point mutation, gene reorganization sudden change, changes of G-belemnoid, non-cytopathogenic M transgenation, ambisense RNA sudden change, the G transgenation of brachymemma, the G gene inserts sudden change and genetically deficient sudden change, and wherein these two kinds are suddenlyd change and work in coordination with that to weaken VSV pathogenic.
2. the VSV of claim 1, the wherein said pathogenic neurovirulence that also is defined as.
3. the VSV of claim 1, wherein these two kinds of sudden changes are brachymemmas G transgenation and N gene reorganization sudden change.
4. the VSV of claim 3, wherein the G albumen by the G genes encoding of brachymemma has last 20 amino acid whose disappearances of C-terminal.
5. the VSV of claim 3, wherein the G albumen by the G genes encoding of brachymemma has last 28 amino acid whose disappearances of C-terminal.
6. the VSV of claim 3, wherein the G albumen by the G genes encoding of brachymemma has the kytoplasm stern construction territory of being made up of 1 amino acid.
7. the VSV of claim 3, wherein the G albumen by the G genes encoding of brachymemma has the kytoplasm stern construction territory of being made up of 9 amino acid.
8. the VSV of claim 3, wherein with wild-type VSV genome 3 '-NPMGL-5 ' compares, described N gene reorganizes into 3 '-PNMGL-5 ' or 3 '-PMNGL-5 ', wherein N is the gene of coding nucleocapsid protein, P is the proteic gene of coding phosphor, M is the gene of coding stromatin, and G is the gene that coding adheres to glycoprotein, and L is the proteic gene of RNA polymerase that coding depends on RNA.
9. the VSV of claim 3, it comprises the genome that is selected from following sudden change: 3 '-PNMG (ct-1)L-5 ', 3 '-PNMG (ct-9)L-5 ', 3 '-PMNG (ct-1)L-5 ' and 3 '-PMNG (ct-9)L-5 ', wherein N is the gene of coding nucleocapsid protein, and P is the proteic gene of coding phosphor, and M is the gene of coding stromatin, G (ct-1)Be that G is the amino acid whose genes that adhere to glycoprotein of coding last 28 C-terminals of disappearance, G (ct-9)Be the amino acid whose genes that adhere to glycoprotein of coding last 20 C-terminals of disappearance, and L is the proteic gene of RNA polymerase that coding depends on RNA.
10. the VSV of claim 9, the genome of wherein said sudden change be 3 '-PMNG (ct-1)L-5 '.
11. the VSV of claim 9, the genome of wherein said sudden change be 3 '-PNMG (ct-1)L-5 '.
12. the VSV of claim 9 also comprises the sudden change of the 3rd class in its genome, wherein this sudden change is ts sudden change, ambisense RNA sudden change, non-cytopathogenic M transgenation, genetically deficient sudden change, gene insertion sudden change or point mutation.
13. the VSV of claim 1, the LD of the wherein said VSV that encephalic ground is injected in age in 4-week female Swiss-Webster mouse 50100 times of the wild-type VSV that injects for encephalic ground in age in 4-week female Swiss-Webster mouse.
14. the VSV of claim 1, the LD of the wherein said VSV that encephalic ground is injected in age in 4-week female Swiss-Webster mouse 501,000 times of the wild-type VSV that injects for encephalic ground in age in 4-week female Swiss-Webster mouse.
15. the VSV of claim 1, the LD of the wherein said VSV that encephalic ground is injected in age in 4-week female Swiss-Webster mouse 5010,000 times of the wild-type VSV that injects for encephalic ground in age in 4-week female Swiss-Webster mouse.
16. the VSV of claim 1, the LD of the wherein said VSV that encephalic ground is injected in age in 4-week female Swiss-Webster mouse 50100,000 times of the wild-type VSV that injects for encephalic ground in age in 4-week female Swiss-Webster mouse.
17. the VSV of claim 1, wherein these two kinds of sudden changes are brachymemmas G transgenation and non-cytopathogenic M transgenation.
18. the VSV of claim 17, wherein the G albumen by the G genes encoding of brachymemma has last 20 amino acid whose disappearances of C-terminal.
19. the VSV of claim 17, wherein the G albumen by the G genes encoding of brachymemma has last 28 amino acid whose disappearances of C-terminal.
20. the VSV of claim 17, wherein the G albumen by the G genes encoding of brachymemma has the kytoplasm stern construction territory of being made up of 1 amino acid.
21. the VSV of claim 17, wherein the G albumen by the G genes encoding of brachymemma has the kytoplasm stern construction territory of being made up of 9 amino acid.
22. the VSV of claim 17, the non-cytopathogenic (M of wherein said M gene (ncp)) sudden change be in the methionine(Met) 33 of M gene and the sudden change at methionine(Met) 51 places.
23. the VSV of claim 17, it comprises 3 '-NPM (ncp)G (ct-1)L-5 ' or 3 '-NPM (ncp)G (ct-9)The genome of the sudden change of L-5 '.
24. the VSV of claim 17 also comprises the 3rd class sudden change in its genome, wherein this sudden change is that ts sudden change, ambisense RNA sudden change, gene reorganization sudden change, genetically deficient sudden change, gene insert that sudden change is inserted in sudden change, G gene, the G-belemnoid becomes or point mutation.
25. the VSV of claim 1, wherein these two kinds of sudden changes are ts N transgenation (N (ts)) and tsL transgenation (L (ts)).
26. the VSV of claim 25, also comprise 3 '-N (ts)PMGL (ts)-5 ' the genome of sudden change.
27. the VSV of claim 25, also comprise the sudden change of the 3rd class in its genome, wherein this sudden change is G transgenation, G gene insertion sudden change and the genetically deficient sudden change of point mutation, gene reorganization sudden change, the change of G-belemnoid, non-cytopathogenic M transgenation, ambisense RNA sudden change, brachymemma.
28. being the G-belemnoid, the VSV of claim 1, wherein a kind of mutation type become.
29. genetic modification VSV carrier, it comprises at least 2 kinds of dissimilar sudden changes in its genome, described sudden change is selected from ts sudden change, point mutation, gene reorganization sudden change, the change of G-belemnoid, non-cytopathogenic M transgenation, ambisense RNA sudden change, the G transgenation of brachymemma, the G gene inserts sudden change and genetically deficient is suddenlyd change, with at least one foreign rna sequence, the non-essential VSV genome area that duplicates is inserted or replaced to described foreign rna sequence, and wherein collaborative to weaken VSV pathogenic for these two kinds sudden changes.
30. the carrier of claim 29, the wherein said pathogenic neurovirulence that also is defined as.
31. the carrier of claim 29, wherein said foreign rna also are defined as open reading-frame (ORF) (ORF).
32. the carrier of claim 29, wherein these two kinds of sudden changes are brachymemmas G transgenation and N gene reorganization sudden change.
33. the carrier of claim 32, wherein the G albumen by the G genes encoding of brachymemma has last 20 amino acid whose disappearances of C-terminal.
34. the carrier of claim 32, wherein the G albumen by the G genes encoding of brachymemma has last 28 amino acid whose disappearances of C-terminal.
35. the carrier of claim 32, wherein the G albumen by the G genes encoding of brachymemma has the kytoplasm stern construction territory of being made up of 1 amino acid.
36. the carrier of claim 32, wherein the G albumen by the G genes encoding of brachymemma has the kytoplasm stern construction territory of being made up of 9 amino acid.
37. the carrier of claim 32, wherein with wild-type VSV genome 3 '-NPMGL-5 ' compares, described N gene reorganizes into 3 '-PNMGL-5 ' or 3 '-PMNGL-5 ', wherein N is the gene of coding nucleocapsid protein, P is the proteic gene of coding phosphor, M is the gene of coding stromatin, and G is the gene that coding adheres to glycoprotein, and L is the proteic gene of RNA polymerase that coding depends on RNA.
38. the carrier of claim 32, it comprises the genome that is selected from following sudden change: 3 '-PNMG (ct-1)L-5 ', 3 '-PNMG (ct-9)L-5 ', 3 '-PMNG (ct-1)L-5 ' and 3 '-PMNG (ct-9)L-5 ', wherein N is the gene of coding nucleocapsid protein, and P is the proteic gene of coding phosphor, and M is the gene of coding stromatin, G (ct-1)Be the amino acid whose genes that adhere to glycoprotein of coding last 28 C-terminals of disappearance, G (ct-9)Be the amino acid whose genes that adhere to glycoprotein of coding last 20 C-terminals of disappearance, and L is the proteic gene of RNA polymerase that coding depends on RNA.
39. the carrier of claim 38, the genome of wherein said sudden change be 3 '-PMNG (ct-1)L-5 '.
40. the carrier of claim 38, the genome of wherein said sudden change be 3 '-PNMG (ct-1)L-5 '.
41. the carrier of claim 38 also comprises the sudden change of the 3rd class in its genome, wherein this sudden change is ts sudden change, point mutation, ambisense RNA sudden change, genetically deficient sudden change, gene insertion sudden change or non-cytopathogenic M transgenation.
42. the carrier of claim 32, the LD of the wherein said VSV that encephalic ground is injected in age in 4-week female Swiss-Webster mouse 50100 times of the wild-type VSV that injects for encephalic ground in age in 4-week female Swiss-Webster mouse.
43. the carrier of claim 32, the LD of the wherein said VSV that encephalic ground is injected in age in 4-week female Swiss-Webster mouse 501,000 times of the wild-type VSV that injects for encephalic ground in age in 4-week female Swiss-Webster mouse.
44. the carrier of claim 32, the LD of the wherein said VSV that encephalic ground is injected in age in 4-week female Swiss-Webster mouse 5010,000 times of the wild-type VSV that injects for encephalic ground in age in 4-week female Swiss-Webster mouse.
45. the carrier of claim 32, the LD of the wherein said VSV that encephalic ground is injected in age in 4-week female Swiss-Webster mouse 50100,000 times of the wild-type VSV that injects for encephalic ground in age in 4-week female Swiss-Webster mouse.
46. the carrier of claim 32, wherein said foreign rna is selected from: the HIV gene, the HTLV gene, the SIV gene, the RSV gene, the PIV gene, the HSV gene, the CMV gene, the Epstein-Barr virus gene, the varicella zoster virus gene, the mumps virus gene, the Measles virus gene, influenza virus gene, virogene of poliomyelitis, the rhinovirus gene, hepatitis A virus genes, hepatitis B virogene, hepatitis c virus gene, the Norwalk virus gene, the togavirus gene, the Alphavirus gene, rubella virus genome, the rabies virus gene, the Marburg virus gene, the Ou Baola virogene, the papillomatosis virus gene, the polyomavirus gene, between matter pneumonitis virus gene, the coronavirus gene, cholera vibrio gene, the streptococcus pneumoniae gene, the streptococcus pyogenes gene, the helicobacter pylori gene, the streptococcus agalactiae gene, meningitis naphthalene Se Shi coccus gene, gonorrhoea naphthalene Se Shi coccus gene, the corynebacterium diphtheriae gene, the clostridium tetani gene, the Bordetella pertussis gene, the hemophilus gene, the chlamydiaceae gene, bacillus coli gene, the gene of the Codocyte factor, the gene of the auxiliary epi-position of coding T-, the gene of coding CTL epi-position, the gene of the gene of coding adjuvant and coding cofactor.
47. the carrier of claim 46, wherein said HIV gene is selected from: gag, env, pol, vif, nef, tat, vpr, rev or vpu.
48. the carrier of claim 47, wherein said HIV gene is gag.
49. the carrier of claim 48, the genome of wherein said sudden change be 3 '-gag 1-PNMG (ct-1)L-5 ', 3 '-gag 1-PNMG (ct-9)L-5 ', 3 '-gag 1-PMNG (ct-1)L-5 ', 3 '-gag 1-PMNG (ct-9)L-5 ', 3 '-PNMG (ct-1)L-gag 5-5 ', 3 '-PNMG (ct-9)L-gag 5-5 ', 3 '-PMNG (ct-1)L-gag 5-5 ' or 3 '-PMNG (ct-9)L-gag 5-5 '.
50. the carrier of claim 29, wherein these two kinds of sudden changes are brachymemmas G transgenation and non-cytopathogenic M transgenation.
51. the carrier of claim 50, wherein the G albumen by the G genes encoding of brachymemma has last 28 amino acid whose disappearances of C-terminal.
52. the carrier of claim 50, wherein the G albumen by the G genes encoding of brachymemma has the kytoplasm stern construction territory of being made up of 1 amino acid.
53. the carrier of claim 50, the non-cytopathogenic (M of wherein said M gene (ncp)) sudden change be in the methionine(Met) 33 of M gene and the sudden change at methionine(Met) 51 places.
54. the carrier of claim 50, it comprises 3 '-NPM (ncp)G (ct-1)L-5 ' or 3 '-NPM (ncp)G (ct-9)The genome of the sudden change of L-5 '.
55. the carrier of claim 50 also comprises the 3rd class and suddenlys change in its genome, wherein this sudden change is ts sudden change, point mutation, gene reorganization sudden change, the change of G-belemnoid, ambisense RNA sudden change, G gene insertion sudden change and genetically deficient sudden change.
56. the carrier of claim 50, wherein said foreign rna is selected from: the HIV gene, the HTLV gene, the SIV gene, the RSV gene, the PIV gene, the HSV gene, the CMV gene, the Epstein-Barr virus gene, the varicella zoster virus gene, the mumps virus gene, the Measles virus gene, influenza virus gene, virogene of poliomyelitis, the rhinovirus gene, hepatitis A virus genes, hepatitis B virogene, hepatitis c virus gene, the Norwalk virus gene, the togavirus gene, the Alphavirus gene, rubella virus genome, the rabies virus gene, the Marburg virus gene, the Ou Baola virogene, the papillomatosis virus gene, the polyomavirus gene, between matter pneumonitis virus gene, the coronavirus gene, cholera vibrio gene, the streptococcus pneumoniae gene, the streptococcus pyogenes gene, the helicobacter pylori gene, the streptococcus agalactiae gene, meningitis naphthalene Se Shi coccus gene, gonorrhoea naphthalene Se Shi coccus gene, the corynebacterium diphtheriae gene, the clostridium tetani gene, the Bordetella pertussis gene, the hemophilus gene, the chlamydiaceae gene, bacillus coli gene, the gene of the Codocyte factor, the gene of the auxiliary epi-position of coding T-, the gene of coding CTL epi-position, the gene of the gene of coding adjuvant and coding cofactor.
57. the carrier of claim 56, wherein said HIV gene is selected from: gag, env, pol, vif, nef, tat, vpr, rev or vpu.
58. the carrier of claim 57, wherein said HIV gene is gag.
59. the carrier of claim 58, wherein Tu Bian genome be 3 '-gag 1-NPM (ncp)G (ct-1)L-5 ', 3 '-gag 1-NPM (ncp)G (ct-9)L-5 ', 3 '-NPM (ncp)G (ct-1)L-gag 5-5 ' or 3 '-NPM (ncp)G (ct-9)L-gag 5-5 '.
60. the carrier of claim 29, wherein these two kinds of sudden changes are ts N transgenation (N (ts)) and tsL transgenation (L (ts)).
61. the carrier of claim 60, it comprises 3 '-N (ts)PMGL (ts)-5 ' the genome of sudden change.
62. the carrier of claim 60, also comprise the sudden change of the 3rd class in its genome, wherein this sudden change is G transgenation, G gene insertion sudden change and the genetically deficient sudden change of point mutation, gene reorganization sudden change, the change of G-belemnoid, non-cytopathogenic M transgenation, ambisense RNA sudden change, brachymemma.
63. the carrier of claim 60, wherein said foreign rna is selected from: the HIV gene, the HTLV gene, the SIV gene, the RSV gene, the PIV gene, the HSV gene, the CMV gene, the Epstein-Barr virus gene, the varicella zoster virus gene, the mumps virus gene, the Measles virus gene, influenza virus gene, virogene of poliomyelitis, the rhinovirus gene, hepatitis A virus genes, hepatitis B virogene, hepatitis c virus gene, the Norwalk virus gene, the togavirus gene, the Alphavirus gene, rubella virus genome, the rabies virus gene, the Marburg virus gene, the Ou Baola virogene, the papillomatosis virus gene, the polyomavirus gene, between matter pneumonitis virus gene, the coronavirus gene, cholera vibrio gene, the streptococcus pneumoniae gene, the streptococcus pyogenes gene, the helicobacter pylori gene, the streptococcus agalactiae gene, meningitis naphthalene Se Shi coccus gene, gonorrhoea naphthalene Se Shi coccus gene, the corynebacterium diphtheriae gene, the clostridium tetani gene, the Bordetella pertussis gene, the hemophilus gene, the chlamydiaceae gene, bacillus coli gene, the gene of the Codocyte factor, the gene of the auxiliary epi-position of coding T-, the gene of coding CTL epi-position, the gene of the gene of coding adjuvant and coding cofactor.
64. the carrier of claim 63, wherein said HIV gene is selected from: gag, env, pol, vif, nef, tat, vpr, rev or vpu.
65. the carrier of claim 64, wherein said HIV gene is gag.
66. the carrier of claim 65, the genome of wherein said sudden change be 3 '-gag 1-N (ts)PMGL (ts)-5 ' or 3 '-N (ts)PMGL (ts)-gag 5-5 '.
67. the carrier of claim 29, wherein these two kinds of sudden changes are that the G belemnoid becomes G (stem)With gene reorganization sudden change.
68. the carrier of claim 67, the genome of wherein said sudden change be 3 '-gag 1-NPMG (stem)L-5 '
69. the carrier of claim 68, also comprise the sudden change of the 3rd class in its genome, wherein this sudden change is G transgenation, G gene insertion sudden change and the genetically deficient sudden change of point mutation, ts sudden change, gene reorganization sudden change, non-cytopathogenic M transgenation, ambisense RNA sudden change, brachymemma.
70. the carrier of claim 67, wherein said foreign rna is selected from: the HIV gene, the HTLV gene, the SIV gene, the RSV gene, the PIV gene, the HSV gene, the CMV gene, the Epstein-Barr virus gene, the varicella zoster virus gene, the mumps virus gene, the Measles virus gene, influenza virus gene, virogene of poliomyelitis, the rhinovirus gene, hepatitis A virus genes, hepatitis B virogene, hepatitis c virus gene, the Norwalk virus gene, the togavirus gene, the Alphavirus gene, rubella virus genome, the rabies virus gene, the Marburg virus gene, the Ou Baola virogene, the papillomatosis virus gene, the polyomavirus gene, between matter pneumonitis virus gene, the coronavirus gene, cholera vibrio gene, the streptococcus pneumoniae gene, the streptococcus pyogenes gene, the helicobacter pylori gene, the streptococcus agalactiae gene, meningitis naphthalene Se Shi coccus gene, gonorrhoea naphthalene Se Shi coccus gene, the corynebacterium diphtheriae gene, the clostridium tetani gene, the Bordetella pertussis gene, the hemophilus gene, the chlamydiaceae gene, bacillus coli gene, the gene of the Codocyte factor, the gene of the auxiliary epi-position of coding T-, the gene of coding CTL epi-position, the gene of the gene of coding adjuvant and coding cofactor.
71. immunogenic composition, it comprises the genetic modification VSV carrier of immunogenicity dosage, described carrier comprises at least 2 kinds of dissimilar sudden changes in its genome, described sudden change is selected from ts sudden change, point mutation, gene reorganization sudden change, the change of G-belemnoid, non-cytopathogenic M transgenation, ambisense RNA sudden change, the G transgenation of brachymemma, the G gene inserts sudden change and genetically deficient is suddenlyd change, with at least one foreign rna sequence, the non-essential VSV genome area that duplicates is inserted or replaced to described foreign rna sequence, and wherein collaborative to weaken VSV pathogenic for these two kinds sudden changes.
72. the immunogenic composition of claim 71, the wherein said pathogenic neurovirulence that also is defined as.
73. the immunogenic composition of claim 71, wherein said foreign rna also are defined as open reading-frame (ORF) (ORF).
74. the immunogenic composition of claim 71, wherein these two kinds of sudden changes are brachymemmas G transgenation and N gene reorganization sudden change.
75. the immunogenic composition of claim 74, wherein the G albumen by the G genes encoding of brachymemma has last 20 amino acid whose disappearances of C-terminal.
76. the immunogenic composition of claim 74, wherein the G albumen by the G genes encoding of brachymemma has last 28 amino acid whose disappearances of C-terminal.
77. the immunogenic composition of claim 74, wherein the G albumen by the G genes encoding of brachymemma has the kytoplasm stern construction territory of being made up of 1 amino acid.
The immunogenic composition of 78 claims 74, wherein the G albumen by the G genes encoding of brachymemma has the kytoplasm stern construction territory of being made up of 9 amino acid.
79. the immunogenic composition of claim 74, wherein with wild-type VSV genome 3 '-NPMGL-5 ' compares, described N gene reorganizes into 3 '-PNMGL-5 ' or 3 '-PMNGL-5 ', wherein N is the gene of coding nucleocapsid protein, P is the proteic gene of coding phosphor, M is the gene of coding stromatin, and G is the gene that coding adheres to glycoprotein, and L is the proteic gene of RNA polymerase that coding depends on RNA.
80. the former composition of the immunity of claim 74, it comprises the genome that is selected from following sudden change: 3 '-PNMG (ct-1)L-5 ', 3 '-PNMG (ct-9)L-5 ', 3 '-PMNG (ct-1)L-5 ' and 3 '-PMNG (ct-9)L-5 ', wherein N is the gene of coding nucleocapsid protein, and P is the proteic gene of coding phosphor, and M is the gene of coding stromatin, G (ct-1)Be the amino acid whose genes that adhere to glycoprotein of coding last 28 C-terminals of disappearance, G (ct-9)Be the amino acid whose genes that adhere to glycoprotein of coding last 20 C-terminals of disappearance, and L is the proteic gene of RNA polymerase that coding depends on RNA.
81. the immunogenic composition of claim 80, the genome of wherein said sudden change be 3 '-PMNG (ct-1)L-5 '.
82. the immunogenic composition of claim 80, the genome of wherein said sudden change be 3 '-PNMG (ct-1)L-5 '.
83. the immunogenic composition of claim 80 also comprises the sudden change of the 3rd class in its genome, wherein this sudden change is ts sudden change, point mutation, ambisense RNA sudden change, genetically deficient sudden change, gene insertion sudden change or non-cytopathogenic M transgenation.
84. the immunogenic composition of claim 71, wherein said foreign rna is selected from: the HIV gene, the HTLV gene, the SIV gene, the RSV gene, the PIV gene, the HSV gene, the CMV gene, the Epstein-Barr virus gene, the varicella zoster virus gene, the mumps virus gene, the Measles virus gene, influenza virus gene, virogene of poliomyelitis, the rhinovirus gene, hepatitis A virus genes, hepatitis B virogene, hepatitis c virus gene, the Norwalk virus gene, the togavirus gene, the Alphavirus gene, rubella virus genome, the rabies virus gene, the Marburg virus gene, the Ou Baola virogene, the papillomatosis virus gene, the polyomavirus gene, between matter pneumonitis virus gene, the coronavirus gene, cholera vibrio gene, the streptococcus pneumoniae gene, the streptococcus pyogenes gene, the streptococcus agalactiae gene, the helicobacter pylori gene, meningitis naphthalene Se Shi coccus gene, gonorrhoea naphthalene Se Shi coccus gene, the corynebacterium diphtheriae gene, the clostridium tetani gene, the Bordetella pertussis gene, the hemophilus gene, the chlamydiaceae gene, bacillus coli gene, the gene of the Codocyte factor, the gene of the auxiliary epi-position of coding T-, the gene of coding CTL epi-position, the gene of the gene of coding adjuvant and coding cofactor.
85. the immunogenic composition of claim 84, wherein said HIV gene is selected from: gag, env, pol, vif, nef, tat, vpr, rev or vpu.
86. the immunogenic composition of claim 85, wherein said HIV gene is gag.
87. the immunogenic composition of claim 86, the genome of wherein said sudden change be 3 '-gag 1-PNMG (ct-1)L-5 ', 3 '-gag 1-PNMG (ct-9)L-5 ', 3 '-gag 1-PMNG (ct-1)L-5 ', 3 '-gag 1-PMNG (ct-9)L-5 ', 3 '-PNMG (ct-1)L-gag 5-5 ', 3 '-PNMG (ct-9)L-gag 5-5 ', 3 '-PMNG (ct-1)L-gag 5-5 ' or 3 '-PMNG (ct-9)L-gag 5-5 '.
88. the immunogenic composition of claim 71, wherein said composition is used by being selected from following approach: in intravenous, intracutaneous, subcutaneous, intramuscular, endoperitoneal, per os, rectum, the nose, mouthful, vagina and exsomatize.
89. the immunogenic composition of claim 71, wherein these two kinds of sudden changes are brachymemmas G transgenation and non-cytopathogenic M transgenation.
90. the immunogenic composition of claim 89, wherein the G albumen by the G genes encoding of brachymemma has last 28 amino acid whose disappearances of C-terminal.
91. the immunogenic composition of claim 89, wherein the G albumen by the G genes encoding of brachymemma has the kytoplasm stern construction territory of being made up of 1 amino acid.
92. the immunogenic composition of claim 89, the non-cytopathogenic (M of wherein said M gene (ncp)) sudden change be in the methionine(Met) 33 of M gene and the sudden change at methionine(Met) 51 places.
93. the immunogenic composition of claim 89, it comprises 3 '-NPM (ncp)G (ct-1)L-5 ' or 3 '-NPM (ncp)G (ct-9)The genome of the sudden change of L-5 '.
94. the immunogenic composition of claim 89 also comprises the 3rd class and suddenlys change in its genome, wherein this sudden change is ts sudden change, point mutation, gene reorganization sudden change, the change of G-belemnoid, ambisense RNA sudden change, G gene insertion sudden change and genetically deficient sudden change.
95. the immunogenic composition of claim 89, wherein said foreign rna is selected from: the HIV gene, the HTLV gene, the SIV gene, the RSV gene, the PIV gene, the HSV gene, the CMV gene, the Epstein-Barr virus gene, the varicella zoster virus gene, the mumps virus gene, the Measles virus gene, influenza virus gene, virogene of poliomyelitis, the rhinovirus gene, hepatitis A virus genes, hepatitis B virogene, hepatitis c virus gene, the Norwalk virus gene, the togavirus gene, the Alphavirus gene, rubella virus genome, the rabies virus gene, the Marburg virus gene, the Ou Baola virogene, the papillomatosis virus gene, the polyomavirus gene, between matter pneumonitis virus gene, the coronavirus gene, cholera vibrio gene, the streptococcus pneumoniae gene, the streptococcus pyogenes gene, the helicobacter pylori gene, the streptococcus agalactiae gene, meningitis naphthalene Se Shi coccus gene, gonorrhoea naphthalene Se Shi coccus gene, the corynebacterium diphtheriae gene, the clostridium tetani gene, the Bordetella pertussis gene, the hemophilus gene, the chlamydiaceae gene, bacillus coli gene, the gene of the Codocyte factor, the gene of the auxiliary epi-position of coding T-, the gene of coding CTL epi-position, the gene of the gene of coding adjuvant and coding cofactor.
96. the immunogenic composition of claim 95, wherein said HIV gene is selected from: gag, env, pol, vif, nef, tat, vpr, rev or vpu.
97. the immunogenic composition of claim 96, wherein said HIV gene is gag.
98. the immunogenic composition of claim 97, the genome of wherein said sudden change be 3 '-gag 1-NPM (ncp)G (ct-1)L-5 ', 3 '-gag 1-NPM (ncp)G (ct-9)L-5 ', 3 '-NPM (ncp)G (ct-1)L-gag 5-5 ' or 3 '-NPM (ncp)G (ct-9)L-gag 5-5 '.
99. the immunogenic composition of claim 71, wherein these two kinds of sudden changes are ts N transgenation (N (ts)) and ts L transgenation (L (ts)).
100. the immunogenic composition of claim 99, it comprises 3 '-N (ts)PMGL (ts)-5 ' the genome of sudden change.
101. the immunogenic composition of claim 99, also comprise the sudden change of the 3rd class in its genome, wherein this sudden change is G transgenation, G gene insertion sudden change and the genetically deficient sudden change of point mutation, gene reorganization sudden change, the change of G-belemnoid, non-cytopathogenic M transgenation, ambisense RNA sudden change, brachymemma.
102. the immunogenic composition of claim 99, wherein said foreign rna is selected from: the HIV gene, the HTLV gene, the SIV gene, the RSV gene, the PIV gene, the HSV gene, the CMV gene, the Epstein-Barr virus gene, the varicella zoster virus gene, the mumps virus gene, the Measles virus gene, influenza virus gene, virogene of poliomyelitis, the rhinovirus gene, hepatitis A virus genes, hepatitis B virogene, hepatitis c virus gene, the Norwalk virus gene, the togavirus gene, the Alphavirus gene, rubella virus genome, the rabies virus gene, the Marburg virus gene, the Ou Baola virogene, the papillomatosis virus gene, the polyomavirus gene, between matter pneumonitis virus gene, the coronavirus gene, cholera vibrio gene, the streptococcus pneumoniae gene, the streptococcus pyogenes gene, the helicobacter pylori gene, the streptococcus agalactiae gene, meningitis naphthalene Se Shi coccus gene, gonorrhoea naphthalene Se Shi coccus gene, the corynebacterium diphtheriae gene, the clostridium tetani gene, the Bordetella pertussis gene, the hemophilus gene, the chlamydiaceae gene, bacillus coli gene, the gene of the Codocyte factor, the gene of the auxiliary epi-position of coding T-, the gene of coding CTL epi-position, the gene of the gene of coding adjuvant and coding cofactor.
103. the immunogenic composition of claim 102, wherein said HIV gene is selected from: gag, env, pol, vif, nef, tat, vpr, rev or vpu.
104. the immunogenic composition of claim 103, wherein said HIV gene is gag.
105. the immunogenic composition of claim 104, the genome of wherein said sudden change be 3 '-gag 1-N (ts)PMGL (ts)-5 ' or 3 '-N (ts)PMGL (ts)-gag 5-5 '.
106. the immunogenic composition of claim 71, wherein these two kinds of sudden changes are G (stem)Sudden change and gene reorganization sudden change.
107. the immunogenic composition of claim 106, the genome of wherein said sudden change be 3 '-gag 1-NPMG (stem)L-5 '.
108. the immunogenic composition of claim 106, also comprise the sudden change of the 3rd class in its genome, wherein this sudden change is G transgenation, G gene insertion sudden change and the genetically deficient sudden change of point mutation, ts sudden change, gene reorganization sudden change, non-cytopathogenic M transgenation, ambisense RNA sudden change, brachymemma.
109. the carrier of claim 108, wherein said foreign rna is selected from: the HIV gene, the HTLV gene, the SIV gene, the RSV gene, the PIV gene, the HSV gene, the CMV gene, the Epstein-Barr virus gene, the varicella zoster virus gene, the mumps virus gene, the Measles virus gene, influenza virus gene, virogene of poliomyelitis, the rhinovirus gene, hepatitis A virus genes, hepatitis B virogene, hepatitis c virus gene, the Norwalk virus gene, the togavirus gene, the Alphavirus gene, rubella virus genome, the rabies virus gene, the Marburg virus gene, the Ou Baola virogene, the papillomatosis virus gene, the polyomavirus gene, between matter pneumonitis virus gene, the coronavirus gene, cholera vibrio gene, the streptococcus pneumoniae gene, the streptococcus pyogenes gene, the helicobacter pylori gene, the streptococcus agalactiae gene, meningitis naphthalene Se Shi coccus gene, gonorrhoea naphthalene Se Shi coccus gene, the corynebacterium diphtheriae gene, the clostridium tetani gene, the Bordetella pertussis gene, the hemophilus gene, the chlamydiaceae gene, bacillus coli gene, the gene of the Codocyte factor, the gene of the auxiliary epi-position of coding T-, the gene of coding CTL epi-position, the gene of the gene of coding adjuvant and coding cofactor.
110. the method for directed toward bacteria infection immunity mammalian hosts, it comprises the genetic modification VSV carrier of using immunogenicity dosage, described carrier comprises: (a) at least two kinds of dissimilar sudden changes in its genome, this sudden change is selected from: ts sudden change, point mutation, gene reorganization sudden change, changes of G-belemnoid, non-cytopathogenic M transgenation, ambisense RNA sudden change, the G transgenation of brachymemma, G gene insert sudden change and genetically deficient sudden change, and wherein these two kinds are suddenlyd change and work in coordination with that to weaken VSV pathogenic; (b) at least one foreign rna sequence, the non-essential VSV genome area that duplicates is inserted or replaced to described foreign rna sequence, and wherein this RNA coding is selected from following bacterioprotein: vibrio cholerae albumen, pneumonia streptococcus mycoprotein, streptococcus pyogenes albumen, streptococcus agalactiae albumen, helicobacter pylori protein, meningitis naphthalene Se Shi coccus albumen, gonorrhoea naphthalene Se Shi coccus albumen, corynebacterium diphtheriae albumen, clostridium tetani albumen, Bordetella pertussis albumen, hemophilus albumen, chlamydiaceae albumen and e. coli protein.
111. the method for claim 110, wherein these two kinds of sudden changes are brachymemmas G transgenation and N gene reorganization sudden change.
112. the method for claim 111, wherein the G albumen by the G genes encoding of brachymemma has last 20 amino acid whose disappearances of C-terminal.
113. the method for claim 111, wherein the G albumen by the G genes encoding of brachymemma has last 28 amino acid whose disappearances of C-terminal.
114. the method for claim 111, wherein the G albumen by the G genes encoding of brachymemma has the kytoplasm stern construction territory of being made up of 1 amino acid.
115. the method for claim 111, wherein the G albumen by the G genes encoding of brachymemma has the kytoplasm stern construction territory of being made up of 9 amino acid.
116. the method for claim 111, wherein with wild-type VSV genome 3 '-NPMGL-5 ' compares, described N gene reorganizes into 3 '-PNMGL-5 ' or 3 '-PMNGL-5 '.
117. the method for claim 111, it comprises the genome that is selected from following sudden change: 3 '-PNMG (ct-1)L-5 ', 3 '-PNMG (ct-9)L-5 ', 3 '-PMNG (ct-1)L-5 ' and 3 '-PMNG (ct-9)L-5 '.
118. the method for claim 103 also comprises the sudden change of the 3rd class in its genome, wherein this sudden change is ts sudden change, point mutation, ambisense RNA sudden change, genetically deficient sudden change, gene insertion sudden change or non-cytopathogenic M transgenation.
119. method at virus infection immunity mammalian hosts, it comprises the genetic modification VSV carrier of using immunogenicity dosage, described carrier comprises: (a) at least two kinds of dissimilar sudden changes in its genome, this sudden change is selected from: ts sudden change, point mutation, gene reorganization sudden change, changes of G-belemnoid, non-cytopathogenic M transgenation, ambisense RNA sudden change, the G transgenation of brachymemma, G gene insert sudden change and genetically deficient sudden change, and wherein these two kinds are suddenlyd change and work in coordination with that to weaken VSV pathogenic; (b) at least one foreign rna sequence, the non-essential VSV genome area that duplicates is inserted or replaced to described foreign rna sequence, and wherein this RNA coding is selected from following viral protein: HIV albumen, HTLV albumen, SIV albumen, rsv protein, PIV albumen, HSV albumen, CMV albumen, Epstein-Barr virus albumen, varicella zoster virus albumen, mumps virus albumen, Measles virus albumen, influenza virus protein, poliovirus albumen, rhinovirus albumen, hepatitis A virus albumen, hepatitis b virus protein, hepatitis C virus protein, Norwalk virus albumen, togavirus albumen, Alphavirus albumen, rubella virus protein, rabies virus albumen, the Marburg toxalbumin, the Ou Baola viral protein, papillomavirus protein, polyomavirus albumen, between matter pneumonitis virus albumen and coronavirus albumen.
120. the method for claim 119, wherein said RNA is selected from following HIV gene: gag, env, pol, vif, nef, tat, vpr, rev or vpu.
121. the method for claim 120, wherein said HIV gene is gag.
122. the method for claim 119, wherein these two kinds of sudden changes are brachymemmas G transgenation and N gene reorganization sudden change.
123. the method for claim 122, wherein the G albumen by the G genes encoding of brachymemma has last 20 amino acid whose disappearances of C-terminal.
124. the method for claim 122, wherein the G albumen by the G genes encoding of brachymemma has last 28 amino acid whose disappearances of C-terminal.
125. the method for claim 122, wherein the G albumen by the G genes encoding of brachymemma has the kytoplasm stern construction territory of being made up of 1 amino acid.
126. the method for claim 122, wherein the G albumen by the G genes encoding of brachymemma has the kytoplasm stern construction territory of being made up of 9 amino acid.
127. the method for claim 122, wherein with wild-type VSV genome 3 '-NPMGL-5 ' compares, described N gene reorganizes into 3 '-PNMGL-5 ' or 3 '-PMNGL-5 '.
128. the method for claim 122, it comprises the genome that is selected from following sudden change: 3 '-PNMG (ct-1)L-5 ', 3 '-PNMG (ct-9)L-5 ', 3 '-PMNG (ct-1)L-5 ' and 3 '-PMNG (ct-9)L-5 '.
129. the method for claim 122 also comprises the sudden change of the 3rd class in its genome, wherein this sudden change is ts sudden change, point mutation, ambisense RNA sudden change, genetically deficient sudden change, gene insertion sudden change or non-cytopathogenic M transgenation.
130. the method for claim 122, the genome of wherein said sudden change be 3 '-gag 1-PNMG (ct-1)L-5 ', 3 '-gag 1-PNMG (ct-9)L-5 ', 3 '-gag 1-PMNG (ct-1)L-5 ', 3 '-gag 1-PMNG (ct-9)L-5 ', 3 '-PNMG (ct-1)L-gag 5-5 ', 3 '-PNMG (ct-9)L-gag 5-5 ', 3 '-PMNG (ct-1)L-gag 5-5 ' or 3 '-PMNG (ct-9)L-gag 5-5 '.
131. the method for claim 119, wherein these two kinds of sudden changes are brachymemmas G transgenation and non-cytopathogenic M transgenation.
132. the method for claim 131, wherein the G albumen by the G genes encoding of brachymemma has last 28 amino acid whose disappearances of C-terminal.
133. the method for claim 131, wherein the G albumen by the G genes encoding of brachymemma has the kytoplasm stern construction territory of being made up of 1 amino acid.
134. the method for claim 131, the non-cytopathogenic (M of wherein said M gene (ncp)) sudden change be in the methionine(Met) 33 of M gene and the sudden change at methionine(Met) 51 places.
135. the method for claim 131, it comprises 3 '-NPM (ncp)G (ct-1)L-5 ' or 3 '-NPM (ncp)G (ct-9)The genome of the sudden change of L-5 '.
136. the method for claim 123 also comprises the 3rd class and suddenlys change in its genome, wherein this sudden change is ts sudden change, point mutation, gene reorganization sudden change, the change of G-belemnoid, ambisense RNA sudden change, G gene insertion sudden change and genetically deficient sudden change.
137. the method for claim 123, the genome of wherein said sudden change be 3 '-gag 1-NPM (ncp)G (ct-1)L-5 ', 3 '-gag 1-NPM (ncp)G (ct-9)L-5 ', 3 '-NPM (ncp)G (ct-1)L-gag 5-5 ' or 3 '-NPM (ncp)G (ct-9)L-gag 5-5 '.
138. the method for claim 119, wherein these two kinds of sudden changes are ts N transgenation (N (ts)) and ts L transgenation (L (ts)).
139. the method for claim 138, it comprises 3 '-N (ts)PMGL (ts)-5 ' the genome of sudden change.
140. the method for claim 138, also comprise the sudden change of the 3rd class in its genome, wherein this sudden change is G transgenation, G gene insertion sudden change and the genetically deficient sudden change of point mutation, gene reorganization sudden change, the change of G-belemnoid, non-cytopathogenic M transgenation, ambisense RNA sudden change, brachymemma.
141. the method for claim 139, the genome of wherein said sudden change be 3 '-gag 1-N (ts)PMGL (ts)-5 ' or 3 '-N (ts)PMGL (ts)-gag 5-5 '.
142. the method for claim 119, wherein these two kinds of sudden changes are G (stem)Sudden change and gene reorganization sudden change.
143. the method for claim 142, wherein Tu Bian genome be 3 '-gag 1-NPMG (stem)L-5 '.
144. the method for claim 142, also comprise the sudden change of the 3rd class in its genome, wherein this sudden change is G transgenation, G gene insertion sudden change and the genetically deficient sudden change of point mutation, ts sudden change, gene reorganization sudden change, non-cytopathogenic M transgenation, ambisense RNA sudden change, brachymemma.
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106794240A (en) * 2014-03-01 2017-05-31 普罗菲克图斯生物科学股份有限公司 Restructuring isfahan virus carrier
WO2019061691A1 (en) * 2017-09-26 2019-04-04 王红伟 Cell line capable of implementing induced rescue of rhabdovirus, construction method therefor, and application thereof
CN110577585A (en) * 2018-06-07 2019-12-17 中国医学科学院基础医学研究所 Vesicular stomatitis virus envelope glycoprotein variant, and construction method and application thereof
WO2020029274A1 (en) * 2018-08-10 2020-02-13 苏州奥特铭医药科技有限公司 Preparation method for attenuated baculovirus and application thereof
WO2022033469A1 (en) * 2020-08-14 2022-02-17 上海行深生物科技有限公司 Recombinant oncolytic virus, and construction method therefor and use thereof
WO2024056106A1 (en) * 2022-09-16 2024-03-21 中国科学院广州生物医药与健康研究院 Segmented vesicular stomatitis virus vector, preparation method therefor, and use thereof
WO2024114673A1 (en) * 2022-11-29 2024-06-06 浙江迪福润丝生物科技有限公司 Method for attenuating vesicular stomatitis virus, attenuated virus strain, and use thereof

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106794240A (en) * 2014-03-01 2017-05-31 普罗菲克图斯生物科学股份有限公司 Restructuring isfahan virus carrier
WO2019061691A1 (en) * 2017-09-26 2019-04-04 王红伟 Cell line capable of implementing induced rescue of rhabdovirus, construction method therefor, and application thereof
CN110577585A (en) * 2018-06-07 2019-12-17 中国医学科学院基础医学研究所 Vesicular stomatitis virus envelope glycoprotein variant, and construction method and application thereof
WO2020029274A1 (en) * 2018-08-10 2020-02-13 苏州奥特铭医药科技有限公司 Preparation method for attenuated baculovirus and application thereof
WO2022033469A1 (en) * 2020-08-14 2022-02-17 上海行深生物科技有限公司 Recombinant oncolytic virus, and construction method therefor and use thereof
WO2024056106A1 (en) * 2022-09-16 2024-03-21 中国科学院广州生物医药与健康研究院 Segmented vesicular stomatitis virus vector, preparation method therefor, and use thereof
WO2024114673A1 (en) * 2022-11-29 2024-06-06 浙江迪福润丝生物科技有限公司 Method for attenuating vesicular stomatitis virus, attenuated virus strain, and use thereof

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